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WO2026027759A1 - Agents and methods for targeted delivery to cells - Google Patents

Agents and methods for targeted delivery to cells

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Publication number
WO2026027759A1
WO2026027759A1 PCT/EP2025/072237 EP2025072237W WO2026027759A1 WO 2026027759 A1 WO2026027759 A1 WO 2026027759A1 EP 2025072237 W EP2025072237 W EP 2025072237W WO 2026027759 A1 WO2026027759 A1 WO 2026027759A1
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Prior art keywords
moiety
compound
tag
payload
ethoxy
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Application number
PCT/EP2025/072237
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French (fr)
Inventor
Ugur Sahin
Zhengxin Dong
Farrukh Vohidov
Griffin Robert Uller GILDERSLEEVE
Christian Hotz
Julia KOWNATZKI
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Biontech SE
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Biontech SE
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Publication of WO2026027759A1 publication Critical patent/WO2026027759A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention relates to agents and methods for targeted delivery of payloads to cells. The payload comprises immunomodulators as further specified herein. In some embodiments, the invention involves providing to a subject a compound comprising a payload moiety and at least one tag (e.g., at least two tags) (tag conjugate) and a compound binding to the tag(s) of the tag conjugate and a target antigen, e.g., a cell surface antigen on a target cell, (docking compound).

Description

AGENTS AND METHODS FOR TARGETED DELIVERY TO CELLS
The invention relates to agents and methods for targeted delivery of payloads to cells. The payload comprises immunomodulators as further specified herein. In some embodiments, the invention involves providing to a subject a compound comprising a payload moiety and at least one tag (e.g., at least two tags) (tag conjugate) and a compound binding to the tag(s) of the tag conjugate and a target antigen, e.g., a cell surface antigen on a target cell, (docking compound). In some embodiments, the docking compound comprises a peptide or polypeptide. In some embodiments, the docking compound comprises a binding moiety binding to target cells (primary targeting moiety) and a further binding moiety binding to the tag(s) of the tag conjugate. The tag(s) of the tag conjugate may bind to its (their) binding moiety on the docking compound and the primary targeting moiety of the docking compound may bind to a target antigen on target cells such as an antigen on cancer cells to thereby precisely deliver a payload to the target cells. In some embodiments, the docking compound is provided to a subject by administering nucleic acid, e.g., RNA, encoding the docking compound. In some embodiments, the docking compound is provided to a subject by administering the docking compound. In some embodiments, a preformed complex wherein the docking compound is bound to the tag conjugate is provided to a subject by administration.
In many areas of medical therapy and diagnosis, it is desired to selectively deliver an agent, such as a therapeutic agent (a drug) or a diagnostic agent (e.g. an imaging agent), to a specific cell in the body of a subject such as a patient.
Active targeting of a cell may be achieved by the direct or indirect conjugation of the desired payload to a targeting moiety, which binds to cell surface antigens on the target cell of interest. The targeting moieties are typically constructs that have affinity for cell surface targets, e.g., membrane proteins, and include antibodies or antibody fragments.
The present invention relates to an approach wherein a docking compound that binds to target cells, e.g., by binding to a cell surface antigen, is used. The docking compound further comprises a moiety, which binds to a compound comprising a payload and being equipped with at least one tag (e.g., at least two tags) which is (are) targeted by the moiety on the docking compound. In some embodiments, a docking compound which binds to a tag conjugate comprising a payload is administered in the form of nucleic acid encoding the docking compound for expressing of the docking compound in a subject. The docking compound may bind to target cells, e.g., by binding to a cell surface antigen, thus resulting in targeting of the payload. Common examples for pairs of interacting moieties on the docking compound and on the tag conjugate are antibody/tag systems. The concept described herein allows to use a single type of tag conjugate for targeting a wide range of target cells, i.e., by using a single type of tag conjugate in combination with different docking compounds targeting different primary targets. The concept described herein is of further advantage, as the primary targeting using a single docking compound can be carried out in combination with different tag conjugates comprising different payloads.
Summary
The invention relates to agents and methods for targeted delivery of payloads to cells. The payload comprises an immunomodulator as specified herein. Targeted delivery of a payload is achieved using a tag conjugate described herein comprising the payload and a docking compound binding to the tag conjugate via its tag(s), said docking compound comprising a targeting molecule for targeting an antigen on target cells.
In one aspect, the invention relates to a kit comprising:
(i) a compound comprising a binding moiety binding to a target antigen and a binding moiety for a tag, or a nucleic acid encoding said compound; and
(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist.
In some embodiments, the STING agonist has the following Formula (XX): wherein:
Ring A is selected from the group consisting of
G and Gi are independently N, CH, or C-X1-R2; or when G and Gi are each C-X1-R2, the R2 groups are optionally linked to form L2;
G' and G2 are independently N or CH;
X is N-R, O, or S;
X' is N or CH; Xi is CH2, 0 or S;
R is hydrogen or C1-4 alkyl;
L1 and L2 are each independently C2-4 alkylene or C2-4 alkenylene;
R2 is selected from the group consisting of hydrogen, C2-4 cyclic ether, C1.4 alkylene-(C2-4 cyclic
0 o
H2N^M^ HOAA ether), C3-4 cycloalkyl, C1-4 alkylene-(C34 cycloalkyl), C1-4 alkyl,
Ri and R3 are independently selected from the group consisting of
Ring B is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 2 heteroatoms independently selected from N, 0, and S;
Rs is -OH or -NR9R10;
R9 and Rio are independently selected from hydrogen and C1-6 alkyl;
X2 and X3 are independently NH or S;
Yi and Y2 are independently a 5-membered heteroaryl or heterocyclic ring, wherein the 5- membered heteroaryl or heterocyclic ring (i) has 1 to 4 heteroatoms independently selected from N, O, and S, (ii) is attached to the remainder of the STING agonist via a C ring atom of the 5-membered heteroaryl or heterocyclic ring, and (iii) is optionally substituted with 1 to 4 R21, wherein each R21 is independently C1-4 alkyl (such as methyl, ethyl, propyl, or butyl); Rs, Re, and R7 are independently selected from hydrogen, Ci-e alkyl, C2-6 alkenyl, , or Rs and Re are optionally connected to form a 5- or 6- membered heterocyclic ring;
Ris is -OH or -NR9R10;
Ring C is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 ring heteroatoms independently selected from N, 0, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 4 ring heteroatoms independently selected from N, O, and S; n, p, q, q', t, and v are independently an integer from 2 to 6 (i.e., 2, 3, 4, 5, or 6, such as 2, 3, 4, or 5, e.g., 2, 3, or 4); and k, I, m, o, u, and w are independently an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6, such as 1, 2, 3, 4, or 5, e.g., 1, 2, 3, or 4).
In some preferred embodiments, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX). In some preferred embodiments, the payload moiety comprises a STING agonist moiety of Formula (XX1) as disclosed herein.
Preferred embodiments of the STING agonist of Formula (XX) and/or of the STING agonist moiety of Formula (XX') are disclosed herein and include: (1) any one of Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), (XXK), (XX-I) to (XX-LXII), (XX-1) to (XX-73), (XX- 6'), (XX-18'), (XX-51'), (XX-531), (XX-73'a), and (XX-73'b); (2) the embodiments of substituents specified herein with respect to any one of Formulas (XX), (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), (XXK), and (XX-I) to (XX-LXII) (e.g., that in some embodiments of Formula (XX), G? is CH); and (iii) the embodiments of substituents specified herein with respect to any one of Formulas (XX1), (XXA'), (XXB'), (XXC), (XXD'), (XXE'), (XXF1), (XXG'), (XXH'), (XXJ'), (XXK1), (XX-6'), (XX-18'), (XX-511), (XX-53'), (XX-73'a), and (XX-73'b) (e.g., that in some embodiments of Formula (XX1), one of the monovalent substituents present in the STING agonist of Formula (XX) (e.g., R2 being hydrogen or C1-4 alkyl; or Ri being N(R6)(R?)N(Rs)C(0)-) has been replaced by a corresponding divalent substituent (e.g., R2 has been replaced by R21, wherein R2' is a bond (if R2 was hydrogen) or C1-4 alkylene (if R2 was a C1-4 alkyl); or Ri has been replaced by Rr, wherein Rr is selected from the group consisting of *-N(Re)N(Rs)C(O)-#, *- N (Rs)C(O)-#, and -C(0)-, wherein * represents the attachment point of R2' and Rr, respectively, to the remainder of the compound under (ii); and # represents the attachment point of R2' and Rr, respectively, to the remainder of the STING agonist moiety of Formula (XX')).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXA) as disclosed herein, wherein G is CH, C-SCH3, C-OCH3, or N. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXA), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXA1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXB) as disclosed herein, wherein G is CH, C-SCH3, C-OCH3, or N; Rs is -OH or -NH2; Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXB), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXB1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXC) as disclosed herein, wherein Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXC), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXC).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXD) as disclosed herein, wherein R?r is hydrogen or C1-4 alkyl. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXD), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXD1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXE) as disclosed herein, wherein X is S or O. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXE), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXE').
In some embodiments, the STING agonist of Formula (XX) has Formula (XXF) as disclosed herein, wherein G is CH, C-SCH3, C-OCH3, or N. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXF), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXF').
In some embodiments, the STING agonist of Formula (XX) has Formula (XXG) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXG), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXG1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXH) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXH), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXH1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXJ) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXJ), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXJ').
In some embodiments, the STING agonist of Formula (XX) has Formula (XXK) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXK), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXK1).
In some preferred embodiments, the STING agonist of Formula (XX) has Formula (XXH) or (XXK) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXH') or (XXK1).
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-181) as disclosed herein, wherein R2' is a bond, -(CHz)3-, #- (CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably Rz is -(CH2)3-, #-(CH2)3N(CH3)-*, or #- (CH2)sN(CH3)NH-*, more preferably Rz is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point of Rz to the remainder of the compound under (ii); and * represents the attachment point of Rz to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-511) as disclosed herein, wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-73'a), wherein Rz is a bond, -(CH2)3-, or #-(CH2)3(piperazin-l,4-diyl)- *, preferably Rz is #-(CH2)3(piperazin-l,4-diyl)-* wherein * represents the attachment point of Rz to the remainder of the compound under (ii); and # represents the attachment point of Rz to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-73'b) as disclosed herein, wherein Rr is a bond, -C(O)-, #-C(O)NH- *, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-6') as disclosed herein, wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
In some particualry preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-53') as disclosed herein, wherein Rz is a bond, -(CH2)3-, (CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably Rz is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point of R2- to the remainder of the compound under (ii); and # represents the attachment point of R2' to the remainder of the STING agonist moiety.
In some embodiments, the compound under (ii) comprises one or more than one tag to which the binding moiety for a tag binds.
In some embodiments, the tag is a peptide tag.
In some embodiments, the compound under (ii) comprises a moiety comprising a polymer. In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising a polymer.
In some embodiments, the polymer is not a polymer of proteinogenic amino acids or their D- isomers.
In some embodiments, the polymer is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
In some embodiments, the polymer comprises poly(ethylene glycol) (PEG), or poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
In some embodiments, the polymer comprises at least one poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof. In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of poly(ethylene glycol) (PEG), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the nucleic acid is RNA.
In some embodiments, the compound under (ii) comprises one tag.
In some embodiments, the total number of tags in the compound under (ii) is one.
In some embodiments, the compound under (ii) comprises one or more payload moieties.
In some embodiments, the compound under (ii) comprises one payload moiety.
In some embodiments, the total number of payload moieties in the compound under (ii) is one.
In some embodiments, the compound under (ii) comprises one tag and one payload moiety.
In some embodiments, the total number of tags in the compound under (ii) is one and the total number of payload moieties in the compound under (ii) is one.
In some embodiments, the compound under (ii) comprises one tag and two payload moieties. In some embodiments, the total number of tags in the compound under (ii) is one and the total number of payload moieties in the compound under (ii) is two.
In some embodiments, the compound under (ii) is SD18317 (PIC8) as disclosed herein.
In some embodiments, the compound under (ii) is SD17248 as disclosed herein.
In some embodiments, the compound under (ii) is PIC12 as disclosed herein.
In some embodiments, the compound under (ii) is PIC13 as disclosed herein.
In some embodiments, the compound under (ii) is PIC14 as disclosed herein.
In some embodiments, the compound under (ii) is EX-M74 (PIC15) as disclosed herein.
In some embodiments, the compound under (ii) is EX-M69 (PIC16) as disclosed herein.
In some embodiments, the compound under (ii) is EX-M71 (PIC17) as disclosed herein.
In some embodiments, the compound under (ii) comprises two payload moieties.
In some embodiments, the total number of payload moieties in the compound under (ii) is two.
In some embodiments, the compound under (ii) comprises three payload moieties.
In some embodiments, the total number of payload moieties in the compound under (ii) is three.
In some embodiments, the compound under (ii) comprises four payload moieties.
In some embodiments, the total number of payload moieties in the compound under (ii) is four. In some embodiments, the compound under (ii) comprises one tag and three payload moieties.
In some embodiments, the total number of tags in the compound under (ii) is one and the total number of payload moieties in the compound under (ii) is three.
In some embodiments, the compound under (ii) comprises one tag and four payload moieties. In some embodiments, the total number of tags in the compound under (ii) is one and the total number of payload moieties in the compound under (ii) is four.
In some embodiments, the compound under (ii) comprises two tags and two payload moieties. In some embodiments, the total number of tags in the compound under (ii) is two and the total number of payload moieties in the compound under (ii) is two.
In some embodiments, the compound under (ii) is SD18321 (PIC7) as disclosed herein.
In some embodiments, the compound under (ii) is EX-B122 (PIC 18) as disclosed herein.
In some embodiments, the compound under (ii) is EX-B133 (PIC19) as disclosed herein.
In some embodiments, the compound under (ii) comprises two tags and three payload moieties.
In some embodiments, the total number of tags in the compound under (ii) is two and the total number of payload moieties in the compound under (ii) is three.
In some embodiments, the compound under (ii) comprises two tags and four payload moieties.
In some embodiments, the total number of tags in the compound under (ii) is two and the total number of payload moieties in the compound under (ii) is four.
In some embodiments, the compound under (ii) comprises a linking moiety connecting a tag and a payload moiety.
In some embodiments, the linking moiety is a branched or an unbranched linking moiety.
In some embodiments, the linking moiety comprises a continuous or non-continuous poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound under (ii) comprises the formula:
P-L-T wherein P comprises a payload moiety as specified herein;
T comprises a tag; and
L comprises a linking moiety.
In some embodiments, L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, L comprises the formula [AEEA]U-[L'-[AEEA]V]W, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
L' comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.
In some embodiments, u and v are each integers from 2 to 10.
In some embodiments, u and v are each integers from 2 to 8.
In some embodiments, u and v are each integers of 2, 4 or 6.
In some embodiments, L or L' comprises an amino acid.
In some embodiments, Lor L' comprises the D-isomer of an amino acid.
In some embodiments, Lor L' comprises cysteine or lysine.
In some embodiments, L or L' is connected to a side chain.
In some embodiments, a side chain comprises a functional moiety.
In some embodiments, a functional moiety comprises a solubilizing functional group.
In some embodiments, a functional moiety comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound under (ii) comprises at least two of said tags.
In some embodiments, the total number of tags in the compound under (ii) is two.
In some embodiments, the compound under (ii) comprises one or more payload moieties.
In some embodiments, the total number of payload moieties in the compound under (ii) is one. In some embodiments, the compound under (ii) comprises two or more payload moieties.
In some embodiments, the total number of payload moieties in the compound under (ii) is two.
In some embodiments, the compound under (ii) comprises payload moieties and tags in an unbranched (linear) configuration.
In some embodiments, the compound under (ii) comprises at least two tags and at least one payload moiety in an unbranched (linear) configuration.
In some embodiments, the compound under (ii) comprises at least two tags and at least two payload moieties in an unbranched (linear) configuration.
In some embodiments, the compound under (ii) comprises two tags and one or two payload moieties in an unbranched (linear) configuration.
In some embodiments, the compound under (ii) comprises one or more linking moieties connecting tags and payload moieties in an unbranched (linear) configuration.
In some embodiments, a linking moiety is a branched or unbranched linking moiety.
In some embodiments, at least one linking moiety comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound under (ii) comprises two tags which are connected by a linking moiety
In some embodiments, the linking moiety connecting the tags comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, a payload moiety is connected to at least one of the tags.
In some embodiments, a payload moiety is connected to a tag by a linking moiety.
In some embodiments, the linking moiety connecting a payload moiety and a tag comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the linking moiety connecting a payload moiety and a tag comprises an enzymatic cleavage site.
In some embodiments, the compound under (ii) comprises the formula:
P-LA-T-LB-T or
P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety as specified herein;
T comprises a tag;
LA comprises a linking moiety;
LB comprises a linking moiety; and
Lc comprises a linking moiety.
In some embodiments, one or more of LA, LB, and Lc comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LB comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Lc comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Ledo not comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Lc comprise an enzymatic cleavage site.
In some embodiments, one or more of LA, LB, and Lc comprises the formula [AEEA]U-[LD- [AEEA]v]w, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
LD comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [LD-[AEEA]V] may be identical or different.
In some embodiments, LB comprises the formula [AEEA]U-[LD-[AEEA]V]W, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof; LD comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [LD-[AEEA]V] may be identical or different.
In some embodiments, u and v are each integers from 2 to 10.
In some embodiments, u and v are each integers from 2 to 8.
In some embodiments, u and v are each integers of 2, 4 or 6.
In some embodiments, LD comprises an amino acid.
In some embodiments, LD comprises the D-isomer of an amino acid.
In some embodiments, LD comprises cysteine or lysine.
In some embodiments, Lois connected to a side chain.
In some embodiments, a side chain comprises a functional moiety.
In some embodiments, a functional moiety comprises a solubilizing functional group.
In some embodiments, the compound under (ii) comprises payload moieties and tags in a branched (non-linear) configuration.
In some embodiments, the compound under (ii) comprises one tag and at least two payload moieties in a branched (non-linear) configuration.
In some embodiments, the compound under (ii) comprises at least two tags and at least one payload moiety in a branched (non-linear) configuration.
In some embodiments, the compound under (ii) comprises at least two tags and at least two payload moieties in a branched (non-linear) configuration.
In some embodiments, the compound under (ii) comprises two tags and one or two payload moieties in a branched (non-linear) configuration.
In some embodiments, the compound under (ii) comprises one or more linking moieties connecting tags and payload moieties in a branched (non-linear) configuration.
In some embodiments, at least one linking moiety is a branched linking moiety.
In some embodiments, at least one linking moiety comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the compound under (ii) comprises two tags which are connected by a linking moiety
In some embodiments, the linking moiety connecting the tags comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, a payload moiety is connected to the linking moiety connecting the tags.
In some embodiments, a payload moiety is connected to the linking moiety connecting the tags by a linking moiety.
In some embodiments, the linking moiety connecting a payload moiety and a linking moiety connecting the tags comprises a continuous or non-continuous poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound under (ii) comprises a main chain connecting two tags and one or more side chains branching from the main chain and connecting one or more payload moieties to the main chain.
In some embodiments, one or more payload moieties are connected to the main chain through a moiety comprising a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, a side chain is connected to the main chain through a branching moiety in the main chain.
In some embodiments, the main chain comprises a continuous or non-continuous poly-2-(2- (2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the main chain comprises two or more poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or derivatives thereof.
In some embodiments, poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or derivatives thereof of the main chain are connected through a moiety comprising a branching moiety.
In some embodiments, the compound under (ii) comprises the formula:
[P-La]2-B-LrT wherein P comprises a payload moiety s specified herein;
B comprises a branching moiety;
T comprises a tag;
La comprises a linking moiety; and
Lt comprises a linking moiety; wherein the different groups [P-La] may be identical or different, and the different groups P may be identical or different.
In some embodiments, the valency of B corresponds to or is greater than 3.
In some embodiments, B comprises an amino acid or bis-amino acid.
In some embodiments, B comprises the D-isomer of an amino acid.
In some embodiments, B comprises cysteine or lysine.
In some embodiments, La comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, cysteine, or a combination thereof.
In some embodiments, La comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, La comprises an enzymatic cleavage site.
In some embodiments, La comprises a moiety which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the moiety which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is an amino acid. In some embodiments, the amino acid which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is cysteine.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is between 2 and 10. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is 2, 4 or 6.
In some embodiments, La is substituted with a solubilizing functional group. In some embodiments, La comprises an amino acid which is substituted with a solubilizing functional group. In some embodiments, the amino acid which is substituted with a solubilizing functional group is lysine or cysteine.
In some embodiments, Lt comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
In some embodiments, Lt comprises one or more selected from the group consisting of a moiety constraining conformation, a moiety for albumin binding, a moiety which increases circulation time, a moiety which reduces renal retention or uptake and an enzymatic cleavage site.
In some embodiments, the compound under (ii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, and EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)). In some preferred embodiments, the compound under (ii) comprises a structure of a compound shown herein as SD18317 (PIC8).
In some embodiments, the compound under (ii) comprises the formula:
[[Pk-Ldn-BHLz-TJo wherein P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety;
L2 comprises a linking moiety; m is an integer from 1 to 4; n is an integer from 1 to 4; and o is an integer from 2 to 4; wherein the different groups [L2-T] may be identical or different, the different groups P may be identical or different, and the different groups [[P]m-Li] may be identical or different.
In some embodiments, the valency of Bi corresponds to or is greater than the sum of n and o.
In some embodiments, Bicomprises an amino acid or bis-amino acid.
In some embodiments, Bi comprises the D-isomer of an amino acid.
In some embodiments, Bi comprises cysteine or lysine.
In some embodiments, Li comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, Li comprises an enzymatic cleavage site.
In some embodiments, L2 comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6. In some embodiments, L2 comprises one or more selected from the group consisting of a moiety constraining conformation, a moiety for albumin binding, a moiety which increases circulation time, a moiety which reduces renal retention or uptake and an enzymatic cleavage site.
In some embodiments, L2 comprises (i) a moiety comprising cysteine and (ii) a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, wherein the poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or derivative thereof and T are linked by the moiety comprising cysteine.
In some embodiments, [[P] m-Li] comprises the formula [[P-Lr]m-B2-Lr], wherein
B2 comprises a branching moiety;
Lr comprises a linking moiety;
Lr comprises a linking moiety; and m is an integer from 1 to 4; wherein the different groups [[P-Lr]m-B2-Lr] may be identical or different, and wherein in a group [[P- Li']m-B2-Li"] the different groups [P-Li ] may be identical or different.
In some embodiments, the valency of B2 corresponds to or is greater than the value of m plus 1.
In some embodiments, B2 comprises an amino acid.
In some embodiments, B2 comprises the D-isomer of an amino acid.
In some embodiments, B2 comprises cysteine or lysine.
In some embodiments, Lr comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, cysteine, or a combination thereof.
In some embodiments, Lr comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, Lr comprises a moiety which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the moiety which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is an amino acid. In some embodiments, the amino acid which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is lysine.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Lr is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Lr is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Li •• is 2, 4 or 6.
In some embodiments, Li- and/or Lr is substituted with a solubilizing functional group.
In some embodiments, Lr and/or Lr comprises an amino acid which is substituted with a solubilizing functional group.
In some embodiments, the amino acid which is substituted with a solubilizing functional group is lysine or cysteine.
In some embodiments, m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3.
In some embodiments, o is 2.
In some embodiments, n is 1 or 2.
In some embodiments, m is 1 or 2.
In some embodiments, o is 2, n is 1 and m is 1.
In some embodiments, o is 2, n is 2 and m is 2.
In some embodiments, the compound under (ii) comprises 1, 2, 3, 4, 5, or 6 molecules of a payload as specified herein, which molecules of a a payload are preferably covalently attached to the compound under (ii). In some embodiments, the compound under (ii) comprises 4 molecules of a payload as specified herein. In some embodiments, the compound under (ii) comprises 2 molecules of a payload as specified herein.
In some embodiments, the compound under (i) comprises one or more binding moieties for the tag. In some embodiments, the compound under (i) comprises a single binding moiety for the tag or at least two, e.g., two, binding moieties for the tag.
In some embodiments, the compound under (ii) comprises the formula:
P-LI-B1-[L2-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety; and
1.2 comprises a linking moiety; wherein the different groups [L2-T] may be identical or different.
In some embodiments, the compound under (ii) comprises the formula:
P-[AEEA]p-Bi-[[AEEA]q-R-[AEEA]r-C-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
R is optional and comprises a moiety constraining conformation;
C is optional and comprises a connecting moiety;
T comprises a tag; p is an integer from 0 to 6; q is an integer from 1 to 4; and r is an integer from 1 to 4; wherein the different groups [[AEEA]q-R-[AEEA]rC-T] may be identical or different.
In some embodiments, the compound under (ii) comprises the formula:
[[P-L1']2-B2-L1"]2-B1-[L2-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
B2 comprises a branching moiety;
T comprises a tag; Li' comprises a linking moiety;
Li" comprises a linking moiety; and
L2 comprises a linking moiety; wherein the different groups [ [P-L1J2-B2-L1-] may be identical or different, wherein in a group [[P-L1J2- B2-L1"] the different groups [P-Li] may be identical or different, and the different groups [L2- T] may be identical or different.
In some embodiments, the compound under (ii) comprises the formula:
[[P-Lij2-B2-[AEEA]5]2-Bi-[[AEEA]t-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
B2 comprises a branching moiety;
T comprises a tag;
Li' comprises a linking moiety; s is an integer from 2 to 8; and t is an integer from 2 to 8; wherein the different groups [[P-LI ]2-B2-[AEEA]S] may be identical or different, wherein in a group [[P- Lr]2-B2-[AEEA]s] the different groups [P-Li ] may be identical or different, and the different groups [[AEEA]t-T] may be identical or different.
In some embodiments, the tag is an ALFA-tag.
In some embodiments, the tag is a cyclic ALFA-tag.
In some embodiments, [AEEA] is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof.
In some embodiments, the compound under (ii) comprises the formula:
P-[AEEA]-Bi-[[AEEA]2-C-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
C is optional and comprises a connecting moiety; and T comprises an ALFA-tag; wherein the different groups [[AEEA]2-C-T] may be identical or different.
In some embodiments, Bi comprises an amino acid. In some embodiments, C comprises an amino acid.
In some embodiments, the compound under (ii) comprises the formula:
P-[AEEA]-BI-[[AEEA]2-C-T]2 wherein
P comprises a payload moiety as specified herein ;
Bi comprises an amino acid, preferably lysine;
C comprises a cysteine moiety; and
T comprises an ALFA-tag; wherein the different groups [[AEEA]2-C-T] may be identical or different.
In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-cyclo(Asp- Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some embodiments, T comprises an ALFA-tag of the formula -Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.
In some embodiments, the compound under (ii) comprises a structure of a compound shown herein as any one of SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), and EX-B122 to EX-B132 (in particular, EX-B122 (PIC18), EX-B133 (PIC19)). In some preferred embodiments, the compound under (ii) comprises a structure of a compound shown herein as SD18321 (PIC7).
In some embodiments, the compound under (ii) comprises at least two chains, wherein each of said chains comprises a tag, and wherein the at least two chains are covalently connected. In some embodiments, the compound under (ii) comprises two chains, wherein each of said chains comprises a tag, and wherein the two chains are covalently connected. In some embodiments, at least one of the chains comprises at least one payload moiety. In some embodiments, each of the chains comprises at least one payload moiety. In some embodiments, the compound under (ii) comprises two chains, wherein each of said chains comprises a tag and a payload moiety, and wherein the two chains are covalently connected. In some embodiments, the compound under (ii) comprises two chains, wherein each of said chains comprises a tag and two payload moieties, and wherein the two chains are covalently connected.
In some embodiments, the tag comprises an ALFA-tag.
In some embodiments, the compound under (ii) comprises two chains, wherein each of said chains comprises an ALFA-tag and two payload moieties, and wherein the two chains are covalently connected.
In some embodiments, the covalent connection comprises a triazole. In some embodiments, the covalent connection comprises a 1,2,3-triazole. In some embodiments, the covalent connection is formed through an intermolecular cycloaddition ("click") reaction between azides and alkynes comprised in the chains to be connected.
In some embodiments, the compound under (ii) comprises two chains, wherein each of said chains comprises an ALFA-tag and two payload moieties, and wherein the two chains are covalently connected through a moiety comprising a 1,2,3-triazole.
In some embodiments, each chain comprises a moiety of the formula:
*-[[AEEA]2-C-T] wherein
C is optional and comprises a connecting moiety;
T comprises a tag; and
* is the attachment point to a moiety that forms a covalent connection to another of the chains.
In some embodiments, the compound under (ii) comprises two chains wherein each chain comprises a moiety of the formula:
*-[[AEEA]2-C-T] wherein
C is optional and comprises a connecting moiety;
T comprises a tag; and
* is the attachment point to a moiety that forms a covalent connection to the other chain.
In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises one or more payload moieties. In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises two payload moieties.
In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises a 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2.
In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises a main chain extending from the attachment point * and comprising two amino acids, wherein the two amino acids are connected through a moiety comprising a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, and wherein the two amino acids are each connected to a payload moiety through their side chains. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2- (2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2. In some embodiments, the amino acid comprises cysteine. In some embodiments, the compound under (ii) comprises two chains wherein each chain comprises a moiety of the formula:
Cys-[AEEA]u-Cys-A-[AEEA]2-C-T wherein
A is optional and comprises a moiety attaching the moiety Cys-[AEEA]U-Cys to the moiety [AEEAh-C-T;
C is optional and comprises a connecting moiety;
T comprises a tag;
Cys is cysteine connected to a payload moiety through its side chain; and u is 1 or 2. In some embodiments, the compound under (ii) comprises two chains wherein each chain comprises a moiety of the formula:
Cys-[AEEA]U-Cys-A-[AEEA]2-C-T wherein
A is optional and comprises a moiety attaching the moiety Cys-[AEEA]U-Cys to the moiety [AEEAh-C-T;
C is optional and comprises a connecting moiety;
T comprises an ALFA-tag;
Cys is cysteine connected to a payload moiety through its side chain; and u is 1 or 2.
In some embodiments, the compound under (ii) comprises two chains wherein each chain comprises a moiety of the formula:
Cys-[AEEA]U-Cys-A-[AEEA]2-C-T wherein
A is optional and comprises a moiety attaching the moiety Cys-[AEEA]U-Cys to the moiety [AEEAh-C-T;
C is optional and comprises a connecting moiety;
T comprises an ALFA-tag;
Cys is cysteine connected to a payload through its side chain; and u is 1 or 2.
In some embodiments, C comprises an amino acid.
In some embodiments, A comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2. In some embodiments, A forms a covalent connection to the other chain. In some embodiments, the covalent connection comprises a triazole. In some embodiments, the covalent connection comprises a 1,2,3-triazole. In some embodiments, the covalent connection is formed through an intermolecular cycloaddition ("click") reaction between azides and alkynes comprised in the chains to be connected.
In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-cyclo(Glu- Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-.
In some embodiments, the compound under (ii) comprises a structure of a compound shown herein as EX-053, EX-054, SD18321 (PIC7), EX-B122 (PIC18), or EX-B133 (PIC19).
In some embodiments, the compound under (ii) comprises a structure of a compound shown herein, in particular of a compound shown in the Examples. In some embodiments, the compound under (ii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), and EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19)), EX-024 to EX-035, EX-032 to EX-035, and EX-047 to EX-054. In some embodiments, the compound under (ii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD1832O (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19). In some preferred embodiments, the compound under (ii) comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8). In some particularly preferred embodiments, the compound under (ii) comprises a structure of a compound shown herein as SD18321 (PIC7). It should be understood that those structures containing a STING agonist as payload may also contain any other STING agonist (in particular any other STING agonist as specified herein, such as any other STING agonist of Formula (XX)) instead of the specific STING agonist shown in these structures. It should further be understood that those structures containing a placeholder reference to a to a toxin or immunomodulator may also contain a STING agonist (in particular a STING agonist as specified herein, such as a STING agonist of Formula (XX)) instead of the placeholder reference shown in these structures. It should further be understood that those structures containing a group other than a STING agonist as payload (e.g., which contain a toxin or a chelator (with or without a radioisotope)) may also contain a STING agonist (in particular a STING agonist as specified herein, such as a STING agonist of Formula (XX)) as payload instead of the group other than a STING agonist. It should further be understood that those structures containing a STING agonist as payload may also contain any other STING agonist instead of the specific STING agonist shown in these structures.
In some embodiments of all aspects and embodiments of the kit described herein, the payload comprises the STING agonist of Formula (XX).
In some embodiments of all aspects and embodiments of the kit described herein, the payload comprises the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all aspects and embodiments of the kit described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
In some embodiments of all aspects and embodiments of the kit described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all aspects and embodiments of the kit described herein, the payload moiety comprises a STING agonist moiety having the Formula (XX-181), (XX-61), (XX-51'), (XX- 53'), (XX-73'a), or (XX-73'b).
In some embodiments of all aspects and embodiments of the kit described herein, the tag is an ALFA-tag.
In some embodiments of all aspects and embodiments of the kit described herein, the tag is a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the kit described herein, the payload comprises the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the kit described herein, the payload comprises the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag. In some embodiments of all aspects and embodiments of the kit described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the kit described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the kit described herein, the payload moiety comprises a STING agonist moiety having the Formula (XX-18'), (XX-61), (XX-511), (XX- 53'), (XX-73'a), or (XX-73'b), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the kit described herein, the compound under (ii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19), or the compound under (ii) is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19). In some preferred embodiments of the kit described herein, the compound under (ii) comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8), or the compound under (ii) is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8). In some particularly preferred embodiments of the kit described herein, the compound under (ii) comprises a structure of a compound shown herein as SD18321 (PIC7), or the compound under (ii) is SD18321 (PIC7).
In one aspect, the invention relates to the compound under (ii) described above.
In one aspect, the invention relates to a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist.
In some embodiments, the STING agonist has the following Formula (XX):
wherein:
Ring A is selected from the group consisting of
G and Gi are independently N, CH, or C-Xi-Rz; or when G and Gi are each C-X1-R2, the Rz groups are optionally linked to form L2;
G' and Gz are independently N or CH;
X is N-R, O, or S;
X' is N or CH;
Xi is CH2, 0 or S;
R is hydrogen or C1-4 alkyl;
L1 and L2 are each independently C2-4 alkylene or C2-4 alkenylene;
Rz is selected from the group consisting of hydrogen, C2-4 cyclic ether, C1-4 alkylene-(C2-4 cyclic o o
Ri and R3 are independently selected from the group consisting of
Ring B is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 2 heteroatoms independently selected from N, O, and S;
Rs is -OH or -NR9R10;
R9 and Rio are independently selected from hydrogen and C1-6 alkyl;
X2 and X3 are independently NH or S;
Yi and Y2 are independently a 5-membered heteroaryl or heterocyclic ring, wherein the 5- membered heteroaryl or heterocyclic ring (i) has 1 to 4 heteroatoms independently selected from N, O, and S, (ii) is attached to the remainder of the STING agonist via a C ring atom of the
5-membered heteroaryl or heterocyclic ring, and (iii) is optionally substituted with 1 to 4 R21, wherein each R21 is independently C1-4 alkyl (such as methyl, ethyl, propyl, or butyl);
Rs, Re, and R7 are independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, , or Rs and Re are optionally connected to form a 5- or 6- membered heterocyclic ring;
Ris is -OH or -NR9R10;
Ring C is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 ring heteroatoms independently selected from N, 0, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 4 ring heteroatoms independently selected from N, O, and S; n, p, q, q', t, and v are independently an integer from 2 to 6 (i.e., 2, 3, 4, 5, or 6, such as 2, 3,
4, or 5, e.g., 2, 3, or 4); and k, I, m, o, u, and w are independently an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6, such as 1, 2, 3, 4, or 5, e.g., 1, 2, 3, or 4).
In some preferred embodiments, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX). In some preferred embodiments, the payload moiety comprises a STING agonist moiety of Formula (XX') as disclosed herein.
Preferred embodiments of the STING agonist of Formula (XX) and/or of the STING agonist moiety of Formula (XX') are disclosed herein and include: (1) any one of Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), (XXK), (XX-I) to (XX-LXII), (XX-1) to (XX-73), (XX- 6'), (XX-18'), (XX-511), (XX-53'), (XX-73'a), and (XX-73'b); (2) the embodiments of substituents specified herein with respect to any one of Formulas (XX), (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), (XXK), and (XX-I) to (XX-LXII) (e.g., that in some embodiments of Formula (XX), G2 is CH); and (iii) the embodiments of substituents specified herein with respect to any one of Formulas (XX'), (XXA1), (XXB'), (XXC), (XXD1), (XXE'), (XXF'), (XXG'), (XXH1), (XXJ'), (XXK1), (XX-61), (XX-18'), (XX-51'), (XX-531), (XX-73'a), and (XX-73'b) (e.g., that in some embodiments of Formula (XX1), one of the monovalent substituents present in the STING agonist of Formula (XX) (e.g., R2 being hydrogen or C1-4 alkyl; or Ri being N(R6)(R?)N(Rs)C(O)-) has been replaced by a corresponding divalent substituent (e.g., R? has been replaced by R2', wherein Rz1 is a bond (if R2 was hydrogen) or C1-4 alkylene (if R2 was a C1-4 alkyl); or Ri has been replaced by Rr, wherein Rr is selected from the group consisting of *-N(Re)N(Rs)C(O)-#, *- N(Rs)C(O)-#, and -C(O)-, wherein * represents the attachment point of R2' and Rr, respectively, to the remainder of the compound under (ii); and # represents the attachment point of R21 and Ri', respectively, to the remainder of the STING agonist moiety of Formula (XX')).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXA) as disclosed herein, wherein G is CH, C-SCH3, C-OCH3, or N. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXA), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXA').
In some embodiments, the STING agonist of Formula (XX) has Formula (XXB) as disclosed herein, wherein G is CH, C-SCHs, C-OCH3, or N; Rs is -OH or -NH2; Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXB), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXB').
In some embodiments, the STING agonist of Formula (XX) has Formula (XXC) as disclosed herein, wherein Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXC), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXC).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXD) as disclosed herein, wherein R21' is hydrogen or C1-4 alkyl. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXD), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXD1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXE) as disclosed herein, wherein X is S or O. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXE), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXE').
In some embodiments, the STING agonist of Formula (XX) has Formula (XXF) as disclosed herein, wherein G is CH, C-SCH3, C-OCH3, or N. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXF), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXF1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXG) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXG), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXG1). In some embodiments, the STING agonist of Formula (XX) has Formula (XXH) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXH), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXH1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXJ) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXJ), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXJ1).
In some embodiments, the STING agonist of Formula (XX) has Formula (XXK) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXK), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXK1).
In some preferred embodiments, the STING agonist of Formula (XX) has Formula (XXH) or (XXK) as disclosed herein. In some preferred embodiments, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK), more preferably, the payload moiety comprised in the compound under (ii) comprises a STING agonist moiety of Formula (XXH') or (XXK1).
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-181) as disclosed herein, wherein Rz1 is a bond, -(CH2)3-, #- (CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably R2' is -(CH2)3-, #-(CH2)3N(CH3)-*, or #- (CH2)3N(CH3)NH-*, more preferably R2' is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point of Rz to the remainder of the compound under (ii); and # represents the attachment point of Rz to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-511) as disclosed herein, wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Ri' to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-73'a), wherein R21 is a bond, -(CH 2)3-, or *-(CH2)3(piperazin-l,4-diyl)- *, preferably R2' is #-(CH2)3(piperazin-l,4-diyl)-* wherein * represents the attachment point of R2' to the remainder of the compound under (ii); and # represents the attachment point of R2' to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-73'b) as disclosed herein, wherein Rr is a bond, -C(O)-, #-C(O)NH- *, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
In some particularly preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-61) as disclosed herein, wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
In some particualry preferred embodiments, the payload moiety comprises a STING agonist moiety having Formula (XX-531) as disclosed herein, wherein R2' is a bond, -(CFhh-, #- (CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably Rr is MCH2)3N(CH3)NH-*, wherein * represents the attachment point of R2' to the remainder of the compound under (ii); and # represents the attachment point of R?' to the remainder of the STING agonist moiety.
In some embodiments, the compound comprises one or more than one tag to which the binding moiety for a tag binds.
In some embodiments, the tag is a peptide tag.
In some embodiments, the compound comprises a moiety comprising a polymer.
In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising a polymer. In some embodiments, the polymer is not a polymer of proteinogenic amino acids or their D- isomers.
In some embodiments, the polymer is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
In some embodiments, the polymer comprises polyethylene glycol) (PEG), or poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
In some embodiments, the polymer comprises at least one poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of poly(ethylene glycol) (PEG), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the payload moiety and the tag or tags are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety.
In some embodiments, the compound comprises one tag.
In some embodiments, the total number of tags in the compound is one.
In some embodiments, the compound comprises one or more payload moieties.
In some embodiments, the compound comprises one payload moiety.
In some embodiments, the total number of payload moieties in the compound is one.
In some embodiments, the compound comprises one tag and one payload moiety.
In some embodiments, the total number of tags in the compound is one and the total number of payload moieties in the compound is one. In some embodiments, the compound comprises one tag and two payload moieties.
In some embodiments, the total number of tags in the compound is one and the total number of payload moieties in the compound is two.
In some embodiments, the compound is SD18317 (PIC8) as disclosed herein.
In some embodiments, the compound is SD17248 as disclosed herein.
In some embodiments, the compound is PIC12 as disclosed herein.
In some embodiments, the compound is PIC13 as disclosed herein.
In some embodiments, the compound is PIC14 as disclosed herein.
In some embodiments, the compound is EX-M69 (PIC16) as disclosed herein.
In some embodiments, the compound is EX-M71 (PIC17) as disclosed herein.
In some embodiments, the compound is EX-M74 (PIC15) as disclosed herein.
In some embodiments, the compound comprises two payload moieties.
In some embodiments, the total number of payload moieties in the compound is two.
In some embodiments, the compound comprises three payload moieties.
In some embodiments, the total number of payload moieties in the compound is three.
In some embodiments, the compound comprises four payload moieties.
In some embodiments, the total number of payload moieties in the compound is four.
In some embodiments, the compound comprises one tag and three payload moieties.
In some embodiments, the total number of tags in the compound is one and the total number of payload moieties in the compound is three.
In some embodiments, the compound comprises one tag and four payload moieties.
In some embodiments, the total number of tags in the compound is one and the total number of payload moieties in the compound is four.
In some embodiments, the compound comprises two tags and two payload moieties.
In some embodiments, the total number of tags in the compound) is two and the total number of payload moieties in the compound is two.
In some embodiments, the compound comprises two tags and three payload moieties.
In some embodiments, the total number of tags in the compound is two and the total number of payload moieties in the compound is three.
In some embodiments, the compound comprises two tags and four payload moieties. In some embodiments, the total number of tags in the compound is two and the total number of payload moieties in the compound is four.
In some embodiments, the compound comprises a linking moiety connecting a tag and a payload moiety.
In some embodiments, the linking moiety is a branched or an unbranched linking moiety.
In some embodiments, the linking moiety comprises a continuous or non-continuous poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound comprises the formula:
P-L-T wherein
P comprises a payload moiety as specified herein;
T comprises a tag; and
L comprises a linking moiety.
In some embodiments, L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, Lcomprises the formula [AEEA]u-[L'-[AEEA]v]w, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
L' comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.
In some embodiments, u and v are each integers from 2 to 10.
In some embodiments, u and v are each integers from 2 to 8.
In some embodiments, u and v are each integers of 2, 4 or 6.
In some embodiments, L or L' comprises an amino acid.
In some embodiments, Lor L' comprises the D-isomer of an amino acid. In some embodiments, Lor L' comprises cysteine or lysine.
In some embodiments, L or L' is connected to a side chain.
In some embodiments, a side chain comprises a functional moiety.
In some embodiments, a functional moiety comprises a solubilizing functional group.
In some embodiments, the compound comprises at least two of said tags.
In some embodiments, the total number of tags in the compound is two.
In some embodiments, the compound comprises one or more payload moieties.
In some embodiments, the total number of payload moieties in the compound is one.
In some embodiments, the compound comprises two or more payload moieties.
In some embodiments, the total number of payload moieties in the compound is two.
In some embodiments, the compound comprises payload moieties and tags in an unbranched (linear) configuration.
In some embodiments, the compound comprises at least two tags and at least one payload moiety in an unbranched (linear) configuration.
In some embodiments, the compound comprises at least two tags and at least two payload moieties in an unbranched (linear) configuration.
In some embodiments, the compound comprises two tags and one or two payload moieties in an unbranched (linear) configuration.
In some embodiments, the compound comprises one or more linking moieties connecting tags and payload moieties in an unbranched (linear) configuration.
In some embodiments, a linking moiety is a branched or unbranched linking moiety.
In some embodiments, at least one linking moiety comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound comprises two tags which are connected by a linking moiety
In some embodiments, the linking moiety connecting the tags comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, a payload moiety is connected to at least one of the tags.
In some embodiments, a payload moiety is connected to a tag by a linking moiety. In some embodiments, the linking moiety connecting a payload moiety and a tag comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the linking moiety connecting a payload moiety and a tag comprises an enzymatic cleavage site.
In some embodiments, the compound comprises the formula:
P-LA-T-LB-T or
P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety as specified herein;
T comprises a tag;
LA comprises a linking moiety;
LB comprises a linking moiety; and
Lc comprises a linking moiety.
In some embodiments, one or more of LA, LB, and Lc comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LB comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Lc comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Ledo not comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Lc comprise an enzymatic cleavage site.
In some embodiments, one or more of LA, LB, and Lc comprises the formula [AEEA]U-[LD- [AEEA]v]w, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
LD comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [LD-[AEEA]V] may be identical or different.
In some embodiments, LB comprises the formula [AEEA]U-[LD-[AEEA]V]W, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
LD comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [LD-[AEEA]V] may be identical or different.
In some embodiments, u and v are each integers from 2 to 10.
In some embodiments, u and v are each integers from 2 to 8.
In some embodiments, u and v are each integers of 2, 4 or 6.
In some embodiments, LD comprises an amino acid.
In some embodiments, LD comprises the D-isomer of an amino acid.
In some embodiments, LD comprises cysteine or lysine.
In some embodiments, LD is connected to a side chain.
In some embodiments, a side chain comprises a functional moiety.
In some embodiments, a functional moiety comprises a solubilizing functional group.
In some embodiments, the compound comprises payload moieties and tags in a branched (non-linear) configuration.
In some embodiments, the compound under (ii) comprises one tag and at least two payload moieties in a branched (non-linear) configuration.
In some embodiments, the compound comprises at least two tags and at least one payload moiety in a branched (non-linear) configuration.
In some embodiments, the compound comprises at least two tags and at least two payload moieties in a branched (non-linear) configuration. In some embodiments, the compound comprises two tags and one or two payload moieties in a branched (non-linear) configuration.
In some embodiments, the compound comprises one or more linking moieties connecting tags and payload moieties in a branched (non-linear) configuration.
In some embodiments, at least one linking moiety is a branched linking moiety.
In some embodiments, at least one linking moiety comprises a continuous or non-continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound comprises two tags which are connected by a linking moiety
In some embodiments, the linking moiety connecting the tags comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, a payload moiety is connected to the linking moiety connecting the tags.
In some embodiments, a payload moiety is connected to the linking moiety connecting the tags by a linking moiety.
In some embodiments, the linking moiety connecting a payload moiety and a linking moiety connecting the tags comprises a continuous or non-continuous poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the compound comprises a main chain connecting two tags and one or more side chains branching from the main chain and connecting one or more payload moieties to the main chain.
In some embodiments, one or more payload moieties are connected to the main chain through a moiety comprising a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, a side chain is connected to the main chain through a branching moiety in the main chain.
In some embodiments, the main chain comprises a continuous or non-continuous poly-2-(2- (2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the main chain comprises two or more poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or derivatives thereof.
In some embodiments, poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moieties or derivatives thereof of the main chain are connected through a moiety comprising a branching moiety.
In some embodiments, the compound comprises the formula:
[P-Lah-B-Lt-T wherein
P comprises a payload moiety s specified herein;
B comprises a branching moiety;
T comprises a tag;
La comprises a linking moiety; and
Lt comprises a linking moiety; wherein the different groups [P-La] may be identical or different, and the different groups P may be identical or different.
In some embodiments, the valency of B corresponds to or is greater than 3.
In some embodiments, B comprises an amino acid or bis-amino acid.
In some embodiments, B comprises the D-isomer of an amino acid.
In some embodiments, B comprises cysteine or lysine.
In some embodiments, La comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, cysteine, or a combination thereof.
In some embodiments, La comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, La comprises an enzymatic cleavage site.
In some embodiments, La comprises a moiety which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the moiety which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is an amino acid. In some embodiments, the amino acid which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is cysteine.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is 2, 4 or 6.
In some embodiments, La is substituted with a solubilizing functional group. In some embodiments, La comprises an amino acid which is substituted with a solubilizing functional group. In some embodiments, the amino acid which is substituted with a solubilizing functional group is lysine or cysteine.
In some embodiments, Lt comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
In some embodiments, Lt comprises one or more selected from the group consisting of a moiety constraining conformation, a moiety for albumin binding, a moiety which increases circulation time, a moiety which reduces renal retention or uptake and an enzymatic cleavage site. In some embodiments, the compound comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, and EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)). In some preferred embodiments, the compound comprises a structure of a compound shown herein as SD18317 (PIC8).
In some embodiments, the compound comprises the formula:
[[P]m-Ll]n-Bl-[L2-T]o wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety;
Lz comprises a linking moiety; m is an integer from 1 to 4; n is an integer from 1 to 4; and o is an integer from 2 to 4; wherein the different groups [Ls-T] may be identical or different, the different groups P may be identical or different, and the different groups [[P]m-Li] may be identical or different.
In some embodiments, the valency of Bi corresponds to or is greater than the sum of n and o. In some embodiments, Bi comprises an amino acid or bis-amino acid.
In some embodiments, Bi comprises the D-isomer of an amino acid.
In some embodiments, Bi comprises cysteine or lysine.
In some embodiments, Li comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, Li comprises an enzymatic cleavage site.
In some embodiments, L2 comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
In some embodiments, L2 comprises one or more selected from the group consisting of a moiety constraining conformation, a moiety for albumin binding, a moiety which increases circulation time, a moiety which reduces renal retention or uptake and an enzymatic cleavage site.
In some embodiments, L2 comprises (i) a moiety comprising cysteine and (ii) a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, wherein the poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or derivative thereof and T are linked by the moiety comprising cysteine.
In some embodiments, [[P]m-Li] comprises the formula [[P-Lijm-Bz-Lr], wherein
B2 comprises a branching moiety;
Li- comprises a linking moiety;
Lr comprises a linking moiety; and m is an integer from 1 to 4; wherein the different groups [[P-Lr]m-B2-Lr] may be identical or different, and wherein in a group [[P- Lr]m-B2-Li"] the different groups [P-Lr] may be identical or different.
In some embodiments, the valency of B2 corresponds to or is greater than the value of m plus 1.
In some embodiments, B2 comprises an amino acid.
In some embodiments, B2 comprises the D-isomer of an amino acid.
In some embodiments, B2 comprises cysteine or lysine. In some embodiments, Lr comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, cysteine, or a combination thereof.
In some embodiments, Lr comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, Lr comprises a moiety which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the moiety which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is an amino acid.
In some embodiments, the amino acid which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is lysine.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Lr is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Lr is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Lr is 2, 4 or 6.
In some embodiments, Lr and/or Lr is substituted with a solubilizing functional group.
In some embodiments, Lr and/or Lr1 comprises an amino acid which is substituted with a solubilizing functional group.
In some embodiments, the amino acid which is substituted with a solubilizing functional group is lysine or cysteine.
In some embodiments, m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3.
In some embodiments, o is 2.
In some embodiments, n is 1 or 2.
In some embodiments, m is 1 or 2.
In some embodiments, o is 2, n is 1 and m is 1. In some embodiments, o is 2, n is 2 and m is 2.
In some embodiments, the payload moiety comprises 1, 2, 3, 4, 5, or 6 molecules of a payload as specified herein, which molecules of a payload are preferably covalently attached to the compound. In some embodiments, the compound comprises 4 molecules of a payload as specified herein. In some embodiments, the compound comprises 2 molecules of a payload as specified herein.
In some embodiments, the compound comprises the formula:
P-LI-BI-[L2-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety; and
L2 comprises a linking moiety; wherein the different groups [L2-T] may be identical or different.
In some embodiments, the compound comprises the formula:
P-[AEEA]p-Bi-[[AEEA]q-R-[AEEA]r-C-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
R is optional and comprises a moiety constraining conformation;
C is optional and comprises a connecting moiety;
T comprises a tag; p is an integer from 0 to 6; q is an integer from 1 to 4; and r is an integer from 1 to 4; wherein the different groups [[AEEA]q-R-[AEEA]r-C-T] may be identical or different.
In some embodiments, the compound comprises the formula: [[P-Li-]2-B2-Lr]2-Bi-[L2-T]2 wherein P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
B2 comprises a branching moiety;
T comprises a tag;
Lr comprises a linking moiety;
Li " comprises a linking moiety; and
L2 comprises a linking moiety; wherein the different groups [[P-Lr]2-B2-Lr] may be identical or different, wherein in a group [[P-L1J2- B2-L1 '] the different groups [P-Lr] may be identical or different, and the different groups [L2- T] may be identical or different.
In some embodiments, the compound comprises the formula:
[[P-Li']2-B2-[AEEA]s]2-Bi-[[AEEA]t-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
B2 comprises a branching moiety;
T comprises a tag;
Lr comprises a linking moiety; s is an integer from 2 to 8; and t is an integer from 2 to 8; wherein the different groups [[P-Lr]2-B2-[AEEA]S] may be identical or different, wherein in a group [[P- Lr]2-B2-[AEEA]S] the different groups [P-Lr] may be identical or different, and the different groups [[AEEA]t-T] may be identical or different.
In some embodiments, the tag is an ALFA-tag.
In some embodiments, the tag is a cyclic ALFA-tag.
In some embodiments, [AEEA] is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof.
In some embodiments, the compound comprises the formula:
P-[AEEA]-BI-[[AEEA]2-C-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
C is optional and comprises a connecting moiety; and
T comprises an ALFA-tag; wherein the different groups [[AEEA] 2-C-T] may be identical or different.
In some embodiments, Bi comprises an amino acid. In some embodiments, C comprises an amino acid.
In some embodiments, the compound comprises the formula:
P-[AEEA]-BI-[[AEEA]2-C-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises an amino acid, preferably lysine;
C comprises a cysteine moiety; and
T comprises an ALFA-tag; wherein the different groups [[AEEA]2-C-T] may be identical or different.
In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-cyclo(Asp- Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some embodiments, T comprises an ALFA-tag of the formula -Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.
In some embodiments, the compound comprises a structure of a compound shown herein as any one of SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), and EX-B133 (PIC19)). In some preferred embodiments, the compound comprises a structure of a compound shown herein as SD18321 (PIC7).
In some embodiments, the compound comprises at least two chains, wherein each of said chains comprises a tag, and wherein the at least two chains are covalently connected. In some embodiments, the compound comprises two chains, wherein each of said chains comprises a tag, and wherein the two chains are covalently connected. In some embodiments, at least one of the chains comprises at least one payload moiety. In some embodiments, each of the chains comprises at least one payload moiety. In some embodiments, the compound comprises two chains, wherein each of said chains comprises a tag and a payload moiety, and wherein the two chains are covalently connected. In some embodiments, the compound comprises two chains, wherein each of said chains comprises a tag and two payload moieties, and wherein the two chains are covalently connected.
In some embodiments, the tag comprises an ALFA-tag.
In some embodiments, the compound comprises two chains, wherein each of said chains comprises an ALFA-tag and two payload moieties, and wherein the two chains are covalently connected.
In some embodiments, the covalent connection comprises a triazole. In some embodiments, the covalent connection comprises a 1,2,3-triazole. In some embodiments, the covalent connection is formed through an intermolecular cycloaddition ("click") reaction between azides and alkynes comprised in the chains to be connected.
In some embodiments, the compound comprises two chains, wherein each of said chains comprises an ALFA-tag and two payload moieties, and wherein the two chains are covalently connected through a moiety comprising a 1,2,3-triazole.
In some embodiments, each chain comprises a moiety of the formula:
*-[[AEEA]2-C-T] wherein
C is optional and comprises a connecting moiety;
T comprises a tag; and
* is the attachment point to a moiety that forms a covalent connection to another of the chains.
In some embodiments, the compound comprises two chains wherein each chain comprises a moiety of the formula:
*-[[AEEAh-C-T] wherein
C is optional and comprises a connecting moiety;
T comprises a tag; and
* is the attachment point to a moiety that forms a covalent connection to the other chain. In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises one or more payload moieties. In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises two payload moieties.
In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises a 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2.
In some embodiments, the moiety that forms a covalent connection to another of the chains or the moiety that forms a covalent connection to the other chain comprises a main chain extending from the attachment point * and comprising two amino acids, wherein the two amino acids are connected through a moiety comprising a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, and wherein the two amino acids are each connected to a payload moiety through their side chains. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2- (2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2. In some embodiments, the amino acid comprises cysteine. In some embodiments, the compound comprises two chains wherein each chain comprises a moiety of the formula:
Cys-[AEEA]U-Cys-A-[AEEA]2-C-T wherein
A is optional and comprises a moiety attaching the moiety Cys-[AEEA]U-Cys to the moiety [AEEA]2-C-T;
C is optional and comprises a connecting moiety;
T comprises a tag; Cys is cysteine connected to a payload moiety through its side chain; and u is 1 or 2.
In some embodiments, the compound comprises two chains wherein each chain comprises a moiety of the formula:
Cys-[AEEA]U-Cys-A-[AEEA]2-C-T wherein
A is optional and comprises a moiety attaching the moiety Cys-[AEEA]U-Cys to the moiety [AEEA]2-C-T;
C is optional and comprises a connecting moiety;
T comprises an ALFA-tag;
Cys is cysteine connected to a payload moiety through its side chain; and u is 1 or 2.
In some embodiments, the compound comprises two chains wherein each chain comprises a moiety of the formula:
Cys-[AEEA]U-Cys-A-[AEEA]2-C-T wherein
A is optional and comprises a moiety attaching the moiety Cys-[AEEA]U-Cys to the moiety [AEEA]2-C-T;
C is optional and comprises a connecting moiety;
T comprises an ALFA-tag;
Cys is cysteine connected to a payload through its side chain; and u is 1 or 2.
In some embodiments, C comprises an amino acid.
In some embodiments, A comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10 such as 2, 4 or 6, in particular 2. In some embodiments, A forms a covalent connection to the other chain. In some embodiments, the covalent connection comprises a triazole. In some embodiments, the covalent connection comprises a 1,2,3-triazole. In some embodiments, the covalent connection is formed through an intermolecular cycloaddition ("click") reaction between azides and alkynes comprised in the chains to be connected.
In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-cyclo(Glu- Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some embodiments, T comprises an ALFA-tag of the formula -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-.
In some embodiments, the compound comprises a structure of a compound shown herein as EX-053, EX-054, SD18321 (PIC7), EX-B122 (PIC18), or EX-B133 (PIC19). In some preferred embodiments, the compound comprises a structure of a compound shown herein as SD18321 (PIC7).
In some embodiments, the compound comprises a structure of a compound shown herein, in particular of a compound shown in the Examples. In some embodiments, the compound comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX- M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19), EX-024 to EX-035, EX-032 to EX-035, and EX- 047 to EX-054. In some preferred embodiments, the compound comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8). In some particularly preferred embodiments, the compound comprises a structure of a compound shown herein as SD18321 (PIC7). It should be understood that those structures containing a STING agonist as payload may also contain any other STING agonist (in particular a STING agonist as specified herein, such as a STING agonist of Formula (XX)) instead of the specific STING agonist shown in these structures. It should further be understood that those structures containing a placeholder reference to a to a toxin or immunomodulator may also contain a STING agonist (in particular a STING agonist as specified herein, such as a STING agonist of Formula (XX)) instead of the placeholder reference shown in these structures. It should further be understood that those structures containing a group other than a STING agonist as payload (e.g., which contain a toxin or a chelator (with or without a radioisotope)) may also contain a STING agonist (in particular a STING agonist as specified herein, such as a STING agonist of Formula (XX)) as payload instead of the group other than a STING agonist. It should further be understood that those structures containing a STING agonist as payload may also contain any other STING agonist instead of the specific STING agonist shown in these structures.
In some embodiments of all aspects and embodiments of the compound described herein, the payload comprises the STING agonist of Formula (XX).
In some embodiments of all aspects and embodiments of the compound described herein, the payload comprises the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all aspects and embodiments of the compound described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
In some embodiments of all aspects and embodiments of the compound described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all aspects and embodiments of the compound described herein, the payload moiety comprises a STING agonist moiety having the Formula (XX-181), (XX-6'), (XX- 51'), (XX-531), (XX-73'a), or (XX-73'b).
In some embodiments of all aspects and embodiments of the compound described herein, the tag is an ALFA-tag.
In some embodiments of all aspects and embodiments of the compound described herein, the tag is a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the compound described herein, the payload comprises the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the compound described herein, the payload comprises the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag. In some embodiments of all aspects and embodiments of the compound described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the compound described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the compound described herein, the payload moiety comprises a STING agonist moiety having the Formula (XX-181), (XX-6'), (XX- 51'), (XX-53'), (XX-73'a), or (XX-73'b), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag. In some embodiments of all aspects and embodiments of the compound described herein, the compound described herein comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19), or the compound described herein is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD1832O (PIC1O), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19). In some preferred embodiments of the compound described herein, the compound described herein comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8), or the compound described herein is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8). In some particularly preferred embodiments, the compound described herein comprises a structure of a compound shown herein as SD18321 (PIC7), or the compound described herein is SD18321 (PIC7).
In one aspect, the invention relates to a method for treating a subject having a disease, disorder or condition characterized by cells expressing a target antigen, comprising:
(i) providing to the subject a compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag; (ii) allowing the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag to become associated with cells expressing the target antigen; and
(iii) administering to the subject a compound described above, e.g., the compound under (ii) described above for the kit of the present invention, comprising one or more tags to which the binding moiety for a tag binds.
In some embodiments, the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is provided to the subject by administering to the subject RNA encoding a polypeptide comprising a binding moiety binding to the target antigen and a binding moiety for a tag; and allowing expression of the polypeptide by cells in the subject.
In some embodiments, the cells expressing the polypeptide are transfected with the RNA.
In some embodiments, the RNA is administered as particulate formulation such as formulated as lipid nanoparticles.
In some embodiments, the cells expressing the polypeptide secrete the polypeptide.
In some embodiments, the cells expressing the polypeptide express the polypeptide such that it is released into the bloodstream.
In some embodiments, the target antigen is a cell surface antigen.
In some embodiments, the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is a fusion polypeptide comprising the binding moiety binding to the target antigen and the binding moiety for a tag.
In some embodiments, the binding moiety binding to the target antigen comprises an antibody or an antibody derivative.
In some embodiments, the binding moiety for a tag comprises an antibody or an antibody derivative.
In some embodiments, the antibody derivative is an antibody fragment.
In some embodiments, the disease, disorder or condition is cancer.
In some embodiments, the cells expressing a target antigen are diseased cells.
In some embodiments, the cells expressing a target antigen are cancer cells.
In some embodiments, the target antigen is a tumor antigen. In some embodiments, the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag comprises a single binding moiety for the tag or at least two, e.g., two, binding moieties for the tag.
In some embodiments of all aspects and embodiments of the method described herein, the payload comprises the STING agonist of Formula (XX).
In some embodiments of all aspects and embodiments of the method described herein, the payload comprises the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all aspects and embodiments of the method described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
In some embodiments of all aspects and embodiments of the method described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all aspects and embodiments of the method described herein, the payload moiety comprises a STING agonist moiety having the Formula (XX-18'), (XX-6'), (XX- 51'), (XX-53'), (XX-73'a), or (XX-73'b).
In some embodiments of all aspects and embodiments of the method described herein, the tag is an ALFA-tag.
In some embodiments of all aspects and embodiments of the method described herein, the tag is a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the method described herein, the payload comprises the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the method described herein, the payload comprises the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the method described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag. In some embodiments of all aspects and embodiments of the method described herein, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all aspects and embodiments of the method described herein, the payload moiety comprises a STING agonist moiety having the Formula (XX-18'), (XX-6'), (XX- 51'), (XX-531), (XX-73'a), or (XX-73'b), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag. In some embodiments of all aspects and embodiments of the method described herein, the compound comprising one or more tags to which the binding moiety for a tag binds (i.e., the compound to be administered under (iii) in the method described herein) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19), EX-024 to EX-035, EX-032 to EX-035, and EX-047 to EX-054. In some embodiments of the method described herein, the compound to be administered under (iii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19), or the compound to be administered under (iii) is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19). In some preferred embodiments of the method described herein, the compound to be administered under (iii) comprises a structure of a compound shown herein as anyone of SD18321 (PIC7) and SD18317 (PIC8), or the compound to be administered under (iii) is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8). In some particularly preferred embodiments of the method described herein, the compound to be administered under (iii) comprises a structure of a compound shown herein as SD18321 (PIC7), or the compound to be administered under (iii) is SD18321 (PIC7). Brief description of the drawings
Figure 1: Plasma stability results of (a) EX-004, and (b) EX-007. All tested compounds were more stable in human plasma.
Figure 2: Association and dissociation curves of BisALFA conjugates in affinity and avidity measurements. Time was plotted on the X-axis while the response of the affinity measurement was plotted on the left Y-axis, the response in the avidity setup was plotted on the right Y-axis. Affinity curves are depicted as dotted lines while avidity binding curves are shown as a plain line. The pairs correspond to Table 2.
Figure 3: Association and dissociation curves of BisALFA conjugates in affinity and avidity measurements. Time was plotted on the X-axis while the response of the affinity measurement was plotted on the left Y-axis, the response in the avidity setup was plotted on the right Y-axis. Affinity curves are depicted as dotted lines while avidity binding curves are shown as a plain line. The pairs correspond to Table 3. For the high affinity pair, affinity measurements on linear NbALFA was included as reference, with the response plotted on a second left Y axis.
Figure 4: Denaturation curve of the bivalent ProteinDocker in the presence of different concentrations of the BisALFA conjugate EX-036. The first derivative of the ratio of the fluorescence signal at 350 nm and 330 nm was plotted against the temperature. Minima and maxima reflect TM values. While the plots in the top row show a clear minimum at ~54°C, which reflects the TM value of NbALFA, no such minima were observed in the lower two plots where at least equimolar concentrations of BisALFA were applied to the ProteinDocker.
Figure 5: Size exclusion analysis of bivalent ProteinDocker with different concentrations of the BisALFA conjugate EX-036. The top graph represents a 10-fold molecular excess of the BisALFA conjugate, while the middle one shows an equimolar BisALFA concentration to the ProteinDocker. In the lower plot, BisALFA was added to a 10-fold lower molecular concentration compared to the ProteinDocker. As a dotted line, uncomplexed Protein Docker was always overlayed as a comparison.
Figure 6: Peptide 29 Cold unlabelled Chemical Purity by HPLC.
Figure 7: Radio-HPLC chromatogram of Ga-68-DOTA-peptide 29.
Figure 8: Radio TLC Peptide 29 Ga-68 labelled.
Figure 9: Peptide 31 Cold unlabelled Chemical Purity by HPLC.
Figure 10: Radio-HPLC chromatogram of Ga-68-DOTA-peptide 31.
Figure 11: Peptide 31 Ga-68 labelled Radio TLC.
Figure 12: Stability of Peptide 29 Ga-68 labelled over 7hrs.
Figure 13: Stability of Peptide 31 Ga-68 labelled over 7 hrs.
Figure 14: Radio-HPLC chromatogram of ln-111-DOTA-peptide 29.
Figure 15: Peptide 29 In-111 labelled Radio TLC
Figure 16: Radio-HPLC chromatogram of ln-111-DOTA-peptide 31
Figure 17: Peptide 31 In-111 labelled Radio TLC.
Figure 18: Peptide 29 In-111 labelled Stability over 24 hrs.
Figure 19: Peptide 31 In-111 labelled Stability over 24 hrs. Figure 20: Dose response curve for cytotox readout 73 hours after treatment is shown for antigen negative ES-2 and antigen positive ES-2-antigen+ cells. Bivalent ProteinDocker only and ProteinDocker + EX-036 serve as controls for ProteinDocker-mediated effects. Specificity of the ProteinDocker + BisALFA complex is evaluated using antigen-negative cells or a ProteinDocker with irrelevant target. EC50 values are calculated and shown in the legend table.
Figure 21: Components of RiboDocker immunomodulators.
Figure 22: Co-culture assay for analysing Fc-mediated activity.
Figure 23: Biolayer interferometric avidity measurements of BisALFA-STING agonists to NbALFA. Sensorgrams of the avidity-driven interaction of SD17453 (A) or SD17516 (B) to NbALFA are shown. A 1:1 serial dilution of the BisALFA-STING agonists, starting at 2.5 nM down to 39.0625 pM were applied to immobilized NbALFA for 900 sec. The shift in wavelength based on the NbALFA-ALFA binding is depicted on the Y axis. After 900 sec (vertical line) dissociation was initiated and measured for the remaining 1500 sec. The acquired datapoints are depicted as a continuous line while the global fitting, based on a the global 1:1 Langmuir binding model, is depicted as a dotted line. Kinetic interaction parameters derived from this fitting are depicted in C.
Figure 24: Complex formation assessment using Dynamic light scattering. ProteinDocker comprising two NbALFA VHH moieties were incubated without a BisALFA STING agonist peptide (no peptide) or with 10-fold excess of SD17516 or SD17453. Intensities observed at higher radii indicate the formation of larger molecular complexes.
Figure 25: Biodistribution of Ga68-DOTA-bisALFA with and without RiboDocker
Figure 26: Biodistribution of Cu64-DOTA-bisALFA with RiboDocker Figure 27: Pretargeting approach using monovalent RiboDocker and fluorophore-labeled monovalent ALFA (monoALFA) conjugate
Figure 28: In vitro activity of pre-complexes using a co-culture of an antigen+ tumor cell line, which was transfected to express the antigen, and THP-1 Dual reporter cells indicating IRF and NFB induction as described in the THP-1 co-culture assay protocol.
Figure 29: In vitro activity of pre-complexes using a co-culture of a second antigen+ tumor cell line, which endogenously expresses the antigen, and THP-1 Dual reporter cells indicating IRF and NFB induction as described in the THP-1 co-culture assay protocol.
Figure 30: In vitro activity of two STING-targeting small molecule payloads in THP-1 Dual reporter cells.
Figure 31: In vitro activity of pre-complexes using a co-culture of two antigen+ tumor cell lines and THP-1 Dual reporter cells indicating IRF and NFB induction as described in the THP-1 coculture assay protocol. A) The antigen+ tumor cell line used was transfected with the antigen. B) The antigen+ tumor cell line used endogenously expresses the antigen. C) The antigen+ tumor cell line used was transfected to express the antigen.
Figure 32: Anti-tumoral efficacy of pre-complexes consisting of the tumor-antigen targeted ProteinDocker and either SD17453 (A) or SD17516 (B) compared to complexes with a nonbinding ProteinDocker. A+B) Tumor growth depicted as Mean + SEM. C) Growth of single tumors.
Figure 33: Anti-tumoral efficacy of pre-complexes consisting of the tumor-antigen targeted ProteinDocker and either PIC7 or PIC8 compared to complexes with a non-binding ProteinDocker. A) Tumor growth depicted as Mean + SEM. B) Growth of single tumors. Figure 34: In vivo tolerability of the pre-complexes of ProteinDocker and immune-conjugate in the murine B16F10 melanoma model stably transfected to overexpress the model antigen. C57BI/6JRjx mice were injected subcutaneously with B16F10 cells. Different pre-complexes with antigen binding ProteinDocker and one pre-complex with a non-binder were injected intravenously on day 16 after tumor inoculations. Plasma was taken 4 and 24 hours after i.v. injection to analyze systemic cytokine induction (IFNa, IFN P, IL-6 and IP-10).
Figure 35: In vivo tolerability of the pre-complexes of ProteinDocker and immune-conjugate in the murine B16F10 melanoma model stably transfected to overexpress the model antigen. C57BI/6JRjx mice were injected subcutaneously with B16F10 cells. Different pre-complexes with antigen binding ProteinDocker and one pre-complex with a non-binder were injected intravenously on day 16 after tumor inoculations. Plasma was taken 4 and 24 hours after i.v. injection to analyze systemic cytokine induction (IFNa, IFN P, IL-6 and IP-10).
Figure 36: Anti-tumoral efficacy of the RiboDocker mRNA LNP and immune-conjugate in the human ovarian cancer model OV90 which inherently expresses the target antigen. Athymic Nude-Foxnlnu mice were injected subcutaneously with the tumor cell line. RiboDocker mRNA LNPs were injected intraveneously on days 11, 18, and 25 after tumor inoculation. PIC7 was administered intraveneously 24 hours after injection of the mRNA LNP. Tumor size was monitored using caliper measurement until mice reached exclusion criteria. A) Tumor growth depicted as Mean + SEM. B) Growth of single tumors.
Figure 37: Anti-tumoral efficacy of the RiboDocker mRNA LNP and immune-conjugate in the teratocarcinoma model PA-1 which inherently expresses the target antigen. Athymic Nude- Foxnlnu mice were injected subcutaneously with the tumor cell line. RiboDocker mRNA LNPs were injected intraveneously on days 16, 23, and 30 after tumor inoculation. PIC7 was administered intraveneously 24 hours after injection of the mRNA LNP. Tumor size was monitored using caliper measurement until mice reached exclusion criteria. A) Tumor growth depicted as Mean + SEM. B) Growth of single tumors. Detailed description
Although the present disclosure is further described in more detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
The practice of the present disclosure will employ, unless otherwise indicated, conventional chemistry, biochemistry, pharmaceutical, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated feature, element, member, integer or step or group of features, elements, members, integers or steps but not the exclusion of any other feature, element, member, integer or step or group of features, elements, members, integers or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps and those that do not materially affect the basic and novel characteristic(s) of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members, integers, or steps. The term "comprising" encompasses the term "consisting essentially of" which, in turn, encompasses the term "consisting of". Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of" or "consisting of". Likewise, at each occurrence in the present application, the term "consisting essentially of" may be replaced with the term "consisting of".
The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context.
The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
The term "optional" or "optionally" as used herein means that the subsequently described event, circumstance or condition may or may not occur, and that the description includes instances where said event, circumstance, or condition occurs and instances in which it does not occur.
Where used herein, "and/or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and/or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.
In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±10%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.9%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.8%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.7%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.6%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.5%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.4%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.3%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.2%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.1%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.05%. In some embodiments, "about" indicates deviation from the indicated numerical value by ±0.01%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.
Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
In the following, definitions and embodiments will be provided which apply to all aspects of the present disclosure. Terms which are defined in the following have the meanings as defined unless otherwise indicated. Any undefined terms have their art recognized meanings.
Terms such as "reduce" or "inhibit" as used herein means the ability to cause an overall decrease, for example, of about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, or about 75% or greater, in the level. The term "inhibit" or similar phrases includes a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.
Terms such as "enhance" as used herein means the ability to cause an overall increase, or enhancement, for example, by at least about 5% or greater, about 10% or greater, about 15% or greater, about 20% or greater, about 25% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 75% or greater, or about 100% or greater in the level. "Physiological pH" as used herein refers to a pH of about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.
As used in the present disclosure, "% w/v" refers to weight by volume percent, which is a unit of concentration measuring the amount of solute in grams (g) expressed as a percent of the total volume of solution in milliliters (mL).
As used in the present disclosure, "% by weight" refers to weight percent, which is a unit of concentration measuring the amount of a substance in grams (g) expressed as a percent of the total weight of the total composition in grams (g).
As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.
As used in the present disclosure, "mol % of the total lipid" is defined as the ratio of the number of moles of one lipid component to the total number of moles of all lipids, multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid- like material.
The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ionic species in a particular solution and their respective charges. Thus, ionic strength I is represented mathematically by the formula: in which c is the molar concentration of a particular ionic species and z the absolute value of its charge. The sum 1 is taken over all the different kinds of ions (i) in solution.
According to the disclosure, the term "ionic strength" in some embodiments relates to the presence of monovalent ions. Regarding the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents is, in some embodiments, sufficiently low so as to prevent degradation of a nucleic acid. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of the phosphodiester bonds between nucleotides such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 pM or less. In some embodiments, there are no or essentially no free divalent ions.
"Osmolality" refers to the concentration of a particular solute expressed as the number of osmoles of solute per kilogram of solvent.
The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing it and then reducing the surrounding pressure (e.g., below 15 Pa, such as below 10 Pa, below 5 Pa, or 1 Pa or less) to allow the frozen medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze- drying" are used herein interchangeably.
The term "spray-drying" refers to spray-drying a substance by mixing (heated) gas with a fluid that is atomized (sprayed) within a vessel (spray dryer), where the solvent from the formed droplets evaporates, leading to a dry powder.
The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state such as its original liquid state.
The term "recombinant" in the context of the present disclosure means "made through genetic engineering". In some embodiments, a "recombinant object" in the context of the present disclosure is not occurring naturally.
The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring. The term "found in nature" means "present in nature" and includes known objects as well as objects that have not yet been discovered and/or isolated from nature, but that may be discovered and/or isolated in the future from a natural source.
As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to temperatures from at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures will include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C and 22°C.
The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given with respect to the EDTA disodium salt.
The term "cryoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the freezing stages.
The term "lyoprotectant" relates to a substance that is added to a formulation in order to protect the active ingredients during the drying stages.
According to the present disclosure, the term "peptide" refers to substances which comprise about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100 or about 150, consecutive amino acids linked to one another via peptide bonds. The term "polypeptide" refers to large peptides, in particular peptides having at least about 151 amino acids. "Peptides" and "polypeptides" are both protein molecules. Thus, the terms "peptide", "protein" and "polypeptide" are used herein usually as synonyms.
Peptides and polypeptides disclosed herein may comprise a linear or a cyclized peptide sequence.
In some embodiments, the peptides disclosed herein comprises at least one cyclic portion, i.e., a polypeptide chain that contains a circular sequence of bonds that is referred to herein as a "cyclic peptide." The circular sequence can occur through a connection between the amino and carboxyl ends of the peptide; a connection between the amino end and a side chain; a connection between the carboxyl end and a side chain; or a connection between two side chains including sulfur groups of two cysteine amino acids by forming a disulfide bond, or more complicated arrangements.
In some embodiments, the peptides and polypeptides disclosed herein are composed of naturally occurring amino acids, non-naturally occurring amino acids, amino acid derivatives and non-amino acid components, or a mixture thereof. In some embodiments, the peptides and polypeptides disclosed herein comprise amino acid mimetics and amino acid analogs. In some embodiments, the peptides and polypeptides disclosed herein comprise non-naturally occurring amino acid sequences that are resistant to enzymatic cleavage.
In some embodiments, one or more positions of a peptide or polypeptide disclosed herein are substituted with a non-naturally occurring amino acid. In some embodiments, the substituted amino acid is chemically related to the original residue (e.g., aliphatic, charged, basic, acidic, aromatic, hydrophilic) or an isostere of the original residue.
In its broadest sense, as used herein, the term "amino acid" refers to a compound and/or substance that can be, is, or has been incorporated into a peptide, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid. In some embodiments, an amino acid is a D-amino acid. In some embodiments, an amino acid is an L-amino acid. "Standard amino acid" or "proteinogenic amino acid" refers to any of the twenty standard L- amino acids commonly found in naturally occurring peptides and polypeptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a peptide or polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a peptide or polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a peptide or polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments it may be used to refer to an amino acid residue of a peptide or polypeptide.
The following table lists the 20 natural amino acids and their abbreviations:
Generally, amino acids are L-amino acids while D-amino acids are denoted by the prefix "D". The prefix "homo" or "h" designates an a-amino acid that is otherwise similar to one of the common ones, but that contains one more methylene group in the carbon chain.
As used herein, "Orn" means ornithine or 2,5-diaminopentanoic acid, "Dab" means 2,4- diaminobutanoic acid, "Dap" means 2,3-diaminopropanoic acid, "hLys" means 2,7- diaminoheptanoic acid, "hCys" means 2-amino-4-mercaptobutanoic acid, and "Pen" means penicillamine or 2-amino-3-methyl-3-sulfanylbutanoic acid.
It may also be possible to include non-peptide linkages and other chemical modification. For example, part or all of the peptide or polypeptide may be synthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995) Trends BiotechnoLI3:132-4). A peptide or polypeptide may include one or more (e.g., all) non-hydrolyzable bonds. Many non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds. Exemplary non- hydrolyzable bonds include -[CHzNHj- reduced amide peptide bonds, -[COCH2]- ketomethylene peptide bonds, -(CH(CN)NH]- (cyanomethylene)amino peptide bonds, - [CH2CH(OH)]- hydroxyethylene peptide bonds, -[CH2O]- oxymethylene peptide bonds, and - [CH2S]- thiomethylene peptide bonds (see e.g., U.S. Pat. No. 6,172,043). The term "amide" as used herein, represents a group of formula "-NHC(O)-".
The term "thioamide" represents a group of formula "-NHC(S)-".
As used herein the term "disulfide bond", "disulfide bridge" or "disulfide" includes the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a second thiol group.
The term "ether" refers to a group or compound having an oxygen between two carbon atoms. The term "cyclic ether" refers to a hetercyclic ring which contains only oxygen atoms as ring heteroatoms. Depending on the size of the heterocyclic ring, a cyclic ether may contain 1, 2, or 3 oxygen atoms as ring heteroatoms (e.g., 3-membered and higher rings may contain 1 oxygen atom; 5-membered and higher rings may contain 2 oxygen atoms; and 7-membered and higher rings may contain 3 oxygen atoms, wherein optionally every two oxygen ring atoms are spaced by at least one carbon ring atoms). Examples of cyclic ethers include oxirane, oxirene, furan, tetrahydrofuran, 1,3-dioxolane, 1,3-dioxole, pyran, tetrahydropyran, 1,3- dioxane, and 1,4-dioxane.
The term "amino" refers to to the the moiety -NHz.
The term "thioether" refers to a group or compound having a sulfur between two carbon atoms.
The term "halo" or "halogen" refers to fluoro, chloro, bromo, or iodo.
The term "ester" refers a compound derived from an carboxylic acid and an alcohol by linking with formal loss of water the hydroxyl group of the -C(=O)OH group in the former and a hydroxy group of the latter. Thus, the term refers to the group -C(O)O-.
The term "thioester" refers to the group -C(O)S- or -C(S)O-.
The term "triazole" refers to chemical compounds that incorporate in their structure any heterocyclic structure having a five-membered ring of two carbon atoms and three nitrogen atoms (e.g., 1,2,3-triazole).
The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or protein the term "portion" thereof may designate a continuous or a discontinuous fraction of said structure.
The terms "part" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or protein refers to a continuous element of said structure. When used in context of a composition, the term "part" means a portion of the composition. For example, a part of a composition may be any portion from 0.1% to 99.9% (such as 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%) of said composition.
"Fragment", with reference to an amino acid sequence (peptide or polypeptide), relates to a part of an amino acid sequence, i.e., a sequence which represents the amino acid sequence shortened at the N-terminus and/or C-terminus. A fragment shortened at the C-terminus (N- terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C- terminal fragment) is obtainable, e.g., by translation of a truncated open reading frame that lacks the 5'-end of the open reading frame, as long as the truncated open reading frame comprises a start codon that serves to initiate translation. A fragment of an amino acid sequence comprises, e.g., at least 50 %, at least 60 %, at least 70 %, at least 80%, at least 90% of the amino acid residues from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from an amino acid sequence. A fragment of an amino acid sequence comprises, e.g., a sequence of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55, consecutive amino acids of the amino acid sequence.
"Variant," as used herein and with reference to an amino acid sequence (peptide or polypeptide), is meant an amino acid sequence that differs from a parent amino acid sequence by virtue of at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild type (WT) amino acid sequence, or may be a modified version of a wild type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference as compared to the parent amino acid sequence, e.g., from 1 to about 20 amino acid differences, such as from 1 to about 10 or from 1 to about 5 amino acid differences compared to the parent.
By "wild type" or "WT" or "native" as used herein and with reference to an amino acid sequence (peptide or polypeptide) is meant an amino acid sequence that is found in nature, including allelic variations. A wild type amino acid sequence, peptide or polypeptide has an amino acid sequence that has not been intentionally modified.
For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide or polypeptide) may comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, in particular those which are naturally occurring. The term "variant" includes, in particular, fragments of an amino acid sequence. Amino acid insertion variants comprise insertions of single or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted into a particular site in an amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants comprise amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions may be in any position of the protein. Amino acid deletion variants that comprise the deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C- terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Preference is given to the modifications being in positions in the amino acid sequence which are not conserved between homologous peptides or polypeptides and/or to replacing amino acids with other ones having similar properties. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
In some embodiments, the degree of similarity, such as identity between a given amino acid sequence and an amino acid sequence which is a variant of said given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given for an amino acid region which is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given, e.g., for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments continuous amino acids. In some embodiments, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. The alignment for determining sequence similarity, such as sequence identity can be done with art known tools, such as using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
"Sequence similarity" indicates the percentage of amino acids that either are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
The terms "% identical" and "% identity" or similar terms are intended to refer, in particular, to the percentage of nucleotides or amino acids which are identical in an optimal alignment between the sequences to be compared. Said percentage is purely statistical, and the differences between the two sequences may be but are not necessarily randomly distributed over the entire length of the sequences to be compared. Comparisons of two sequences are usually carried out by comparing the sequences, after optimal alignment, with respect to a segment or "window of comparison", in order to identify local regions of corresponding sequences. The optimal alignment for a comparison may be carried out manually or with the aid of algorithms, e.g., the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, the local homology algorithm by Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search algorithm by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the United States National Center for Biotechnology Information (NCBI) website (e.g., at blast. ncbi.nlm. nih.gov/Blast. cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC =align2seq). In some embodiments, the algorithm parameters used for BLASTN algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 28; (iii) Max matches in a query range set to 0; (iv) Match/Mismatch Scores set to 1, -2; (v) Gap Costs set to Linear; and (vi) the filter for low complexity regions being used. In some embodiments, the algorithm parameters used for BLASTP algorithm on the NCBI website include: (i) Expect Threshold set to 10; (ii) Word Size set to 3; (iii) Max matches in a query range set to 0; (iv) Matrix set to BLOSUM62; (v) Gap Costs set to Existence: 11 Extension: 1; and (vi) conditional compositional score matrix adjustment.
Percentage identity is obtained by determining the number of identical positions at which the sequences to be compared correspond, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence) and multiplying this result by 100.
In some embodiments, the degree of similarity or identity is given for a region which is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments continuous nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence. Homologous amino acid sequences exhibit according to the disclosure at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and, e.g., at least 95%, at least 98 or at least 99% identity of the amino acid residues. The amino acid sequence variants described herein may readily be prepared by the skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or polypeptides having substitutions, additions, insertions or deletions, is described in detail in Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook et al. (1989), eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012, for example. Furthermore, the peptides, polypeptides and amino acid variants described herein may be readily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.
In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant exhibiting one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., it is functionally equivalent. With respect to sequences of binding agents such as antibodies, one particular function is one or more binding activities displayed by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, in particular refers to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence and that is still capable of fulfilling one or more of the functions of the parent molecule or sequence, e.g., binding to a target molecule. In some embodiments, the modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, e.g., function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the parent molecule or sequence. However, in other embodiments, function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.
An amino acid sequence (peptide or polypeptide) "derived from" a designated amino acid sequence (peptide or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, the amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof. For example, it will be understood by one of ordinary skill in the art that the sequences suitable for use herein may be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences.
In some embodiments, "isolated" means removed (e.g., purified) from the natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide or polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid, peptide or polypeptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid, peptide or polypeptide can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
The term "bind" or "binding" relates to the non-covalent interaction with a target. In some embodiments, the term "bind" or "binding" relates to a specific binding. By the term "specific binding" or "specifically binds", as used herein, is meant a molecule such as an antibody which recognizes a specific target molecule, but does not substantially recognize or bind other molecules in a sample or in a subject. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
In some instances, the terms "specific binding" or "specifically binds", can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the antibody, will reduce the amount of labeled A bound to the antibody.
As used herein, the terms "binding" or "capable of binding" typically is a binding with an affinity corresponding to a KD of about 10~7 M or less, such as about 10'8 M or less, such as about 10~9 M or less, about IO 10 M or less, or about 1011 M or even less, when determined using Bio-Layer Interferometry (BLI), or, for instance, when determined using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument. In some embodiments, a binding moiety or agent binds to a predetermined target with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its affinity for binding to a non-specific target (e.g., BSA, casein).
The term "kd" (sec -1), as used herein, refers to the dissociation rate constant of a particular interaction, e.g., antibody-antigen interaction. Said value is also referred to as the kotf value.
The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction, e.g., antibody-antigen interaction.
Generally, the terms "bind" or "binding" and "target" or "targeting" are used interchangeably herein.
The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids, e.g., DNA and/or RNA, into a cell. For purposes of the present disclosure, the term "transfection" also includes the introduction of a nucleic acid into a cell or the uptake of a nucleic acid by such cell, wherein the cell may be present in a subject, e.g., a patient, or the cell may be in vitro, e.g., outside of a patient. Thus, according to the present disclosure, a cell for transfection of a nucleic acid described herein can be present in vitro or in vivo, e.g. the cell can form part of an organ, a tissue and/or the body of a patient. According to the disclosure, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. RNA can be transfected into cells to transiently express its coded protein. Since the nucleic acid introduced in the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted through mitosis or degraded. Cells allowing episomal amplification of nucleic acids greatly reduce the rate of dilution. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection, for example. Generally, cells that are transfected with nucleic acid encoding a docking compound are transiently transfected with nucleic acid encoding the docking compound. RNA can be transfected into cells to transiently express its coded protein. As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.
The term "fusion protein" as used herein refers to a polypeptide or protein comprising two or more subunits. Preferably, the fusion protein is a translational fusion between the two or more subunits. The translational fusion may be generated by genetically engineering the coding nucleotide sequence for one subunit in a reading frame with the coding nucleotide sequence of a further subunit. Subunits may be interspersed by a linker.
As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.
As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.
The term "autologous" is used to describe anything that is derived from the same subject. For example, "autologous transplant" refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier which otherwise results in rejection.
The term "allogeneic" is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical.
The term "syngeneic" is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e., identical twins or animals of the same inbred strain, or their tissues.
The term "heterologous" is used to describe something consisting of multiple different elements. As an example, the transfer of one individual's bone marrow into a different individual constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject.
According to various embodiments of the present disclosure, a nucleic acid encoding a peptide or polypeptide is taken up by or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject, resulting in expression of said peptide or polypeptide. The cell may, e.g., express the encoded peptide or polypeptide intracellularly (e.g. in the cytoplasm and/or in the nucleus), may secrete the encoded peptide or polypeptide, and/or may express it on the surface. In some embodiments, if the encoded peptide or polypeptide is a docking compound, the cell secretes the encoded peptide or polypeptide. According to the present disclosure, terms such as "nucleic acid expressing" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, e.g. within a cell, can be expressed to produce said peptide or polypeptide.
The term "expression" as used herein includes the transcription and/or translation of a particular nucleotide sequence.
In the context of the present disclosure, the term "transcription" relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA (especially mRNA). Subsequently, the RNA may be translated into peptide or polypeptide.
With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.
A medical preparation, in particular kit, described herein may comprise instructional material or instructions. As used herein, "instructional material" or "instructions" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the present disclosure. The instructional material of the kit of the present disclosure may, for example, be affixed to a container which contains the compositions/formulations of the present disclosure or be shipped together with a container which contains the compositions/formulations. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compositions be used cooperatively by the recipient.
The term "average diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the polydispersity index (PDI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here "average diameter", "diameter" or "size" for particles is used synonymously with this value of the Zaverage-
In some embodiments, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter". Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles. The term "alkyl" refers to a monovalent radical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms, abbreviated as C1-12 alkyl, (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, abbreviated as Cuo alkyl), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1-methylethyl), butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "alkylene" refers to a divalent radical of a saturated straight or branched hydrocarbon. Preferably, the alkylene comprises from 1 to 12 (such as 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CHs)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3-propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3- butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1,1-iso-butylene, 1,2- iso-butylene, and 1,3-iso-butylene), the pentylene isomers (e.g., 1,1-pentylene, 1,2- pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso-pentylene, 1,1-sec-pentyl, 1,1-neo-pentyl), the hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4- hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1-isohexylene), the heptylene isomers (e.g., 1,1- heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7- heptylene, and 1,1-isoheptylene), the octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3- octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1- isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2-butylene can also be called butan-l,2-diyl). A "substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "alkenyl" refers to a monovalent radical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividingthe number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms, 2 to 4 carbon atoms, 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 or 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 12, abbreviated as C2-12 alkenyl, (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1- butenyl, 2-butenyl, 3-butenyl, 2-methyl-l-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3- heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5- octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6- nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6- decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4- undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7- dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. If an alkenyl group is attached to a nitrogen atom, it is preferred that the double bond is not alpha to the nitrogen atom. A "substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "alkenylene" refers to a divalent radical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has
1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably
2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen-l,2-diyl, vinylidene (also called ethenylidene), l-propen-l,2-diyl, l-propen-l,3-diyl, l-propen-2,3-diyl, allylidene, 1-buten- 1,2-diyl, l-buten-l,3-diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, l-buten-3,4- diyl, 2-buten-l,2-diyl, 2-buten-l,3-diyl, 2-buten-l,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4-diyl, 2- buten-3,4-diyl, and the like. If an alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. A "substituted alkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be 2 to 20, typically six to twenty, often 2 to 10, such as 2 to 8, 2 to 6, or 2 to 4. Alkynyl groups can optionally have one or more carbon carbon double bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds. Exemplary alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4- pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 3- heptynyl, 4-heptynyl, 5-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 3-octynyl, 4-octynyl, 5- octynyl, 6-octynyl, 7-octynyl, 1-nonylyl, 2-nonynyl, 3-nonynyl, 4-nonynyl, 5-nonynyl, 6- nonynyl, 7-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl, 3-decynyl, 4-decynyl, 5-decynyl, 6- decynyl, 7-decynyl, 8-decynyl, 9-decynyl, and the like. If an alkynyl group is attached to a nitrogen atom, it is preferred that the triple bond is not alpha to the nitrogen atom. A "substituted alkynyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "cycloalkyl" or "cyclic alkyl" represents cyclic non-aromatic versions of "alkyl", "alkenyl", and "alkynyl" with preferably 3 to 20 ring-forming carbon atoms, such as 3 to 15, 3 to 10, 3 to 8 , 3 to 6, 3 to 5, 4 to 6, or 5 or 6 ring-forming carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ring-forming carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbon atoms), more preferably 3 to 7 ring-forming carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptenyl, cycloheptatrienyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, norbornyl, norpinyl, norcarnyl, oradamantly. The cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted cycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein or an oxo (i.e., =0) or sulfido (i.e., =S) group.
The term "cycloalkylene" represents cyclic non-aromatic versions of "alkylene" and may be saturated or unsaturated. A cycloalkylene is a geminal, vicinal or isolated divalent radical. In certain embodiments, the cycloalkylene (i) is monocyclic or polycyclic (such as bi- or tricyclic) and/or (ii) is 3- to 14-membered {i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, such as 3- to 12-membered or 3- to 10-membered). In one embodiment the cycloalkylene is a mono-, bi- or tricyclic 3- to 14-membered {i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14- membered, such as 3- to 12-membered or 3- to 10-membered) cycloalkylene. Generally, instead of using the ending "ylene" for cycloalkylene moieties as specified above, one can also use the ending "diyl" {e.g., 1,2-cyclopropylene can also be called cyclopropan-l,2-diyl). Exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclobutenylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene {e.g., tricyclo[3.3.1.13'7]decan-2,2-diyl). A "substituted cycloalkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "aryl" refers to a monovalent radical of an aromatic cyclic hydrocarbon. Preferably, the aryl group contains 3 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., pheny!) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes. A "substituted aryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "arylene" refers to a divalent radical of an aryl as specified above. Preferably, the arylene group contains 3 to 20 carbon atoms which can be arranged in one ring (e.g., phenylene), or two or more condensed rings (e.g., naphthylene). Exemplary arylene groups are derived from cyclopropenylium, cyclopentadienyl, benzene, indene, naphthalene, azulene, fluorene, anthracene, or phenanthracene by removing two hydrogen atoms. Preferably, "arylene" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenylene and naphthylene. A "substituted arylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an arylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the arylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl (= furyl), thienyl, oxazolyl, isoxazolyl, oxadiazolyl (such as 1,2,5-oxadiazolyl (= furazanyl)), pyrrolyl (= pyrryl), imidazolyl, pyrazolyl, triazolyl (including 1,2,4-triazolyl and 1,2,3-triazolyl), tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridinyl (= pyridyl), pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H- indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl (= quinolyl), isoquinolinyl (= isoquinolyl), benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "heteroarylene" refers to a divalent radical of a heteroaryl as specified above, a heteroaryl group as defined above in which one hydrogen atom has been removed resulting in a diradical. Exemplary heteroarylene groups include furanylene, thienylene, oxazolylene, isoxazolylene, oxadiazolylene (1,2,5- and 1,2,3-), pyrrolylene, imidazolylene, pyrazolylene, triazolylene (1,2,3- and 1,2,4-), thiazolylene, isothiazolylene, thiadiazolylene (1,2,3- and 1,2,5- ), pyridinylene, pyrimidinylene, pyrazinylene, triazinylene (1,2,3-, 1,2,4-, and 1,3,5-), benzofuranylene (1- and 2-), indolylene, isoindolylene, benzothienylene (1- and 2), 1H- indazolylene, benzimidazolylene, benzoxazolylene, indoxazinylene, benzisoxazolylene, benzothiazolylene, benzisothiazolylene, benzotriazolylene, quinolinylene, isoquinolinylene, benzodiazinylene, quinoxalinylene, quinazolinylene, benzotriazinylene (1,2,3- and 1,2,4- benzotriazinyl), pyridazinylene, phenoxazinylene, thiazolopyridinylene, pyrrolothiazolylene, phenothiazinylene, isobenzofuranylene, chromenylene, xanthenylene, phenoxathiinylene, pyrrolizinylene, indolizinylene, indazolylene, purinylene, quinolizinylene, phthalazinylene, napht hyridinylene (1,5-, 1,6-, 1,7-, 1,8-, and 2,6-), cinnolinylene, pteridinylene, carbazolylene, phenanthridinylene, acridinylene, perimidinylene, phenanthrolinylene (1,7-, 1,8-, 1,10-, 3,8-, and 4,7-), phenazinylene, oxazolopyridinylene, isoxazolopyridinylene, pyrrolooxazolylene, and pyrrolopyrrolyl. Exemplary 5- or 6-memered heteroarylene groups include furanylene, thienylene, oxazolylene, isoxazolylene, oxadiazolylene (1,2,5- and 1,2,3-), pyrrolylene, imidazolylene, pyrazolylene, triazolylene (1,2,3- and 1,2,4-), thiazolylene, isothiazolylene, thiadiazolylene (1,2,3- and 1,2,5-), pyridylene, pyrimidinylene, pyrazinylene, triazinylene (1,2,3-, 1,2,4-, and 1,3,5-), and pyridazinylene. A "substituted heteroarylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroarylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroarylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein.
The term "heterocyclyl" or "heterocyclic ring" means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorous, or sulfur, preferably O, S, or N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms; e.g., a monocyclic heterocyclyl group may contain 3, 4, 5, 6, or 7 (preferably 5, 6, or 7) ring atoms, whereas a bicyclic heterocyclyl group may contain 7 to 10 (such as 8, 9, or 10) ring atoms. Preferably, in each ring of the heterocyclyl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein or an oxo or sulfido group.
The term "heterocycloalkylene" or "divalent heterocyclic ring" as used herein means a heterocyclyl group as defined above which contains at least one ring heteroatom (such as those selected from the group consisting of O, S, N, B, Si, and P) and in which one hydrogen atom has been removed resulting in a geminal, vicinal or isolated divalent radical. In some embodiments, the heteroatoms of the heterocycloalkylene group are selected from the group consisting of O, S, and N. For example, the heterocycloalkylene may be O/S- heterocycloalkylene, such as O-heterocycloalkylene. In some embodiments, in each ring of the heterocycloalkylene group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The heterocycloalkylene may be monocyclic or polycyclic (such as bi- or tricyclic). In some embodiments, the heterocycloalkylene is a mono-, bi- or tricyclic 4- to 14-membered (i.e., 4-, 5-, 6-, 7-, 8-, 9-, 10- , 11-, 12-, 13-, or 14-membered, such as 4- to 12-membered or 4- to 10-membered) heterocycloalkylene. The term "heterocycloalkylene" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above- mentioned heteroaryl groups (preferably partially or completely hydrogenated forms of the above-mentioned mono-, bi-, or tricyclic heteroaryl groups) in which one hydrogen atom has been removed from the same carbon atom resulting in a geminal divalent radical. Thus, in some embodiments, a heterocycloalkylene is saturated or unsaturated (i.e., it contains one or more double bonds within the ring) but cannot be aromatic. Exemplary heterocycloalkylene groups include pyrrolidinylene, imidazolidinylene, pyrazolidinylene, piperidinylene, piperazinylene (such as 1,4-piperazinylen which is also designated as piperazin-1, 4-diyl), indolinylene, isoindolinylene, etc. A "substituted heterocycloalkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocycloalkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). Preferably, the substituent other than hydrogen is a 1st level substituent as specified herein or an oxo or sulfido group.
The expression "partially hydrogenated form" of an unsaturated compound or group as used herein means that part of the unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group without removing all unsaturated moieties. The phrase "completely hydrogenated form" of an unsaturated compound or group is used herein interchangeably with the term "perhydro" and means that all unsaturation has been removed by formally adding hydrogen to the initially unsaturated compound or group. For example, partially hydrogenated forms of a 5-membered heteroaryl group (containing 2 double bonds in the ring, such as furan) include dihydro forms of said 5-membered heteroaryl group (such as 2,3-dihydrofuran or 2,5-dihydrofuran), whereas the tetrahydro form of said 5-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is a completely hydrogenated (or perhydro) form of said 5-membered heteroaryl group. Likewise, for a 6-membered heteroaryl group having 3 double bonds in the ring (such as pyridyl), partially hydrogenated forms include di- and tetrahydro forms (such as di- and tetrahydropyridyl), whereas the hexahydro form (such as piperidinyl in case of the heteroaryl pyridyl) is the completely hydrogenated (or perhydro) derivative of said 6-membered heteroaryl group. Consequently, a hexahydro form of an aryl or heteroaryl can only be considered a partially hydrogenated form according to the present disclosure if the aryl or heteroaryl contains at least 4 unsaturated moieties consisting of double and triple bonds between ring atoms.
The term "aromatic" as used in the context of hydrocarbons means that the whole molecule has to be aromatic. For example, if a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure. Likewise, if a bi- or polycyclic aryl (such as naphthyl) is hydrogenated the resulting hydrogenated bi- or polycyclic structure (such as 1,2- dihydronaphthyl) is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic). A similar distinction is made within the present application between heteroaryl and heterocyclyl. For example, indolinyl, i.e., a dihydro variant of indolyl, is classified as heterocyclyl for the purposes of the present disclosure, since only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.
Typical 1st level substituents are preferably selected from the group consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2-Z(CH3)Z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1.3 alkyl is methyl, ethyl, propyl or isopropyl. Particularly preferred 1st level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3, such as halogen (e.g., F, Cl, or Br), and -CF3. Additional typical 1st level substituents of non-aromatic ring moieties (such as cycloalkyl or heterocyclyl moieties) are oxo or sulfido.
It is intended that all compounds shown herein (e.g., tag conjugates and docking compounds) and all formulas shown herein (in particular, any of Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), (XXK), (XX-I) to (XX-LXII), (XX-1) to (XX-73), (XX'), (XXA'), (XXB'), (XXC), (XXD'), (XXE1), (XXF'), (XXG'), (XXH'), (XXJ'), (XXK'), (XX-61), (XX-18'), (XX-51'), (XX-531), (XX-73'a), (XX-73'b), SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19)), EX-024 to EX- 035, EX-032 to EX-035, and EX-047 to EX-054) encompass not only the compounds/formulas as depicted but also their tautomers, solvates (e.g., hydrates), salts (in particular, pharmaceutically acceptable salts), crystalline forms, non-crystalline, unlabeled forms, isotopically labeled forms, diastereomers, enantiomers, and any combinations thereof (unless this is clearly contracdicted by the specific compound/formula).
"Isomers" are compounds having the same molecular formula but differ in structure ("structural isomers") or in the geometrical (spatial) positioning of the functional groups and/or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers which are non- superimposable mirror-images of each other. A "racemic mixture" or "racemate" contains a pair of enantiomers in equal amounts and is denoted by the prefix (±). "Diastereomers" are stereoisomers which are non-superimposable and which are not mirror-images of each other. "Tautomers" are structural isomers of the same chemical substance that spontaneously and reversibly interconvert into each other, even when pure, due to the migration of individual atoms or groups of atoms; i.e., the tautomers are in a dynamic chemical equilibrium with each other. An example of tautomers are the isomers of the keto-enol-tautomerism. "Conformers" are stereoisomers that can be interconverted just by rotations about formally single bonds, and include, in particular, those leading to different 3-dimentional forms of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.
The term "solvate" as used herein refers to an addition complex of a dissolved material in a solvent (such as an organic solvent (e.g., an aliphatic alcohol (such as methanol, ethanol, n- propanol, isopropanol), acetone, acetonitrile, ether, and the like), water or a mixture of two or more of these liquids), wherein the addition complex exists in the form of a crystal or mixed crystal. The amount of solvent contained in the addition complex may be stoichiometric or non-stoichiometric. A "hydrate" is a solvate, wherein the solvent is water.
Docking compound
According to the disclosure, a payload is delivered specifically to a target cell by providing a docking compound with a moiety that binds to a target on target cells, e.g., an antigen on target cells, and a moiety that binds to a compound which carries the payload (tag conjugate). The target on target cells is also referred to herein as "primary target".
A "docking compound" is used to form a connection, such as a non-covalent connection, between a primary target, e.g., a target cell or an antigen on target cells, and the docking compound. The docking compound may form a connection, such as a non-covalent or covalent connection, to a compound comprising a payload to be delivered to a target cell (tag conjugate). The tag conjugate comprises one or more tags for binding by the docking compound which are covalently attached to the payload.
In some embodiments, a docking compound comprises a "primary targeting moiety", e.g., a moiety targeting a cell surface antigen on target cells, that is capable of binding to the primary target of interest, e.g., a cell surface antigen on target cells. A "primary targeting moiety" as used herein relates to the part of the docking compound which binds to a primary target. Such targeting moieties are typically moieties that have affinity for cell surface targets. These moieties can be any peptide or protein (e.g. antibodies or antibody fragments) binding to the primary target. Particular embodiments of suitable primary targeting moieties for use herein include cell surface antigen binding moieties, such as antibodies, antibody fragments and DARPins. Other examples of primary targeting moieties are peptides or proteins which bind to a receptor.
A primary targeting moiety preferably binds with high specificity and/or high affinity and the bond with the primary target is preferably stable within the body.
In order to allow specific targeting of primary targets, the primary targeting moiety of the docking compound can comprise compounds including but not limited to antibodies, antibody fragments, e.g. F(ab')2, Fab, scFV, VHH domains, and other proteins or peptides.
According to some embodiments, the primary target is a cell surface antigen such as a cancer antigen, and suitable primary targeting moieties include but are not limited to, peptides and polypeptides targeting the cell surface antigen, e.g., antibodies, antibody fragments and DARPins.
According to some embodiments, the primary target is a receptor and suitable primary targeting moieties include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g., a receptor binding peptide in the case of receptor binding protein ligands.
According to some embodiments, the primary target and primary targeting moiety are selected so as to result in the specific or increased targeting of certain cells, e.g., diseased cells, such as cells involved in and characteristic for a disease such as cancer, an inflammation, an infection, a cardiovascular disease, e.g. thrombus, atherosclerotic lesion, hypoxic site, e.g. stroke, tumor, cardiovascular disorder, brain disorder, apoptosis, and angiogenesis. This can be achieved by selecting primary targets with cell-specific expression. For example, cancer antigens, e.g., those described herein, may be expressed in cancer cells while they are not expressed or expressed in a lower amount in normal non-cancerous cells.
The docking compound further comprises a group which serves as a binding moiety for a respective tag of a tag conjugate. The moiety of the docking compound binding to the tag conjugate and the primary targeting moiety are linked to each other, preferably by a covalent linkage.
According to some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody. In some embodiments, the docking compound comprises a binding domain binding to a primary target and a binding domain binding to a tag conjugate. In some embodiments, the docking compound comprises an antibody or antibody fragment binding to a primary target and an antibody or antibody fragment binding to a tag conjugate. In some embodiments, at least one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, each binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, at least one binding domain comprises a single-domain antibody such as a VHH. In some embodiments, each binding domain comprises a single-domain antibody such as a VHH. In some embodiments, one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody and the other binding domain comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a primary target comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a tag conjugate comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a tag conjugate comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody and the binding domain binding to a tag conjugate comprises a single-domain antibody such as a VHH.
In some embodiments, the docking compound comprises one binding domain binding to a primary target and one binding domains binding to a tag conjugate. In some embodiments, the docking compound comprises one binding domain binding to a primary target and two binding domains binding to a tag conjugate. In some embodiments, the docking compound comprises two binding domains binding to a primary target and two binding domains binding to a tag conjugate.
In some embodiments, the docking compound comprises an antibody wherein the Fab fragments of the antibody are replaced by VHHs binding to a primary target and being N- terminally linked to the remaining part of the heavy chains. In some embodiments, the heavy chains are C-terminally linked to VHHs binding to a tag on a tag conjugate.
In some embodiments, the docking compound comprises a full-length antibody binding to a primary target. In some embodiments, the heavy chains of the full-length antibody are C- terminally linked to VHHs binding to a tag on a tag conjugate.
In some embodiments, the docking compound comprises a fusion protein which comprises a binding domain binding to a primary target and a binding domain binding to a tag conjugate. In some embodiments, the docking compound comprises a single peptide chain. In some embodiments, the single peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., antibody or antibody fragment, binding to a tag conjugate. In some embodiments, the docking compound comprises two peptide chains which may be covalenty linked, e.g., by one or more disulfide bonds, wherein each peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., antibody or antibody fragment, binding to a tag conjugate. In some embodiments, the antibody fragments are VHH, scFv, or a mixture thereof. In different embodiments, the docking compound or a peptide chain of a docking compound comprises one of the following structures (from N- to C-terminus):
VHH (a tag conjugate)-optional linker-VHH (a primary target) VHH (a primary targetj-optional linker-VHH (a tag conjugate) VHH (a tag conjugatej-optional linker-scFv (a primary target) scFv (a primary targetj-optional linker-VHH (a tag conjugate) VHH (a primary targetj-optional linker-scFv (a tag conjugate) scFv (a tag conjugatej-optional linker-VHH (a primary target) scFv (a tag conjugatej-optional linker-scFv (a primary target) scFv (a primary target)-optional linker-scFv (a tag conjugate)
The present disclosure provides in one aspect, a docking compound as described herein. In some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody, wherein one specificity binds (monovalently, bivalenty or with even more valency) to an epitope tag, e.g., an ALFA-tag and the other specificity binds (monovalently, bivalenty or with even more valency) to a primary target, e.g., a cell surface antigen on target cells. In some embodiments, the specificity which binds to an epitope tag is an antibody or antibody fragment such as an NbALFA-nanobody (NbALFA). In some embodiments, the specificity which binds to a primary target is an antibody, antibody fragment or DARPin. In some embodiments, the moiety targeting a primary target of the docking compound is selected from the group consisting of an anti-primary target DARPin, an anti-primary target VHH and an anti-primary target scFv and/or the moiety binding to a tag conjugate of the docking compound is an NbALFA-nanobody (NbALFA). In some embodiments, the docking compound has a structure selected from the group consisting of NbALFA x antiprimary target DARPin, NbALFA x anti-primary target VHH and NbALFA x anti-primary target scFv. In some embodiments, the primary target is a cancer antigen. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-cancer antigen VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-cancer antigen scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-cancer antigen DARPin. In some embodiments, a docking compound may be provided by administering to a subject nucleic acid encoding the docking compound and allowing expression of the docking compound by cells of the subject. Delivery of nucleic acid encoding a docking compound to target cells for expression may be effected by using particles comprising the nucleic acid. The particles may comprise a targeting molecule that binds to a target, e.g., an antigen on target cells, for expression. In some embodiments, the docking compound is secreted from the cells expressing the nucleic acid. In some embodiments, the docking compound comprises a signal peptide, e.g., an N-terminal signal peptide, which allows secretion of the docking compound from the cell expressing the nucleic acid. In some embodiments, the cells expressing the nucleic acid are the same cells as those to which a payload is to be delivered herein. In some embodiments, the cells expressing the nucleic acid are different to the cells to which a payload is to be delivered herein. In some embodiments, the cells expressing the nucleic acid are liver cells. In some embodiments, the cells expressing the nucleic acid secrete the docking compound into the bloodstream. In some preferred embodiments, the nucleic acid encoding the docking compound is RNA. The RNA-encoded docketing compound is also called "RiboDocker" herein.
Tag conjugate
The tag conjugate described herein comprises a payload to be delivered and one or more tags for binding by the docking compound. The one or more tags of the tag conjugate thus are the part of the tag conjugate that forms the binding partner for the docking compound. Generally, the one or more tags of the tag conjugate are covalently attached to the payload moiety in a manner such that they are available for binding to the docking compound.
A tag conjugate described herein comprises at least one tag, e.g., at least two tags.
In some embodiments, a tag conjugate described herein comprises a single tag, preferably a single ALFA-tag and preferably comprises a single payload moiety as specified herein. In some embodiments, a tag conjugate described herein comprises a single tag, preferably a single ALFA-tag and comprises two payload moieties as specified herein.
In some embodiments, a tag conjugate described herein comprises 2, 3, 4, 5, 6, 7, 8 or even more tags, which tags may be identical or different. In some embodiments, the tags of a tag conjugate described herein are identical. In some embodiments, a tag conjugate described herein comprises partially or completely different tags, but the one or more binding moieties for a tag of a docking compound bind to the different tags. In some embodiments, each of the tags of a tag conjugate described herein comprises an ALFA-tag. In some embodiments, a tag conjugate described herein comprises one tag, preferably one ALFA-tag. In some embodiments, a tag conjugate described herein comprises two tags, preferably two ALFA- tags. In some embodiments, a tag conjugate described herein comprises two identical tags, preferably two identical ALFA-tags.
In some embodiments, the one or more tags of the tag conjugate comprise a peptide or protein (e.g., peptide tags).
In some embodiments, the one or more tags of the tag conjugate comprise a peptide or protein (e.g., peptide tags) and are chemically linked, e.g., through a linker, to the payload moiety.
A tag conjugate further comprises a moiety, termed "payload moiety" or "payload" herein, that is capable of attracting, providing or bringing about the desired effect, e.g., therapeutic effect. The tags and the payload moiety may be covalently or non-covalently linked.
In some embodiments, the tag conjugate comprises a polymer. In some embodiments, the payload moiety of the tag conjugate and the one or more tags of the tag conjugate are connected through a linker comprising the polymer.
In some embodiments, the polymer is not a polymer of proteinogenic amino acids or their D- isomers. In some embodiments, the polymer is a hydrophilic polymer. In some embodiments, the polymer portion of the tag conjugate contributes to conferring stealth properties on the tag conjugate. In some embodiments, the plasmatic half-life of the tag conjugate described herein is greater than 2 hours, e.g., between 3 and 10 hours. This characteristic advantageously allows the tag conjugate to accumulate at the target cells and to liberate therein their contents (payload) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.
The term "stealth" is used herein to describe the ability of the particles described herein not to be detected and then sequestered and/or degraded, or to be hardly detected and then sequestered and/or degraded, and/or to be detected and then sequestered and/or degraded late, by the immune system of the host to which they are administered.
Macrophages constitute one of the most important components of the immune system and play a predominant role in eliminating foreign particles, including liposomes and other colloidal particles, from the blood circulation. At the molecular level, the clearance of particles takes place in two steps: opsonization by the depositing of serum proteins (or "opsonins") at the surface of the particles followed by recognition and capture of the opsonized particles by macrophages.
Modification of the surface of particles with chains of hydrophilic and flexible polymers, e.g., polymers of the polyethylene glycol) type, confers them a steric protection by preventing the opsonins reaching the surface of the particles.
In some embodiments, the polymer for use herein is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof).
In some embodiments, a polymer is designed to sterically stabilize a tag conjugate by forming a protective hydrophilic layer. In some embodiments, a polymer can reduce association of a tag conjugate with serum proteins and/or the resulting uptake by the reticuloendothelial system when such tag conjugates are administered in vivo.
In some embodiments, the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG is unsubstituted. In some embodiments, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. In some embodiments, the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.
In some embodiments, the PEG moiety has a molecular weight of 1000 or more. In some embodiments, the PEG moiety comprises 10 units or more of formula (O-CH2-CH2)n. In some embodiments, the PEG comprises from 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.
In some embodiments, the PEG comprises "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons.
In some embodiments, PEG2000, PEG3000 and PEG5000 are used as the polymer. In some embodiments, a pSar comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units.
In some embodiments, a pSar comprises the structure of the following general formula: wherein s is the number of sarcosine units.
In some embodiments, the POX and/or POZ polymer comprises between 2 and 200, between 2 and 190, between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70 POX and/or POZ repeating units.
In some embodiments, the POX and/or POZ polymer comprises the following general formula: wherein a is an integer between 1 and 2; Rn is alkyl, in particular C1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; and m refers to the number of POX and/or POZ repeating units.
In some embodiments, the POX and/or POZ polymer is a polymer of POX and comprises repeating units of the following general formula: In some embodiments, the POX and/or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula:
In any of the above embodiments of formulas, m (i.e., the number of repeating units in the polymer) preferably is between 2 and 190, such as between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70. In certain embodiments, m is
2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.
In some embodiments, the POX and/or POZ polymer is a copolymer comprising repeating units of the following general formulas: wherein the number of repeating units shown on the left in the copolymer is 1 to 199; the number of repeating units of formula on the right in the copolymer is 1 to 199; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 200.
In some embodiments, the number of repeating units of formula on the left in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; the number of repeating units of formula on the right in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50. In some of the above embodiments, Rn at each occurrence (i.e., in each repeating unit) may be the same alkyl group (e.g., Rn may be methyl in each repeating unit). In some alternative embodiments, Rn in at least one repeating unit differs from Rn in another repeating unit (e.g., for at least one repeating unit Rn is one specific alkyl (such as ethyl), and for at least one different repeating unit Rn is a different specific alkyl (such as methyl)). For example, each Rn may be selected from two different alkyl groups (such as methyl and ethyl) and not all Rn are the same alkyl.
In any of the above embodiments, Rn preferably is methyl or ethyl, more preferably methyl. Thus, in some embodiments, each Rn is methyl or each Rn is ethyl. In some alternative embodiments, Rn is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit Rn is methyl, and in at least one repeating unit Rn is ethyl.
In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof.
In some embodiments, the polymer comprises the following general formula: wherein
X2 and X1 taken together are optionally substituted amide, optionally substituted thioamide ester, or thioester;
Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is the number of repeating units, e.g., 1 to 100.
In some embodiments,
(i) when X1 is -C(O)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(O)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(O)- then X2 is -O-;
(vi) when X1 is -O- then X2 is -C(O)-; (vii) when X1 is -C(S)- then X2 is -0-;
(viii) when X1 is -0- then X2 is -C(S)-;
(ix) when X1 is -C(0)- then X2 is -S-; or
(x) when X1 is -S- then X2 is -C(0)-; wherein R1 is hydrogen or Ci-8 alkyl; preferably
(i) when X1 is -C(0)- then X2 is -NR1-;
(ii) when X1 is -NR1- then X2 is -C(0)-;
(iii) when X1 is -C(S)- then X2 is -NR1-;
(iv) when X1 is -NR1- then X2 is -C(S)-;
(v) when X1 is -C(0)- then X2 is -0-; or
(vi) when X1 is -0- then X2 is -C(0)-; wherein R1 is hydrogen or Ci-8 alkyl.
In some embodiments, X1 is -C(0)- and X2 is -NR1-, wherein R1 is hydrogen or Ci-8 alkyl. In some embodiments, X1 is -C(0)- and X2 is -NR1-, wherein R1 is hydrogen or methyl. In some embodiments, X1 is -C(0)- and X2 is -NR1-, wherein R1 is hydrogen.
In some embodiments, Y is -CH2- or -(CHih-- In some embodiments, Y is -CH?-.
In some embodiments, the polymer comprises the following general formula: wherein
R1 is hydrogen or C1-8 alkyl; z is 2 to 24; and n is the number of repeating units, e.g., 1 to 100.
In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.
In some embodiments, the polymer comprises the following general formula: R1 is hydrogen or Ci-s alkyl; and n is the number of repeating units, e.g., 1 to 100.
In some embodiments of the above formulas, R1 is hydrogen or methyl. In some embodiments, R1 is hydrogen.
In some embodiments, the polymer comprises the following general formula: wherein n is the number of repeating units, e.g., 1 to 100.
In some embodiments of the above formulas, n is 2 to 50. In some embodiments, n is 4 to 25. In some embodiments, n is 6 to 20. In some embodiments, n is 8 to 16. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, n is
2, 4, 6, 8, 10, or 12. In some embodiments, n is 2, 4, or 6.
In some embodiments, a tag conjugate comprises one or more tags and one or more payload moieties in an unbranched arrangement.
In some embodiments, a tag conjugate comprises the formula:
P-L-T wherein
P comprises a payload moiety as specified herein;
T comprises a tag; and
L comprises a linking moiety.
In some embodiments, L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, a tag conjugate comprises the formula:
P-LA-T-LB-T or
P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety as specified herein;
T comprises a tag;
LA comprises a linking moiety; LB comprises a linking moiety; and
Lc comprises a linking moiety.
In some embodiments, one or more of LA, LB, and Lc comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LB comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2- (2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises the following general formula: wherein n is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.
In some embodiments, a tag conjugate comprises the formula:
P-LA-T-LB-T or
P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety as specified herein;
T comprises a tag;
LA comprises a linking moiety;
LB comprises a linking moiety; and
Lc comprises a linking moiety.
In some embodiments, one or more of LA, LB, and Lc comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LB comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, LA and/or Lc comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Ledo not comprise a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, LA and/or Lc comprise an enzymatic cleavage site.
In some embodiments, one or more of LA, LB, and Lc comprises the formula [AEEA]U-[LD- [AEEA]V]W, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
LD comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [LD-[AEEA]V] may be identical or different.
In some embodiments, LB comprises the formula [AEEA]U-[LD-[AEEA]V]W, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
LD comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [LD-[AEEA]V] may be identical or different.
In some embodiments, u and v are each integers from 2 to 10.
In some embodiments, u and v are each integers from 2 to 8.
In some embodiments, u and v are each integers of 2, 4 or 6.
In some embodiments, LD comprises an amino acid.
In some embodiments, LD comprises the D-isomer of an amino acid.
In some embodiments, LD comprises cysteine or lysine.
In some embodiments, Lois connected to a side chain.
In some embodiments, a side chain comprises a functional moiety. In some embodiments, a functional moiety comprises a solubilizing functional group.
In some embodiments, a tag conjugate comprises a branching moiety to which the tags and the payload moieties are connected through a linking moiety.
The term "branching moiety" refers to a chemical moiety, e.g., an amino acid or polymer such as a peptide, that provides the functional groups for linking to the tags, e.g., via a linking moiety comprising a polymer as described herein, and provides the functional group(s) for linking to the payload moiety/moieties, e.g., via a linking moiety which may comprise a polymer as described herein. Thus, a tag conjugate described herein may comprise several arms which are connected by a branching moiety. In some embodiments, a tag conjugate described herein comprises two arms ending in a tag, and one or more arms ending in a payload moiety.
In some embodiments, the term "branching moiety" refers to a chemical moiety in an arm ending in a payload moiety which provides the functional groups for linking, e.g., via a linking moiety, more than one payload moiety and/or further functional groups to the arm.
In some embodiments, at least one of the linking moieties connecting a tag to the branching moiety comprises a polymer as described above. In some embodiments, each of the linking moieties connecting a tag to the branching moiety comprises a polymer as described above. In some embodiments, at least one of the linking moieties connecting a payload moiety to the branching moiety comprises a polymer as described above. In some embodiments, each of the linking moieties connecting a payload moiety to the branching moiety comprises a polymer as described above.
In some embodiments, a tag conjugate comprises the formula:
[P-La]2-B-Lt-T wherein
P comprises a payload moiety as specified herein;
B comprises a branching moiety;
T comprises a tag;
La comprises a linking moiety; and
Lt comprises a linking moiety; wherein the different groups [P-La] may be identical or different, and the different groups P may be identical or different.
In some embodiments, the valency of B corresponds to or is greater than 3. In some embodiments, B comprises an amino acid or bis-amino acid.
In some embodiments, B comprises the D-isomer of an amino acid.
In some embodiments, B comprises cysteine or lysine.
In some embodiments, La comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, cysteine, or a combination thereof.
In some embodiments, La comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, La comprises an enzymatic cleavage site.
In some embodiments, La comprises a moiety which is substituted by a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the moiety which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is an amino acid. In some embodiments, the amino acid which is substituted by a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is cysteine.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in La is 2, 4 or 6.
In some embodiments, La is substituted with a solubilizing functional group. In some embodiments, La comprises an amino acid which is substituted with a solubilizing functional group. In some embodiments, the amino acid which is substituted with a solubilizing functional group is lysine or cysteine.
In some embodiments, Lt comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
In some embodiments, Lt comprises one or more selected from the group consisting of a moiety constraining conformation, a moiety for albumin binding, a moiety which increases circulation time, a moiety which reduces renal retention or uptake and an enzymatic cleavage site.
In some embodiments, a tag conjugate comprises the formula:
[[P]m-Li]n-Bi-[L2-T]0 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety;
L2 comprises a linking moiety; m is an integer from 1 to 4; n is an integer from 1 to 4; and o is an integer from 2 to 4; wherein the different groups [L2-T] may be identical or different, the different groups P may be identical or different, and the different groups [[P]m-Li] may be identical or different. in some embodiments, the valency of Bi corresponds to or is greater than the sum of n and o. In some embodiments, Bi comprises an amino acid or bis-amino acid. In some embodiments, bivalent amino acid conjugates are obtainable via an intermolecular cycloaddition ("click") reaction using amino acid derived azides and alkynes as building blocks.
In some embodiments, Bi comprises the D-isomer of an amino acid.
In some embodiments, Bi comprises cysteine or lysine. In some embodiments, Li comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, L? comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2- (2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises the following general formula: wherein n is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.
In some embodiments, L2 comprises (i) a moiety comprising cysteine and (ii) a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, wherein the poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or derivative thereof and T are linked by the moiety comprising cysteine.
In some embodiments, [[P]m-Li] comprises the formula [[P-Li']m-B2-Lr], wherein
B2 comprises a branching moiety;
Lr comprises a linking moiety;
Lr comprises a linking moiety; and m is an integer from 1 to 4; wherein the different groups [[P-Li ]m-B2-Lr ] may be identical or different, and wherein in a group [[P- Li ]m-B2-Li") the different groups [P-Lr] may be identical or different.
In some embodiments, the valency of B2 corresponds to or is greater than the value of m plus 1.
In some embodiments, B2 comprises an amino acid. In some embodiments, B2 comprises the D-isomer of an amino acid.
In some embodiments, B2 comprises cysteine or lysine.
In some embodiments, Li- comprises a 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof, cysteine, or a combination thereof.
In some embodiments, Lr comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
In some embodiments, the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof in Li " is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6. in some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2- (2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments, the poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof comprises the following general formula: wherein n is between 2 and 30, e.g., between 2 and 10, such as 2, 4 or 6.
In some embodiments of the formula [[P]m-Li]n-Bi-[L2-T]0, or [[P-Li’]m-B2-Li"]n-Bi-[L2-T]0, m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3.
In some embodiments, o is 2.
In some embodiments, n is 1 or 2.
In some embodiments, m is 1 or 2.
In some embodiments, o is 2, n is 1 and m is 1. In these embodiments, the docking compound may comprise a single binding moiety for the tag.
In some embodiments, the tag conjugate comprises the formula:
P-LI-BI-[L2-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety; T comprises a tag;
Li comprises a linking moiety; and
L? comprises a linking moiety; wherein the different groups [L2-T] may be identical or different.
In some embodiments, the tag conjugate comprises the formula:
P-[AEEA]p-Bi-[[AEEA]q-R-[AEEA]r-C-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
R is optional and comprises a moiety constraining conformation;
C is optional and comprises a connecting moiety;
T comprises a tag; p is an integer from 0 to 6; q is an integer from 1 to 4; and r is an integer from 1 to 4; wherein the different groups [[AEEA]q-R-[AEEA]r-C-T] may be identical or different.
In some embodiments, o is 2, n is 2 and m is 2. In these embodiments, the docking compound may comprise at least two binding moieties for the tag, e.g., two binding moieties for the tag. In some embodiments, the tag conjugate comprises the formula: [[P-L1']2-B2-L1"]2-B1-[L2-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
B2 comprises a branching moiety;
T comprises a tag;
Lr comprises a linking moiety;
Li" comprises a linking moiety; and
L2 comprises a linking moiety; wherein the different groups [[P-L1J2-B2-L1"] may be identical or different, wherein in a group [[P-LI ]2- B2-L1 "] the different groups [P-Li ] may be identical or different, and the different groups [L2- T] may be identical or different.
In some embodiments, the tag conjugate comprises the formula: [[P-Lr]2-B2-[AEEA]5]2-B1-[[AEEA]t-T]2 wherein
P comprises a payload moiety as specified herein;
Bi comprises a branching moiety;
B2 comprises a branching moiety;
T comprises a tag;
Li- comprises a linking moiety; s is an integer from 2 to 8; and t is an integer from 2 to 8; wherein the different groups [(P-Lr]2-B2-[AEEA]S] may be identical or different, wherein in a group [[P- LI']2-B2-[AEEA]S] the different groups [P-Li ] may be identical or different, and the different groups [[AEEA]t-T] may be identical or different.
In some embodiments of all tag conjugates described above, the payload comprises the STING agonist of Formula (XX).
In some embodiments of all tag conjugates described above, the payload comprises the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all tag conjugates described above, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
In some embodiments of all tag conjugates described above, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK).
In some embodiments of all tag conjugates described above, the payload moiety comprises a STING agonist moiety having the Formula (XX- 18’), (XX-6'), (XX-511), (XX-531), (XX-73'a), or (XX- 73'b).
In some embodiments of all tag conjugates described above, the tag is an ALFA-tag, e.g., a cyclic ALFA-tag.
In some embodiments of all tag conjugates described above, the payload comprises the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all tag conjugates described above, the payload comprises the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag. In some embodiments of all tag conjugates described above, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all tag conjugates described above, the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH) or (XXK), and the tag is an ALFA- tag, preferably a cyclic ALFA-tag.
In some embodiments of all tag conjugates described above, the payload moiety comprises a STING agonist moiety having the Formula (XX-181), (XX-6'), (XX-51'), (XX-53’), (XX-73'a), or (XX- 73'b), and the tag is an ALFA-tag, preferably a cyclic ALFA-tag.
In some embodiments of all tag conjugates described above, the tag conjugate comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19), EX-024 to EX-035, EX-032 to EX-035, and EX-047 to EX-054. In some embodiments, the tag conjugate comprises a structure of a compound shown herein as any one of SD18317 (PICS), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX- 6133 (PIC19), or the tag conjugate is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19). In some preferred embodiments, the tag conjugate comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8), or the tag conjugate is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8). In some particularly preferred embodiments, the tag conjugate comprises a structure of a compound shown herein as SD18321 (PIC7), or the tag conjugate is SD18321 (PIC7).
The present disclosure provides in one aspect, a tag conjugate as described above. Interacting moieties on the tag conjugate and on the docking compound
In some embodiments, the moiety on the tag conjugate, i.e., the one or more tags, and the moiety on the docking compound interacting with each other non-covalently bind to each other.
In some embodiments, the moieties on the tag conjugate and on the docking compound interacting with each other bind to each other under physiological conditions.
In some embodiments, the moieties on the tag conjugate and on the docking compound interacting with each other are antigen/antibody systems.
In some embodiments, the moiety of the tag conjugate binding to the docking compound comprises a peptide or protein, e.g., a peptide tag, and the moiety of the docking compound binding to the tag conjugate comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.
In some embodiments, the moieties on the tag conjugate and on the docking compound interacting which each other comprise an epitope tag/binder system.
As used herein, an "epitope tag" refers to a stretch of amino acids to which an antibody or proteinaceous molecule with antibody-like function can bind.
In some embodiments, the epitope tag comprises an ALFA-tag. In some embodiments, the epitope tag/binder system comprises an ALFA-tag and an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.
The use of an ALFA-tag / ALFA-specific sdAb system is particularly advantageous in the context of the present disclosure. It will be possible to select an ALFA-tag with a suitable affinity to the sdAb for the respective application.
In embodiments where the tag conjugate comprises a total number of one tag, it is preferred that the tag is a high affinity tag, such as a high affinity ALFA-tag. For example, in applications which profit from a tight binding between the docking compound and the tag conjugate, a high affinity tag, such as a high affinity ALFA-tag, can be selected. In such embodiments, it can also be preferred to use a tag conjugate that has at least two tags and a docking compound that has at least two binding moieties for a tag, wherein the tag is preferably a high affinity tag. The effect of two tags in one compound binding to two binding moieties for a tag in another compound may lead to even tighter binding. This "two-on-two" binding between a docking compound and a tag conjugate can be particularly advantageous in applications, where circulation of free tag-conjugates should be minimized, for example to avoid off-target effects of the free tag-conjugate or to increase the half-live of the tag-conjugate in circulation. Epitope tag/binder systems with suitable affinities are described herein.
In other applications that leverage the avidity effect of the tag conjugates described herein only at the target side, a comparably lower affinity tag (a medium/low affinity tag), such as a medium/low affinity ALFA-tag, may be preferred to increase the avidity effect at the target site, for example at a tumor cell. In such embodiments, it can also be preferred to use a tag conjugate that has at least two tags and a docking compound that has a single binding moiety for a tag, wherein the tag is preferably a medium/low affinity tag. In such embodiments, a target site (e.g., target cells) with a high density of target antigen will lead to binding of docking compounds (e.g., with one tag-binding moiety) in close proximity to each other. Due to this proximity, the tag conjugates comprising two tags will preferably bind to the docking compounds at the target site, due to an avidity effect resulting from the proximity of two tagbinding moieties of two docking compounds at the target site or on the target cells. Binding to single docking compounds at sides of lower docking compound proximity (e.g. in circulation or at sides of target-unspecific docking compound deposition) is less likely. Epitope tag/binder systems that can be used in the context of the present invention taking advantage of the avidity effect are disclosed herein.
One main advantage of this avidity effect is that off-target binding of free tag-conjugates comprising two tags can be minimized while a target-sepcific accumulation of the tagconjugates occurs on target cells with a high density of bound docking compounds comprising one tag-binding moiety.
In some embodiments, the affinity of WT ALFA (Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg- Leu-Thr-Glu-NHz) to NbALFA is used as a reference point. In some embodiments, a high affinity tag (e.g., high affinity Alfa-tag) is characterized by having a value of dissociation from a particular binding partner (e.g., NbAlfa), such as measured by kdis, that, relative to the corresponding value of the corresponding wild-type tag (e.g., Alfa-tag), is 5 or less, e.g., 4 or less, 3 or less, 2 or less, or 1 or less. In other words, in some embodiments, the ratio kdis (high affinity tag)/kdis (wild-type tag) is 5 or less, e.g., 4 or less, 3 or less, 2 or less, or 1 or less. In some embodiments, a medium/low affinity tag (e.g., medium/low affinity Alfa-tag) is characterized by having a value of dissociation from a particular binding partner (e.g., NbAlfa), such as measured by kdis, that, relative to the corresponding value of the corresponding wildtype tag (e.g., Alfa-tag), is more than 5, e.g., more than 6, more than 7, or more than 8. In other words, in some embodiments, the ratio kdis (medium/low affinity tag)/kdiS (wild-type tag) is more than 5, e.g., more than 6, more than 7, or more than 8. In some embodiments, affinities are determined by biolayer interferometry (BLI).
In some embodiments it is also possible to select a tag/binder system with suitable binding properties, such as suitable kdis, that is different from the ALFA-tag / ALFA-specific sdAb system by using WT ALFA as a reference.
Examples of high affinity tags are
- Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2,
Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-(cyclo8)Lys-Arg-Arg-Leu-Thr-Glu-NH2,
- Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-(cyclol3)Glu-NH2, Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2,
- Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclolO)Lys-Arg-Leu-Thr-Glu-NH2.
Examples of medium/low affinity tags are
- Ac-Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2,
- Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Lys-Glu-Leu-Arg-(cyclolO)Glu-Arg-Leu-Thr-Glu-NH2,
- Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2.
Based on the provided examples of high and medium/low affinity tags and the provided method of determining the suitability of an epitope tag/binder system for the respective application a skilled person can select a suitable system.
In some embodiments, an ALFA-tag comprises the amino acid sequence
-AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are:
AAO is Pro or deleted;
AA1 is Ser, Gly, Thr, or Pro;
AA2 is Arg, Gly, Ala, Glu, or Pro;
AA3 is Leu, He, or Vai;
AA4 is Glu or Gin;
AA5 is Glu or Gin;
AA6 is Glu or Gin;
AA7 is Leu, He, or Vai;
AA8 is Arg, Ala, Gin, or Glu; AA9 is Arg, Ala, Gin, or Glu;
AA10 is Arg;
AA11 is Leu;
AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;
AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and
AA14 is Pro or deleted.
In some embodiments, an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE, PSRLEEELRRRLTE, SRLEEELRRRLTEP, and PSRLEEELRRRLTEP.
In some embodiments, an ALFA-tag comprises the cyclized amino acid sequence -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the side-chains of any two of the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 (XI, X2) are connected covalently; and wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 which are not XI and X2 are: AAO is Pro or deleted;
AA1 is Ser, Gly, Thr, or Pro;
AA2 is Arg, Gly, Ala, Glu, or Pro;
AA3 is Leu, lie, or Vai;
AA4 is Glu or Gin;
AA5 is Glu or Gin;
AA6 is Glu or Gin;
AA7 is Leu, lie, or Vai;
AA8 is Arg, Ala, Gin, or Glu;
AA9 is Arg, Ala, Gin, or Glu;
AA10 is Arg;
AA11 is Leu;
AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;
AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and AA14 is Pro or deleted.
In some embodiments, XI and X2 are separated by 2 or 3 amino acids. In some embodiments, AA5 is XI and AA9 is X2, AA5 is XI and AA8 is X2, AA9 is XI and AA13 is X2, AA6 is XI and AA9 is X2, AA9 is XI and AA12 is X2, AA10 is XI and AA13 is X2, AA6 is XI and AA1O is X2 or AA4 is XI and AA8 is X2.
In some embodiments, an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of a. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, b. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, c. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)-AA14-, d. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, e. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-X2)-AA13-AA14-, f. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(Xl-Leu-AA12-X2)-AA14-, g. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-AA9-X2)-Leu-AA12-AA13-AA14-, and h. -AA0-AAl-AA2-AA3-cyclo(Xl-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, wherein the side-chains of Xi and X2 amino acid residues are connected covalently;
AAO is Pro or deleted;
AA1 is Ser, Gly, Thr, or Pro;
AA2 is Arg, Gly, Ala, Glu, or Pro;
AA3 is Leu, lie, or Vai;
AA4 is Glu or Gin;
AA5 is Glu or Gin;
AA6 is Glu or Gin;
AA7 is Leu, He, or Vai;
AA8 is Arg, Ala, Gin, or Glu;
AA9 is Arg, Ala, Gin, or Glu;
AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;
AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and
AA14 is Pro or deleted.
In some embodiments, Xi and X? in the peptides disclosed herein are connected covalently via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and/or 1,2,3-triazole.
In some embodiments, a cyclized amino acid sequence described herein is generated by linking an amino group of a side-chain of one of Xi and X2 to the carboxyl group of a side-chain of the other of Xi and X2 via an amide bond. The amino group of the side chain of an amino acid that possesses a pendant amine group, e.g., lysine or a lysine derivative, and the carboxyl group of the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid or a derivative thereof, can be used to generate a cyclized amino acid sequence via an amide bond.
In some embodiments, a cyclized amino acid sequence described herein is generated by linking a sulfhydryl group of a side-chain of one of Xi and X2 to the sulfhydryl group of a sidechain of the other of Xi and X2 via a disulfide bond. Sulfhydryl group-containing amino acids include cysteine and other sulfhydryl-containing amino acids as Pen.
In some embodiments, Xi and X2 are, independently, selected from the group consisting of Glu, DGIu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when Xi is Glu, DGIu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap, X2 is Glu, DGIu, Asp, or DAsp; and when XI is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.
In some embodiments, Xi is Glu and X2 is Lys. In some embodiments, -cyclo(Glu - Lys)-, - c(Glu - Lys)-, -cyclo(E - K)-, -c(E - K)-, -E K- cyclo, or -cycloE— -cycloK- comprises the following structure:
In some embodiments, Xi is Lys and X2 is Glu. In some embodiments, -cyclo(Lys - Glu)-, - c(Lys - Glu)-, -cyclo(K - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloE- comprises the following structure:
In some embodiments, Xi is Cys and X2 is Cys. In some embodiments, -cyclo(Cys - Cys)-, -cyclo(C C)-, -c(C - C)-, -C — C- cyclo, or -cycloC cycloC- comprises the following structure:
Particular cyclized amino acid sequences of the above-identified generic formulas include, for example,
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-,
-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Asp)-,
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,
-Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-,
-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,
-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leii-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Ar -Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-z
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-z
-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-z
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-z -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-z -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-z -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-;
-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-z -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-z -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-z -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-z -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-z -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-;
-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-z -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclothCys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glii-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-, -Ser-Arg-Leu-Glii-Glu-Glu-Leii-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-;
-Ser-Arg-Leu-Glu-Glu-Glu-Leii-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-,
-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclofCys-Arg-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-;
-Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGlu)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-Pro-;
-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-;
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGIu)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-,
-Ser-Ar -Leu-Glu-cyclo(DGIu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-;
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-;
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGIu)-Arg-Arg-Leu-Thr-Glu-Pro-; -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Ar -Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Ar -Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-z -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-z -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-; -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-; -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu yclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-Pro-; -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg<yclo(Cys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)-Pro-; -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-hCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-DhCys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-Pen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu-Thr-DPen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-Pen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu-Thr-DPen)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-Cys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-Cys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-hCys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu-DhCys)-Glu-Pro-; -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg-Leu-hCys)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Asp)-Glu-Pro-; -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-Pro-; -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-Pro-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclofLys-Arg-Leu-AspJ-Glu-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Asp-Arg-Leu-Lys)-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Glu)-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Glu-Arg-Leu-Lys)-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-Pro-,
-Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-Pro-,
-Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-Pro-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-Pro-, -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.
In some embodiments, the cyclic peptide is attached to a 3-mercaptopropionyl moiety through an a-amine moiety of the leftmost amino acid in the cyclic peptide. In some embodiments, the rightmost amino acid in the cyclic peptide comprises an amide.
In some embodiments, the cyclized amino acid sequence is one selected from the group consisting of
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-,
-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Asp)-,
-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-
-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-,
-Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-
-Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu-
-Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-
-Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-, -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-, -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-, -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)-,
-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)-,
-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu-,
-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-, and -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu-.
In some embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu- Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-. In yet some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)- Arg-Arg-Leu-Thr-Glu-. In still some other embodiments, the cyclized amino acid sequence is - Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)-.
The cyclic peptides may have different cyclic bridging moieties forming the ring structure. Preferably, chemically stable bridging moieties are included in the ring structure such as, for example, an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3-triazole. The following are examples illustrating the variability of bridging moieties in a peptide:
In some embodiments, an ALFA-tag binding moiety comprises an antibody or antibody fragment, e.g., a camelid VHH domain. In some embodiments, an ALFA-tag binding moiety comprises a single-domain antibody (sdAb), NbALFA-nanobody.
In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.
In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTXiSALNAMAMG, wherein Xi is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.
In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTISALNAMAMG, the CDR2 sequence AVSERGNAM, and the CDR3 sequence LEDRVDSFHDY.
In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGNAMYRESV Q.GRFTVTRDFTNKMVSLQ.MDNLKPEDTAVYYCHVLEDRVDSFHDYWGQ.GTQ.VTVSS, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence, or a fragment of said amino acid sequence or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence. In some embodiments, the amino acid sequence comprises CDR1, CDR2 and CDR3 sequences as described above.
In some embodiments, the epitope tag/binder system comprises an epitope tag comprising the sequence PDRVRAVSHWSS (Spot-tag) and the binder comprises a single-domain antibody (sdAb, or nanobody) (Spot-nanobody (14.7 kD)) that specifically binds to the Spot-tag. In some embodiments, following binding of the moieties on the tag conjugate and on the docking compound interacting which each other, a covalent connection is formed. In these embodiments, the system used herein may comprise a Tag/Catcher system forming a covalent bond, e.g., SpyTag/SpyCatcher forming an isopeptide bond.
The SpyTag/SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag spontaneously reacts with the protein SpyCatcher to form an intermolecular isopeptide bond between the pair. Using the Tag/Catcher pair, bioconjugation can be achieved between two recombinant proteins.
In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise Digoxigenin and an antibody, antibody fragment or derivative, e.g., scFv, or any protein binding to Digoxigenin.
In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise caffeine and an antibody, antibody fragment or derivative, e.g., nanobody, binding to caffeine.
In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise GFP and an antibody, antibody fragment or derivative, e.g., nanobody, binding to GFP.
In some embodiments, the interacting moieties on the tag conjugate and on the docking compound comprise biotin and an antibody, antibody fragment or derivative binding to biotin. The present disclosure provides in one aspect, a complex wherein a tag conjugate is bound to a docking compound. Thus, the tag conjugate and the docking compound comprise moieties interacting which each other.
Accordingly, the present disclosure provides in one aspect, a complex comprising:
(i) a compound comprising a binding moiety binding to a target antigen and a binding moiety for a tag (docking compound), and
(ii) a compound comprising a payload moiety and at least one tag, e.g., at least two tags, to which the binding moiety for a tag binds (tag conjugate).
Different embodiments of the tag conjugate and the docking compound which are complexed are described herein.
In some embodiments, the tag conjugate comprises at least one ALFA-tag, e.g., at least two ALFA-tags. In some embodiments, the at least two ALFA-tags may be identical or different. In some embodiments, the tag conjugate comprises at least two identical ALFA-tags. In these embodiments, the moiety binding to a tag conjugate of the docking compound may be a NbALFA-nanobody (NbALFA). In some embodiments, the docking compound comprises at least two moieties binding to a tag of a tag conjugate, e.g., at least two NbALFA-nanobodies (NbALFA). In some embodiments, the docking compound may have a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH and NbALFA x anti-primary target scFv. In some embodiments, the docking compound comprises a full-length anti-primary target antibody comprising two heavy chains and two light chains, wherein NbALFA is linked to the C-terminus of each of the heavy chains.
Payload
In the present disclosure, a payload comprises a Stimulator of Interferon Genes (STING) agonist (i.e., an immunomodulator).
STING is expressed broadly in numerous tissue types, of both immune and non-immune origin, and is required for the type 1 interferon response in both immune and non-immune cells. STING has been shown to directly bind to a variety of different cyclic-di-nucleotides. The substantial pre-clinical anti-tumor activity of STING agonists has led to the development of multiple pharmacologic classes of agents at various stages of being translated into the clinic. The term "STING agonist" as used herein refers to a compound, e.g., a small molecule, which agonizes STING.
In some embodiments, the STING agonist is a compound having the following Formula (XX): wherein:
Ring A is selected from the group consisting of G and Gi are independently N, CH, or C-X1-R2; or when G and Gi are each C-X1-R2, the R2 groups are optionally linked to form L2;
G' and G2 are independently N or CH;
X is N-R, 0, or S;
X' is N or CH;
Xi is CH2, 0 or S;
R is hydrogen or C1-4 alkyl;
L1 and L2 are each independently C2-4 alkylene or C2-4 alkenylene;
R2 is selected from the group consisting of hydrogen, C2-4 cyclic ether, C1-4 alkylene-(C24 cyclic ether), C3-4 cycloalkyl, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl,
O O
Ri and R3 are independently selected from the group consisting of
Ring B is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 2 heteroatoms independently selected from N, O, and S;
Rs is -OH or -NR9R10;
R9 and Rio are independently selected from hydrogen and Ci-e alkyl (preferably, selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl));
X2 and X3 are independently NH or S; Yi and Y2 are independently a 5-membered heteroaryl or heterocyclic ring, wherein the 5- membered heteroaryl or heterocyclic ring (i) has 1 to 4 heteroatoms independently selected from N, O, and S, (ii) is attached to the remainder of the STING agonist via a C ring atom of the
5-membered heteroaryl or heterocyclic ring, and (iii) is optionally substituted with 1 to 4 R21, wherein each R21 is independently C1-4 alkyl (such as methyl, ethyl, propyl, or butyl);
Rs, Re, and R7 are independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, , or Rs and Re are optionally connected to form a 5- or 6- membered heterocyclic ring;
Ris is -OH or -NR9R10;
Ring C is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 ring heteroatoms independently selected from N, 0, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 4 ring heteroatoms independently selected from N, O, and S; n, p, q, q', t, and v are independently an integer from 2 to 6 (i.e., 2, 3, 4, 5, or 6, such as 2, 3, 4, or 5, e.g., 2, 3, or 4); and k, I, m, o, u, and w are independently an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6, such as 1, 2, 3, 4, or 5, e.g., 1, 2, 3, or 4).
In some embodiments of Formula (XX), G2 is CH.
In those embodiments of Formula (XX), where Ring A is , preferably G' is CH, more preferably both of G' and G? are CH.
In some embodiments of Formula (XX), Ring A is selected from the group consisting of wherein R preferably is hydrogen, methyl, ethyl, propyl, or butyl, such as hydrogen. In some preferred embodiments, Ring A is selected from the group consisting of
In some embodiments of Formula (XX), G and Gi are independently N or C-X1-R2. In some preferred embodiments of Formula (XX), each of G and Gi is C-X1-R2, or one of G and Gi is C- X1-R2 and the other of G and Gi is N.
In those embodiments of Formula (XX), where G and/or Gi is C-X1-R2, it is preferred that R2 is selected from the group consisting of hydrogen, C1-4 alkylene-(Ca-4 cycloalkyl), C1-4 alkyl,
In some embodiments of Formula (XX), L2 is ethylene, propylene, butylene, ethenylene, propenylene, or butenylene, preferably propylene; and/or that each Xi is O. In some preferred embodiments of Formula (XX), L2 is ethylene, propylene, butylene, ethenylene, propenylene, or butenylene (preferably propylene), and each Xi is O.
In some embodiments of Formula (XX), one of X2 and X3 is S and the other is NH. In some preferred embodiments of Formula (XX), each of X2 and X3 is NH. In some embodiments of Formula (XX), L1 is ethenylene, propenylene, butenylene, ethylene, propylene, or butylene, preferably ethenylene.
In some embodiments of Formula (XX), L1 is ethenylene, propenylene, butenylene, ethylene, propylene, or butylene, preferably ethenylene; and each of X2 and X3 is NH.
In some embodiments of Formula (XX), Yi and Y2 are independently selected from the group consisting of pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, dioxazolyl, dithiazolyl, and tetrazolyl, each of which is optionally substituted with 1, 2, 3, or 4 (i.e., up to 4) R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently , wherein X5 is S, 0, or NR21', wherein R21' is hydrogen or Ci-4 alkyl (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of Yi and Y2 are oxazolyl which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of
YI and Y2 are , wherein X5 is O.
In those embodiments of Formula (XX), where at least one of Rs, it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1.4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CH3), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XX), Rs, Re, and R7 are independently selected from the group consisting of hydrogen, C1-4 alkyl, , , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each Ris is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NHfCHs), and -NfCHsh (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XX), Rs is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and CM alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NH(CH3), or -N(CHs)2, such as -OH or -NH2.
R8-XB'\4-4U
In those embodiments of Formula (XX), where R2 is , it is preferred that Ring B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rio are independently selected from hydrogen and CM alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NH2, -NH(CHB), or -NfCHsh (such as -OH or -NH2). Preferably, u is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In those embodiments of Formula preferred that R9 is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl) and/or that Rs is hydrogen or Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl), and/or o 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In some embodiments of Formula (XX), at least one of G and Gi is C-X1-R2; R? is selected from the group consisting of hydrogen, C1-4 alkylene-(Cs-4 cycloalkyl), C1.4 alkyl, Rs , and , preferably from the group consisting of hydrogen, C1-4 alkylene-(Cs-4 independently selected from hydrogen, C1-4
, wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or R5 and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XX), Ri and R3 are independently selected from the group consisting of preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XX),
(i) for one of G and Gi being the other of G and Gi may also be C-X1-R2, but for this other of G and Gi R2 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), Ci-4 alkyl, R5 more preferably from the group consisting of hydrogen, Ci-4 alkylene-(Ca-
( other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of , more preferably independently selected from the group consisting
In some preferred embodiments of Formula (XX), either (i) for one of G and Gi being the other of G and Gi may also be C-X1-R2, but for this other of G and Gi R2 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of hydrogen, C1-4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl, Rs and , more preferably from the group consisting of hydrogen, C1-4 alkylene-(Ca- the other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of ^ more preferably independently selected from the group consisting
In some preferred embodiments of Formula (XX):
(1) at least one of G and Gi is C-X1-R2; R2 is selected from the group consisting of hydrogen, Ci-
4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl, from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, Rs
(2) Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring; (3) Ri and R3 are independently selected from the group consisting of , , preferably independently selected from the group consisting of and
(4) either (i) for one of G and Gi being the other of G and Gi may also be C-X1-R2, but for this other of G and Gi R2 is as defined under (1) (i.e., selected from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, defined under (3) (i.e., selected from the group consisting of preferably selected from the group consisting of
In some embodiments of Formula (XX), Xi is CH2 or O. In some embodiments of Formula (XX), Xi is CH2 or S. In some embodiments of Formula (XX), Xi is O or S. In some embodiments of
Formula (XX), Xi is CH2. In some embodiments of Formula (XX), Xi is 0. In some embodiments of Formula (XX), Xi is S.
In some embodiments of Formula (XX), the STING agonist has the following formula (XXA): wherein:
G is CH, C-SCH3, C-OCH3, or N; and the remaining substituents (in particular, Xi, Ri, R2, and R3) are as defined for Formula (XX).
In some embodiments of Formula (XXA), G is CH. In some embodiments of Formula (XXA), G is C-SCH3. In some embodiments of Formula (XXA), G is C-OCH3. In some embodiments of
Formula (XXA), G is N.
In some embodiments of Formula (XXA), R2 is selected from the group consisting of hydrogen, preferably from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl,
In some embodiments of Formula (XXA), Yi and Y2 are independently selected from the group consisting of pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, dioxazolyl, dithiazolyl, and tetrazolyl, each of which is optionally substituted with 1, 2, 3, or 4 (i.e., up to 4) R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently , wherein X5 is S, O, or NR21, wherein R21' is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of Yi and Y2 are oxazolyl which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of
YI and Y2 are t wherein X5 is O.
In those embodiments of Formula (XXA), where at least one of Rs, it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NHz, - NH(CHa), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXA), Rs, Re, and R7 are independently selected from the O group consisting of hydrogen, C1-4 alkyl, , , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CH3), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXA), Rs is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NH(CH3), or -N(CHs)2, such as -OH or -NH2.
R8-ABA-MU
In those embodiments of Formula (XXA), where Rz is y , it is preferred that Ring B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rio are independently selected from hydrogen and CM alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NH2, -NHfCHs), or -N(CH3h (such as -OH or -NH2). Preferably, u is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3. In some embodiments of Formula (XXA), R2 is selected from the group consisting of hydrogen,
Ci~4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, Rs preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or R5 and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXA), Ri and R3 are independently selected from the group consisting of preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XXA),
other of Ri and R3 can be selected from the groups specified for Formula (XX) or (XXA),
In some preferred embodiments of Formula (XXA), either the other of Ri and R3 can be selected from the groups specified for Formula (XX) or (XXA), preferably selected from the group consisting more preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXA):
(1) R2 is selected from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4
(2) Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring; (3) Ri and R3 are independently selected from the group consisting of , preferably independently selected from the group consisting of other of Ri and R3 is as defined under (3) (i.e., selected from the group consisting of
In some embodiments of Formula (XXA), Xi is CHz or O. In some embodiments of Formula
(XXA), Xi is CH2 or S. In some embodiments of Formula (XXA), Xi is O or S. In some embodiments of Formula (XXA), Xi is CH2. In some embodiments of Formula (XXA), Xi is O. In some embodiments of Formula (XXA), Xi is S. In some embodiments of Formula (XX), the STING agonist has the following formula (XXB): wherein:
G is CH, C-SCH3, C-OCH3, or N;
R8 is -OH or -NH2;
Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R2i; and the remaining substituents (in particular, Xi, Ri, R2, and R3) are as defined for Formula (XX) or Formula (XXA).
In some embodiments of Formula (XXB), G is CH. In some embodiments of Formula (XXB), G is C-SCH3. In some embodiments of Formula (XXB), G is C-OCH3. In some embodiments of Formula (XXB), G is N.
In some embodiments of Formula (XXB), R2 is selected from the group consisting of hydrogen, preferably from the group consisting of hydrogen, C1-4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl,
In those embodiments of Formula (XXB), where R2 is , it is preferred that Ring
B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs. Preferably, u is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In some embodiments of Formula (XXB), both of Yi and Ya are oxazolyl which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of Yi and Y2 are , wherein X5 is O.
In those embodiments of Formula (XXB), where at least one of Rs, it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that Ris is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXB), Rs, Re, and R7 are independently selected from the 0 R15< A R15-fc ) — / group consisting of hydrogen, C1-4 alkyl, v y , and , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each Ris is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Cv 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each Ris is independently selected from the group consisting of -OH, -NH2, -NHfCHs), and -N(CH3)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXB), R2 is selected from the group consisting of hydrogen,
C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, Rs preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXB), Ri and R3 are independently selected from the group consisting of preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXB), the other of Ri and R3 can be selected from the groups specified for Formula (XX), (XXA), or (XXB), (ii) one the other of Ri and R3 can be selected from the groups specified for Formula (XX), (XXA), or (XXB), preferably selected from the group consisting preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXB):
(1) R2 is selected from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, R15< A
(2) Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, \ /w , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring;
O
(3) Ri and Rs are independently selected from the group consisting Of
H2NV H0x <
A , and , preferably independently selected from the group consisting of and
(4) either (
(ii) one the o other of Ri and R3 is as defined under (3) (i.e., selected from the group consisting of
In some embodiments of Formula (XXB), Xi is CH2 or O. In some embodiments of Formula
(XXB), Xi is CH2 or S. In some embodiments of Formula (XXB), Xi is 0 or S. In some embodiments of Formula (XXB), Xi is CH2. In some embodiments of Formula (XXB), Xi is O. In some embodiments of Formula (XXB), Xi is S.
In some embodiments of Formula (XX), the STING agonist has the following formula (XXC): wherein:
Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21; and the remaining substituents (in particular, Ri, R3, and R21) are as defined for any one of
Formulas (XX), (XXA), and (XXB).
In some embodiments of Formula (XXC), both of Yi and Y2 are oxazolyl which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of Yi and Y2 are , wherein X5 is 0.
In those embodiments of Formula (XXC), where at least one of Rs, it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -NfCHsh (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXC), Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1,
2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and - NR9R10, wherein preferably R9 and Rioare independently selected from hydrogen and C1.4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CH3), and -NfCHsh (such as from the group consisting of - OH and -NH?).
In some embodiments of Formula (XXC), Ri and R3 are independently selected from the group consisting of preferably independently selected
R7 o R7 O
In some preferred embodiments of Formula (XXC), one of Ri and R3 is Rs the other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of more preferably independently selected from the group
In some embodiments of Formula (XXC), one the other is selected from the group consisting of H2N
In some preferred embodiments of Formula (XXC):
(1) Ri and R3 are independently selected from the group consisting of , preferably independently selected from the group consisting of
(2) R5, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Rs are optionally connected to form a 5 or 6-membered heterocyclic ring; and
(3) one the o other of Ri and R3 is as defined under (1) (i.e., selected from the group consisting of H2N
In some embodiments of Formula (XX), the STING agonist has the following formula (XXD): wherein:
Rzr is hydrogen or C1-4 alkyl; and the remaining substituents (in particular, Ri and R3) are as defined for any one of Formulas (XX), (XXA), (XXB), and (XXC).
In some embodiments of Formula (XXD), R211 is hydrogen, methyl, ethyl, propyl, or butyl, preferably methyl or ethyl, more preferably ethyl. In those embodiments of Formula (XXD), where at least one of Rs, Re, and it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that Ris is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NHfCHs), and -NfCHsh (such as from the group consisting of -OH and -NHz).
In some embodiments of Formula (XXD), Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , , , wherein w is 1 to 6 (i.e., 1,
2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each Ris is independently selected from the group consisting of -OH and - NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CHs), and -NfCHsh (such as from the group consisting of - OH and -NH2).
In some embodiments of Formula (XXD), Ri and R3 are independently selected from the group consisting of preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XXD), one of Ri and R3 is Rs the other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of more preferably independently selected from the group
In some embodiments of Formula (XXD), one
In some preferred embodiments of Formula (XXD):
O
(1) Ri and R3 are independently selected from the group consisting of H2N. 5
, and , preferably independently selected from the group consisting of
(2) Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e.j. 1, 1, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring; and
(3) one the
O other of Ri and R3 is as defined under (1) (i.e., selected from the group consisting of H2N
In some embodiments of Formula (XX), the STING agonist has the following formula (XXE): wherein:
X is S or O; and the remaining substituents (in particular, Xi, Yi, Y2, Ri, R2, R3,) are as defined for Formula (XX).
In some embodiments of Formula (XXE), X is S.
In some embodiments of Formula (XXE), R2 is selected from the group consisting of hydrogen,
C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, , i ,
In some embodiments of Formula (XXE), Yi and Y2 are independently selected from the group consisting of pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, dioxazolyl, dithiazolyl, and tetrazolyl, each of which is optionally substituted with 1, 2, 3, or 4 (i.e., up to 4) R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently wherein X5 is S, O, or NR21, wherein R21' is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of Yi and Y2 are oxazolyl which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of
Yi and Y2 are v 'X * V5 , wherein X5 is O.
In those embodiments of Formula (XXE), where at least one of R5, it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXE), Rs, Re, and R7 are independently selected from the 0 R15$A group consisting of hydrogen, C1-4 alkyl, x , , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CH3>, and -NfCHsh (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXE), Rs is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NHfCHs), or -NfCHsk, such as -OH or -NH2.
In those embodiments of Formula (XXE), where R2 is , it is preferred that Ring
B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rio are independently selected from hydrogen and C1.4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NH2, -NH(CHs), or -N(CHs)2 (such as -OH or -NH2). Preferably, u is 1 to 5 (i.e., 1,
2, 3, 4, or 5), such as 3. In those embodiments of Formula preferred that R9 is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl) and/or that Rs is hydrogen or Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl), and/or o 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In some embodiments of Formula (XXE), R2 is selected from the group consisting of hydrogen,
C1-4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl, from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, R5 preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Rs are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXE), Ri and R3 are independently selected from the group consisting of preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXE), the other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of t preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXE), either (ii) one the other of Ri and Rs can be selected from the groups specified for Formula (XX), (XXA), (XXB),
(XXC), or (XXD), preferably selected from the group consisting
O
HO\ , r , more preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XXE):
(1) R2 is selected from the group consisting of hydrogen, CM alkylene-(Cs-4 cycloalkyl), C1-4 R15< A
(2) Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, ' 'W , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring;
O
(3) Ri and R3 are independently selected from the group consisting of , preferably independently selected from the group consisting of
O other of Ri and R3 is as defined under (3) (i.e., selected from the group consisting of H2N
In some embodiments of Formula (XXE), Xi is CH2 or O. In some embodiments of Formula
(XXE), Xi is CH? or S. In some embodiments of Formula (XXE), Xi is O or S. In some embodiments of Formula (XXE), Xi is CH2. In some embodiments of Formula (XXE), Xi is O. In some embodiments of Formula (XXE), Xi is S.
In some embodiments of Formula (XX), the STING agonist has the following formula (XXF): wherein:
G is CH, C-SCH3, C-OCH3, or N; and the remaining substituents (in particular, Xi, Yi, Y2, Ri, R2, and R3) are as defined for Formula (XX).
In some embodiments of Formula (XXF), G is CH. In some embodiments of Formula (XXF), G is C-SCH3. In some embodiments of Formula (XXF), G is C-OCH3. In some embodiments of Formula (XXF), G is N. In some embodiments of Formula (XXF), G is CH, C-OCH3, or N.
In some embodiments of Formula (XXF), R2 is selected from the group consisting of hydrogen,
C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, preferably from the group consisting of hydrogen, C1-4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl,
In some embodiments of Formula (XXF), Yi and Y2 are independently selected from the group consisting of pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, dioxazolyl, dithiazolyl, and tetrazolyl, each of which is optionally substituted with 1, 2, 3, or 4 (i.e., up to 4) R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some preferred embodiments, Yi and Y2 are independently wherein X5 is S, O, or NR21', wherein R21' is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of Yi and Y2 are oxazolyl which is optionally substituted with 1 or 2 R21 (such as methyl, ethyl, propyl, or butyl). In some particularly preferred embodiments, both of
Yi and Y2 are , wherein X5 is O.
In those embodiments of Formula (XXF), where at least one of R5, Re, and R7 it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and Cu alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -NJCHsh (such as from the group consisting of -OH and -NH2). In some embodiments of Formula (XXF), Rs, Re, and R? are independently selected from the group consisting of hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each Ris is independently selected from the group consisting of -OH, -NHz, -NH(CHs), and -N(CHB)Z (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXF), Rs is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NHfCHs), or -N(CHs)z, such as -OH or -NH2.
In those embodiments of Formula (XXF), where Rz is , it is preferred that Ring
B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rio are independently selected from hydrogen and C1.4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NHz, -NH(CH3), or -N(CHs)2 (such as -OH or -NHz). Preferably, u is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In those embodiments of Formula preferred that R9 is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl) and/or that Rs is hydrogen or Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl), and/or o 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3. In some embodiments of Formula (XXF), Rz is selected from the group consisting of hydrogen,
CI-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, Rs preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or R5 and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXF), Ri and R3 are independently selected from the group consisting of preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XXF), (ii) one the other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of more preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXF), either other of Ri and Rs can be selected from the groups specified for Formula (XX), (XXA), (XXB),
O
(XXC), (XXD), or (XXE), preferably selected from the group consisting of HO\ ' , and , more preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XXF):
(1) R2 is selected from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4
(2) Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring;
O
(3) Ri and R3 are independently selected from the group consisting of
H2N\y h0\/
, and , preferably independently selected from the group consisting of and
(4) either (
(ii) one of Ri the
O other of Ri and R3 is as defined under (3) (i.e. , selected from the group consisting of H2N preferably selected from the group consisting of H2N
In some embodiments of Formula (XXF), Xi is CH2 or O. In some embodiments of Formula (XXF), Xi is CH2 or S. In some embodiments of Formula (XXF), Xi is O or S. In some embodiments of Formula (XXF), Xi is CH2. In some embodiments of Formula (XXF), Xi is O. In some embodiments of Formula (XXF), Xi is S.
In some embodiments of Formula (XX), the STING agonist has the following formula (XXG):
wherein the substituents (in particular, Xi, X2, X3, Ri, R2, and R3) are as defined for Formula
(XX).
In some embodiments of Formula (XXG), one of X2 and X3 is S and the other is NH. In some preferred embodiments of Formula (XXG), each of X2 and X3 is NH.
In some embodiments of Formula (XXG), R2 is selected from the group consisting of hydrogen, preferably from the group consisting of hydrogen, C1-4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl,
In those embodiments of Formula (XXG), where at least one of Rs, Re, and it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXG), Rs, Re, and R7 are independently selected from the
R15<A R15/CY- 7 HO^Ay group consisting of hydrogen, C1-4 alkyl, ' 'w , , and f , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CH3), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXG), Rs is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1.4 alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NH(CHs), or -NfCHsh, such as -OH or -NH2.
In those embodiments of Formula (XXG), where R? is , it is preferred that Ring
B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NH2, -NH(CHs), or -N(CHs)2 (such as -OH or -NH2). Preferably, u is 1 to 5 (i.e., 1,
2, 3, 4, or 5), such as 3.
In those embodiments of Formula preferred that R9 is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl) and/or that Rs is hydrogen or Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl), and/or o 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3. In some embodiments of Formula (XXG), R2 is selected from the group consisting of hydrogen, from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, Rs preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXG), Ri and R3 are independently selected from the group consisting of t preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XXG), (ii) one the other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of other of Ri and R3 can be selected from the groups specified for Formula (XX), (XXA), (XXB),
(XXC), (XXD), (XXE), or (XXF), preferably selected from the group consisting of HOV , and , more preferably independently selected from the group consisting of
In some preferred embodiments of Formula (XXG):
(1) Rz is selected from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4
(2) Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring;
(3) Ri and R3 are independently selected from the group consisting of , , preferably independently selected from the group consisting of and
(4) either (
(ii) one the other of Ri and R3 is as defined under (3) (i.e., selected from the group consisting of
In some embodiments of Formula (XXG), Xi is CH2 or O. In some embodiments of Formula
(XXG), Xi is CH2 or S. In some embodiments of Formula (XXG), Xi is O or S. In some embodiments of Formula (XXG), Xi is CH2. In some embodiments of Formula (XXG), Xi is O. In some embodiments of Formula (XXG), Xi is S.
In some embodiments of Formula (XX), the STING agonist has the following formula (XXH):
wherein the substituents (in particular, Xi, Ri, R2, and R3) are as defined for Formula (XX).
In some embodiments of Formula (XXH), R2 is selected from the group consisting of hydrogen, preferably from the group consisting of hydrogen, C1-4 alkylene-(Ca-4 cycloalkyl), C1-4 alkyl,
In those embodiments of Formula (XXH), where at least one of Rs, Re, and R7 is it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that Ris is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -NfCHsh (such as from the group consisting of -OH and -NH2). In some embodiments of Formula (XXH), Rs, Re, and R7 are independently selected from the O R15<A R^-fcV-7 HO^Ay group consisting of hydrogen, Ci-4 alkyl, ' v — , and ' , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CHa), and -NfCHsh (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXH), Rg is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NH(CHs), or -N(CHs)2, such as -OH or -NH2.
In those embodiments of Formula (XXH), where R2 is , it is preferred that Ring
B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NHz, -NH(CHs), or -N(CHs)2 (such as -OH or -NH2). Preferably, u is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In those embodiments of Formula preferred that R9 is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl) and/or that Rs is hydrogen or Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl), and/or o 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3. In some embodiments of Formula (XXH), R2 is selected from the group consisting of hydrogen,
C1-4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl, preferably from the group consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl,
2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or R5 and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXH), Ri and R3 are independently selected from the group
In some preferred embodiments of Formula (XXH), (ii) one the other of Ri and Ra can be selected from the group consisting
In some preferred embodiments of Formula (XXH), either the other of Ri and R3 can be selected from the group consisting
In some preferred embodiments of Formula (XXH):
(1) R2 is selected from the group consisting of hydrogen, C1-4 alkylene-(Cs-4 cycloalkyl), C1-4 hydrogen, C1-4 alkylene-(Ca-4 cycloalkyl), C1-4 alkyl, Rs R5
(2) R5, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring;
O
(3) Ri and R3 are independently selected from the group consisting of
O other of Ri and R3 is as defined under (3) (i.e., selected from the group consisting of
In some embodiments of Formula (XXH), Xi is CH2 or O. In some embodiments of Formula (XXH), Xi is CH2 or S. In some embodiments of Formula (XXH), Xi is O or S. In some embodiments of Formula (XXH), Xi is CH2. In some embodiments of Formula (XXH), Xi is O. In some embodiments of Formula (XXH), Xi is S.
In some embodiments of Formula (XX), the STING agonist has the following formula (XXJ): wherein the substituents (in particular, Xi, X2, X3, Ri, R2, and R3) are as defined for Formula (XX).
In some embodiments of Formula (XXJ), one of X2 and X3 is S and the other is NH. In some preferred embodiments of Formula (XXJ), each of X2 and X3 is NH.
In some embodiments of Formula (XXJ), each R2 is independently selected from the group consisting of hydrogen, C1.4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, preferably from the group consisting of hydrogen, C1-4 alkylene-(C3-4 of Formula (XXJ), one R2 is C1-4 alkyl, and the other is selected from the group consisting of Ci- 4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, preferably the other is selected from the group consisting
In those embodiments of Formula (XXJ), where at least one of R5, it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -NfCHsh (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXJ), R5, Re, and R7 are independently selected from the group consisting of hydrogen, C1-4 alkyl, ; , , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rware independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CHs), and -N(CHs)2 (such as from the group consisting of -OH and -NH2). In some embodiments of Formula (XXJ), Rs is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NH(CH3), or -N(CH3)2, such as -OH or -NH2.
In those embodiments of Formula (XXJ), where R2 is , it is preferred that Ring B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NH2, -NH(CHs), or -N(CHs)2 (such as -OH or -NH2). Preferably, u is 1 to 5 (i.e., 1,
2, 3, 4, or 5), such as 3.
In those embodiments of Formula preferred that R9 is hydrogen or C1.4 alkyl (such as methyl, ethyl, propyl, or butyl) and/or that Rs is hydrogen or Ci-
4 alkyl (such as methyl, ethyl, propyl, or butyl), and/or o 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In some embodiments of Formula (XXJ), one R2 is C1-4 alkyl, and the other is selected from the group consisting of C1-4 alkylene-(Ca-4 cycloalkyl), C1-4 alkyl, ; and Rs, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXJ), Ri and R3 are independently selected from the group consisting of preferably independently selected
In some preferred embodiments of Formula (XXJ), other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXJ), either
(i) one
(ii) one the other of Ri and R3 can be selected from the groups specified for Formula (XX), (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), or (XXH), preferably selected from the group consisting of more preferably independently selected from the group
In some preferred embodiments of Formula (XXJ):
(1) one R2 is C1-4 alkyl, and the other is selected from the group consisting of C1-4 alkylene-(Cs-
4 cycloalkyl), C14 alkyl,
RM,A
(2) Rs, Re, and R7 are independently selected from hydrogen, C1.4 alkyl, x /w , , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring;
O
(3) Ri and R3 are independently selected from the group consisting of , preferably independently selected from the group consisting of
(i.e., the other R2 is as defined under (1), i.e., the other is C1-4 alkyl); or (ii) one of Ri and R3 is the other of Ri and R3 is as defined under (3) (i.e., selected from the group consisting of
In some embodiments of Formula (XXJ), Xi is CH2 or 0. In some embodiments of Formula (XXJ), Xi is CH2 or S. In some embodiments of Formula (XXJ), Xi is O or S. In some embodiments of
Formula (XXJ), Xi is CH2. In some embodiments of Formula (XXJ), Xi is O. In some embodiments of Formula (XXJ), Xi is S.
In some embodiments of Formula (XX), the STING agonist has the following formula (XXK): wherein the substituents (in particular, Xi, Ri, R2, and R3) are as defined for Formula (XX).
In some embodiments of Formula (XXK), each R2 is independently selected from the group
R7 O R7
A R6 . NA HA R6 A N P<AW consisting of hydrogen, C1-4 alkylene-(C3-4 cycloalkyl), C1-4 alkyl, Rs , Rs preferably from the group consisting of hydrogen, C1-4 alkylene-(C3-4 of Formula (XXK), one R? is C1-4 alkyl, and the other is selected from the group consisting of
In those embodiments of Formula (XXK), where at least one of Rs, Re, and it is preferred that Ring C is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. In those embodiments, it is preferred that R15 is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, - NH(CHs), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXK), Rs, Re, and R7 are independently selected from the 0 R15<A R^-fcV-7 group consisting of hydrogen, Ci-4 alkyl, v v — 7 , and , wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring. In some preferred embodiments, each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rio are independently selected from hydrogen and Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NHfCHs), and -N(CHs)2 (such as from the group consisting of -OH and -NH2).
In some embodiments of Formula (XXK), Rs is -OH or -NR9R10, wherein R9 and Rio are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)). In some preferred embodiments, Rs is -OH, -NH2, -NH(CHs), or -N(CH3)2, such as -OH or -NH2.
In those embodiments of Formula (XXK), where R2 is , it is preferred that Ring
B is selected from the group consisting of phenyl, pyrrolyl, furyl, thienyl, imidazolyl, pyrazolyl, oxathiolyl, isoxathiolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, and piperazinyl, each of which is substituted with Rs, wherein Rs is -OH or -NR9R10 (preferably R9 and Rw are independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., Rs is -OH, -NH2, -NH(CHs), or -N(CHs)2 (such as -OH or -NH2). Preferably, u is 1 to 5 (i.e., 1,
2, 3, 4, or 5), such as 3.
In those embodiments of Formula preferred that R9 is hydrogen or C1-4 alkyl (such as methyl, ethyl, propyl, or butyl) and/or that R5 is hydrogen or Ci- 4 alkyl (such as methyl, ethyl, propyl, or butyl), and/or o 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3.
In some embodiments of Formula (XXK), one R2 is C1-4 alkyl, and the other is selected from the group consisting of C1.4 alkylene-(Cs-4 cycloalkyl), C1-4 alkyl,
preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring.
In some embodiments of Formula (XXK), Ri and R3 are independently selected from the group
In some preferred embodiments of Formula (XXK), other of Ri and R3 can be selected from the groups specified for Formula (XX), preferably selected from the group consisting of more preferably independently selected from the group consisting
In some preferred embodiments of Formula (XXK), either other of Ri and R3 can be selected from the group consisting
In some preferred embodiments of Formula (XXK):
(1) one R2 is C1-4 alkyl, and the other is selected from the group consisting of C1-4 alkylene-(C3-
4 cycloalkyl), C1-4 alkyl,
(2) R5, Re, and R7 are independently selected from hydrogen, C1-4 alkyl, wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4
(i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring;
O
(3) Ri and R3 are independently selected from the group consisting of
(i.e., the other R2 is as defined under (1), i.e., the other is C1-4 alkyl); or (ii) one of Ri and R3 is the other of Ri and R3 is as defined under (3) (i.e., selected from the group consisting
In some embodiments of Formula (XXK), Xi is CH2 or O. In some embodiments of Formula (XXK), Xi is CH2 or S. In some embodiments of Formula (XXK), Xi is O or S. In some embodiments of Formula (XXK), Xi is CH2. In some embodiments of Formula (XXK), Xi is O. In some embodiments of Formula (XXK), Xi is S.
In some embodiments of Formula (XX), the STING agonist has one of the following Formulas (XX-l) to (XX-LXII):
(XX-XXVIII) (XX-XXIX) (XX-XXX)
(XX-XL) (XX-XLI) (XX-XLII)
(XX-LXI) (XX-LXII) wherein the substituents Xi, Rs, Re, R7, m, and n are as defined for any one of Formulas (XX), (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), and (XXK).
In some embodiments of Formulas (XX-I) to (XX-LXII), one or more of the following requirements apply:
(1) Rs, Re, and R7 are independently selected from the group consisting of hydrogen, C1-4 alkyl, wherein w is 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6), preferably 1 to 4 (i.e., 1, 2, 3, or 4); and Ring C is phenylene; or Rs and Re are optionally connected to form a 5- or 6-membered heterocyclic ring; and/or
(2) Each R15 is independently selected from the group consisting of -OH and -NR9R10, wherein preferably R9 and Rioare independently selected from hydrogen and C1-4 alkyl (such as methyl, ethyl, propyl, or butyl)), e.g., each R15 is independently selected from the group consisting of -OH, -NH2, -NH(CH3), and -N(CH3)2 (such as from the group consisting of -OH and -NH2); and/or
(3) Xi is O (in particular, for Formulas (XX-I) and (XX-IV)); and/or
(4) m is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3 (in particular, for Formulas (XX-I), (XX-IX), (XX-X), (XX-XXXI), (XX-XXXII), (XX-XXXIII), (XX-XXXIV), (XX-XLVII), and (XX-L)); or
(5) n is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3 (in particular, for Formulas (XX-IV), (XX-XVII), (XX- XVIII), (XX-XLIII), (XX-XLIV), (XX-XLV), (XX-XLVI), (XX-LIII), and (XX-LVI)).
In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1) and (2) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1) and (3) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1) and (4) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1) and (5) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1), (2), and (3) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1), (2), and (4) apply. In some embodiments of Formulas (XX-I) to (XX- LXII), the above requirements (1), (2), and (5) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1), (2), (3), and (4) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (1), (2), (3), and (5) apply.
In some embodiments of Formulas (XX-I) to (XX-LXII), one or more of the following requirements apply:
(6) the moiety selected from the group consisting of H2NNH-, H2NN(CH3)-, H(CH3)NNH-, H(CH3)NN(CH3)-, H2NN(CH2CH2NH2)-, (4-aminophenyl-CH2-)NHNH-, and H2N-(4- aminophenyl-CH2-)N-; and/or
(7) Xi is O (in particular, for Formulas (XX-I) and (XX-IV)); and/or
(8) m is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3 (in particular, for Formulas (XX-I), (XX-IX), (XX-X), (XX-XXXI), (XX-XXXII), (XX-XXXIII), (XX-XXXIV), (XX-XLVII), and (XX-L)); or
(9) n is 1 to 5 (i.e., 1, 2, 3, 4, or 5), such as 3 (in particular, for Formulas (XX-IV), (XX-XVII), (XX- XVIII), (XX-XLIII), (XX-XLIV), (XX-XLV), (XX-XLVI), (XX-LIII), and (XX-LVI)). In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (6) and (7) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (6) and (8) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (6) and (9) apply, some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (6), (7), and (8) apply. In some embodiments of Formulas (XX-I) to (XX-LXII), the above requirements (6), (7), and (9) apply.
Specific examples of STING agonists of Formula (XX) include the following:
(XX-28) (XX-29)
(XX-72) (XX-73)
Preferred examples of STING agonists of Formula (XX) include any one of Formulas (XX-6), (XX- 16), (XX-17), (XX-18), (XX-19), (XX-20), (XX-21), (XX-22), (XX-29), (XX-30), (XX-50), (XX-51), (XX- 53), (XX-55), (XX-58), (XX-60), (XX-64), (XX-71), (XX-72), and (XX-73), such as any one of Formulas (XX-6), (XX-18), (XX-51), (XX-53), and (XX-73). Particularly preferred examples are
Formulas (XX-18), (XX-51), and (XX-73), such as (XX-18).
In case the STING agonist is (in particular covalently) attached to a different molecule (such as in a tag conjugate as described herein (i.e., in "a compound comprising a payload moiety and a tag, wherein the payload moiety comprises a STING agonist", as disclosed herein or in " (ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload moiety comprises a STING agonist", as disclosed herein)), the STING agonist is preferably a STING agonist moiety.
The term "STING agonist moiety" as used herein refers to a radical (preferably a monovalent radical) of a STING agonist. Preferably, the monovalent radical of a STING agonist is one in which a hydrogen atom, a monovalent functional group (like a hydroxy, amino, or hydrazine group) or a monovalent part of a functional group (like a hydrogen atom of a hydroxy, amino or hydrazine functional group; a hydroxy group of a carboxy, hydroxyamide, or hydroxy functional group; or an amino group of an amide, hydrazine, or amino functional group) present in the STING agonist has been removed and replaced by a bond, wherein the STING agonist moiety preferably still has STING agonistic activity. For example, if a STING agonist has Formula (XX), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XX'): wherein Ring A, X2, X3, Yi, Y2, L1, Gi, G2, and Ri are as defined for Formula (XX), and wherein one of the monovalent substituents present in the STING agonist of Formula (XX) (e.g., R2 being hydrogen or C1-4 alkyl; or Ri being N(R6)(R?)N(Rs)C(O)-) has been replaced by a corresponding divalent substituent (e.g., R2 has been replaced by R2', wherein R2' is a bond (if R2 was hydrogen) or C1-4 alkylene (if R2 was a C1-4 alkyl); or Ri has been replaced by Ri', wherein Ri1 is selected from the group consisting of *-N(R6)N(Rs)C(O)-# (formally obtained by replacing the monovalent substituent R7 by a bond), *-N(Rs)C(O)-# (formally obtained by replacing the monovalent group (Re)(R?)N- by a bond), and -C(O)- (formally obtained by replacing the monovalent group N(R6)(R?)N(Rs)- by a bond), wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload moiety comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload moiety comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rr to the remainder of the STING agonist moiety).
Similar considerations apply to the cases, where the STING agonist has one of the other formulas disclosed herein (e.g., Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), (XXK), (XX-I) to (XX-LXII), and (XX-1) to (XX-73), particularly Formulas (XX-6), (XX-16), (XX- 17), (XX- 18), (XX- 19), (XX-20), (XX-21), (XX-22), (XX-29), (XX-30), (XX-50), (XX-51), (XX-53), (XX- 55), (XX-58), (XX-60), (XX-64), (XX-71), (XX-72), and (XX-73)). For example, if a STING agonist has Formula (XXA), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XXA'): wherein Xi, Yi, Y2, G, Ri, R2, and R3 are as defined for Formula (XXA), and wherein one of the monovalent substituents present in the STING agonist of Formula (XXA) (e.g., R2 being hydrogen or C1-4 alkyl; or Ri being N(R6)(R7)N(Rs)C(O)-) has been replaced by a corresponding divalent substituent (e.g., R2 has been replaced by R2’, wherein R2' is a bond or C1-4 alkylene; or Ri has been replaced by Rr, wherein Ri' is selected from the group consisting of *- N(R6)N(RS)C(O)-#, *-N(RS)C(O)-#, and -C(O)-, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload moiety comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload moiety comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rr to the remainder of the STING agonist moiety). Likewise, if a STING agonist has Formula (XXH), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XXH'):
wherein Xi, Ri, R2, and R3 are as defined for Formula (XXH), and wherein one of the monovalent substituents present in the STING agonist of Formula (XXH) (e.g., R2 being hydrogen or CH alkyl; or Ri being N(RG)(R7)N(RS)C(O)-) has been replaced by a corresponding divalent substituent (e.g., R2 has been replaced by R2', wherein R2' is a bond or C1.4 alkylene; or Ri has been replaced by Ri’, wherein Rr is selected from the group consisting of *- N(R6)N(RS)C(O)-#, *-N(Rs)C(O)-w, and -C(O)-, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rr to the remainder of the STING agonist moiety).
Likewise, if a STING agonist has Formula (XX-18), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XX-181): wherein one of the monovalent substituents of R2 (being -(CHahMCHsjNI-h) present in the
STING agonist of Formula (XX-18) has been replaced by a corresponding divalent substituent
(e.g., R2 has been replaced by R21, wherein R2' is a bond, -(CHih-, #-(CH2)3N(CHs)-*, or #- (CH2)3N(CH3)NH-*, preferably R2' is #-(CH2)3N(CHs)NH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of R21 to the remainder of the STING agonist moiety).
Furthermore, if a STING agonist has Formula (XX-6), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XX-6'): wherein one of the monovalent substituents of Ri (being -C(O)NHNH2) present in the STING agonist of Formula (XX-6) has been replaced by a corresponding divalent substituent (e.g., Ri has been replaced by Rr, wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rr to the remainder of the STING agonist moiety).
Furthermore, if a STING agonist has Formula (XX-51), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XX-51'): wherein one of the monovalent substituents of Ri (being -C(O)NHNH2) present in the STING agonist of Formula (XX-51) has been replaced by a corresponding divalent substituent (e.g., Ri has been replaced by Ri1, wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rr to the remainder of the STING agonist moiety).
Furthermore, if a STING agonist has Formula (XX-53), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XX-53'): wherein one of the monovalent substituents of R2 (being -(CH2)3N (CHs)N H2) present in the STING agonist of Formula (XX-18) has been replaced by a corresponding divalent substituent (e.g., R2 has been replaced by Rz-, wherein R2' is a bond, -(CFhh-, #-(CH2)3N(CH3)-*, or #- (CH2)3N(CH3)NH-*, preferably R2' is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rz to the remainder of the STING agonist moiety).
Furthermore, if a STING agonist has Formula (XX-73), then a corresponding STING agonist moiety (i.e., a STING agonist moiety derived from the STING agonist) may have the following Formula (XX-73'a) or (XX-73'b): wherein either (I) one of the monovalent substituents of R2 (being -(CH2)3N(CH3)NH2) present in the STING agonist of Formula (XX-73) has been replaced by a corresponding divalent substituent (e.g., R2 has been replaced by R2', wherein R2' is a bond, -(CFhh-, or #- (CH2)3(piperazin-l,4-diyl)-*, preferably R2' is #-(CH2)3(piperazin-l,4-diyl)-*) (cf., Formula XX- 73'a), or (II) wherein one of the monovalent substituents of Ri (being -C(O)NHNH2) present in the STING agonist of Formula (XX-73) has been replaced by a corresponding divalent substituent (e.g., Ri has been replaced by Rr, wherein Ri' is a bond, -C(O)-, #-C(O)NH-*, or #- C(O)NHNH-*, preferably Ri1 is #-C(O)NHNH-*) (cf., Formula XX-73'b), wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of R2' and Rr, respectively, to the remainder of the STING agonist moiety).
Thus, in some embodiments, the STING agonist moiety has Formula (XX'), wherein Ring A, X2, X3, Yi, Y2, L, Gi, G2, and Ri are as defined for Formula (XX), and wherein one of the monovalent substituents present in the STING agonist of Formula (XX) has been replaced by a corresponding divalent substituent (formally by removing a hydrogen atom, a monovalent functional group (like a hydroxy, amino, or hydrazine group) or a monovalent part of a functional group (like a hydrogen atom of a hydroxy, amino or hydrazine functional group; a hydroxy group of a carboxy, hydroxyamide, or hydroxy functional group; or an amino group of an amide, hydrazine, or amino functional group) present in the STING agonist of Formula (XX) has been removed and replacing the removed hydrogen atom, monovalent functional group or monovalent part of a functional group by a bond).
In some embodiments of Formula (XX1), the STING agonist has one of the following Formulas (XXA1), (XXB1), (XXC), (XXD1), (XXE1), (XXF'), (XXG1), (XXH'), (XXJ1), or (XXK'):
(XXG1) (XXH1)
(XXJ1) (XXK') wherein Xi, X2, X3, Yi, Y2, G, Ri , R2, R3, and R21' are as defined for Formula (XXA), (XXB), (XXC),
(XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), and (XXK), respectively, and wherein one of the monovalent substituents present in the STING agonist of Formula (XXA), (XXB), (XXC), (XXD),
(XXE), (XXF), (XXG), (XXH), (XXJ), and (XXK), respectively, has been replaced by a corresponding divalent substituent (formally by removing a hydrogen atom, a monovalent functional group (like a hydroxy, amino, or hydrazine group) or a monovalent part of a functional group (like a hydrogen atom of a hydroxy, amino or hydrazine functional group; a hydroxy group of a carboxy, hydroxyamide, or hydroxy functional group; or an amino group of an amide, hydrazine, or amino functional group) present in the STING agonist of Formula (XX) has been removed and replacing the removed hydrogen atom, monovalent functional group or monovalent part of a functional group by a bond).
In some preferred embodiments, the monovalent substituent present in the STING agonist of Formula (XX) (or any one of Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), and (XXK)) which has been replaced by a corresponding divalent substituent present in the STING agonist moiety of Formula (XX') (or (XXA'), (XXB'), (XXC), (XXD1), (XXE'), (XXF'), (XXG'), (XXH'), (XXJ1), and (XXK'), respectively) is selected from the group consisting of Ri, R3, R2 in Gi being C-X1-R2, and R2 in G being C-X1-R2. In those embodiments, it is preferred that:
(1) either Ri is Rr or R3 is R3', wherein Ru and R3' are selected from the group consisting of a bond, -C(O)-, -O-, -N(R5)-, -(CH2)k-, *-OCH2-«, *-N(R5)(CH2)k-# ; *-N(R5)C(O)-#, *-OC(O)-#, *- N(R6)N(R5)-#, *-N(R6)N(R5)C(O)-#, *-N(R6)N(R5)(CH2)k-#, and *-ON(R5)C(O)-#; or
(2) R2 of either Gi being C-X1-R2 or G being C-X1-R2 is R2', wherein Rz1 is selected from the group consisting of a bond, a diradical of a C2-4 cyclic ether, #-Ci-4 alkylene-(diradical of a C2-4 cyclic ether)-*, C3-4 cycloalkylene, #-Ci-4 alkylene-(C3-4 cycloalkylene)-*, C1-4 alkylene, -(CHz)k-, *- C(O)(CH2)k-#, *-O(CH2)n-#, *-N(R5)(CH2)n-#, *-N(R5)C(O)(CH2)k-#, *-OC(O)(CH2)k-#, *- N(R6)N(R5)C(O)(CH2)k *-N(R6)N(R5)(CH2)n-#, *-ON(Rs)C(O)(CH2)k *-(diradical of morpholine)( -(Ring B)(CH2)k-#, *-0-
(Ring B)(CH2)k-#, and *-(Ring B)(CH2)k-#, wherein Rs, Re, R9, Ring B, k, and n are as defined herein with respect to Formula (XX) (or with respect to any one of Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), and (XXK)); * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Ri-, R3', and R2', respectively, to the remainder of the STING agonist moiety.
In some particularly preferred embodiments of the STING agonist moiety of Formula (XX1) (or (XXA'), (XXB'), (XXC), (XXD1), (XXE'), (XXF1), (XXG'), (XXH1), (XXJ'), and (XXK1), respectively), (1) either Ri is Rr or R3 is R3', wherein Rr and R3' are selected from the group consisting of a bond, -C(O)-, -N(R5)-, -(CH2)k-, *-N(R5)(CH2)k-#, *-N(R5)C(O)-#, *-OC(O)-#, *-N(R6)N(R5)-#, *- N(R6)N(R5)C(O)-#, *-N(R6)N(R5)(CH2)k-#, and *-ON(R5)C(O)-#; or (2) R2 of either Gi being C-XI-R2 or G being C-XI-R2 is Rz, wherein R2' is selected from the group consisting of a bond, -(CH2)k-, *-C(O)(CH2)k-#, *-N(R5)(CH2)n-#, *-N(R5)C(O)(CH2)k-#, *-OC(O)(CH2)k-#, *-N(R6)N(R5)C(O)(CH2)k -
(Ring B)(CH2)k-#, and *-(Ring B)(CH2)k-#, wherein Rs, Re, R9, Ring B, k, and n are as defined herein with respect to Formula (XX) (or with respect to any one of Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), and (XXK)); * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and * represents the attachment point of Rr, R3', and R2', respectively to the remainder of the STING agonist moiety.
In some particularly preferred embodiments of the STING agonist moiety of Formula (XX') (or (XXA'), (XXB1), (XXC), (XXD1), (XXE'), (XXF'), (XXG'), (XXH1), (XXJ'), and (XXK'), respectively), (1) either Ri is Rr or R3 is R3', wherein Rr and R3' are selected from the group consisting of a *- N(R6)N(R5)-#, *-N(R6)N(R5)C(O)-#, *-N(R6)N(R5)(CH2)k-#, and *-ON(R5)C(O)-#; or (2) R2 of either Gi being C-X1-R2 or G being C-X1-R2 is Rz, wherein R2' is selected from the group consisting of a *-N(R6)N(R5)C(O)(CH2)k -#, *-N(R6)N(R5)(CH2)n-#, *-ON(R5)C(O)(CH2)k -#, and t wherein R5, Re, k, and n are as defined herein with respect to Formula (XX) (or with respect to any one of Formulas (XXA), (XXB), (XXC), (XXD), (XXE), (XXF), (XXG), (XXH), (XXJ), and (XXK)); * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rr, R3', and R2', respectively to the remainder of the STING agonist moiety.
Preferred STING agonist moieties derived from Formulas (XX-I) to (XX-LXII) and (XX-1) to (XX- 73) are those, wherein either (1) a monovalent substituent corresponding to Ri or R3 (e.g., the group -C(O)N(RS)N(R6)(R7) in Formula (XX-II)) has been replaced by a corresponding divalent group Rr or R3' (e.g., Rr or R3' is a bond, -C(O)-, -C(O)N(Rs)-, or -C(O)N(Rs)N(Re)-); or (2) a monovalent substituent corresponding to R2 (e.g., the group -(CH2)m-C(O)N(Rs)N(R6)(R7) in Formula (XX-I)) has been replaced by a corresponding divalent group R2' (e.g., Rz is a bond, - (CH2)m-, #-(CH2)m-C(O)-*, #-(CH2)m-C(O)N(R5)-*, or #-(CH2)m-C(O)N(R5)N(R6)-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rr, R3', and Rz, respectively to the remainder of the STING agonist moiety. The following table provides specific examples of divalent moieties R1/R3' and R2' derived from corresponding monovalent moieties R1/R3 and R2:
In a particularly preferred embodiment, the STING agonist moiety of Formula (XX1) has the following Formula (XX-18'): wherein R21 is a bond, -(CHzh*, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably R2' is - (CH2)3-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, more preferably R2> is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to " (ii ) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of R2' to the remainder of the STING agonist moiety).
In a further particularly preferred embodiment, the STING agonist moiety of Formula (XX') has the following Formula (XX-61): wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is -C(O)-, #-C(O)NH- *, or #-C(O)NHNH-*, more preferably Ri1 is #-C(O)NHNH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Ra1 to the remainder of the STING agonist moiety).
In a further particularly preferred embodiment, the STING agonist moiety of Formula (XX1) has the following Formula (XX-511): wherein Ri' is a bond, -C(O)-, #-C(O)NH-*, or S-C(O)NHNH-*, preferably Rr is -C(O)-, #-C(O)NH- *, or #-C(O)NHNH-*, more preferably Ri1 is #-C(O)NHNH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of Rz' to the remainder of the STING agonist moiety).
In a further particularly preferred embodiment, the STING agonist moiety of Formula (XX1) has the following Formula (XX-53'):
wherein R2' is a bond, -(CHzh-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably R2' is - (CH2)3-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, more preferably Rz is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and # represents the attachment point of R2' to the remainder of the STING agonist moiety).
In a further particularly preferred embodiment, the STING agonist moiety of Formula (XX') has the following Formula (XX-73'a): wherein R2' is a bond, -(CH2)3-, or #-(CH2)3(piperazin-l,4-diyl)-*, preferably R2' is -(CH2)3- or #- (CH2)3(piperazin-l,4-diyl)-*, more preferably R2' is #-(CH2)3(piperazin-l,4-diyl)-*, wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and * represents the attachment point of R2' to the remainder of the STING agonist moiety). In a further particularly preferred embodiment, the STING agonist moiety of Formula (XX') has the following Formula (XX-73'b): wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is -C(O)-, #-C(O)NH- *, or #-C(O)NHNH-*, more preferably Rr is #-C(O)NHNH-*), wherein * represents the attachment point to the different molecule (in particular, to "a compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist", as disclosed herein, or to "(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist", as disclosed herein); and ** represents the attachment point of R2' to the remainder of the STING agonist moiety).
In some embodiments, a payload to be delivered to a target cell is taken up by the target cell. In some embodiments, a payload is released from the tag conjugate at the target cell, e.g., by extracellular cleavage. In some embodiments, a tag conjugate is cleavable. In some embodiments, a tag conjugate is cleavable in an intracellular environment. In some embodiments, a tag conjugate is cleavable in an extracellular environment. In some embodiments, cleavage results in the release of the payload moiety from the tag conjugate. In some embodiments, a tag conjugate comprises an enzymatic cleavage site for cleaving the payload moiety from the tag conjugate, e.g., a cathepsin B linker, an MMP linker, a legumain linker, a glucosidase linker, or an ester bond.
Embodiments of tag conjugate and docking compound
In some embodiments, the one or more tags of a tag conjugate are ALFA-tags.
In some embodiments, the tag conjugate comprises one ALFA-tag. In these embodiments, the binding moiety for the tag conjugate on the docking compound comprises an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody. In some embodiments, the tag conjugate comprises two ALFA-tags. In some embodiments, the ALFA-tags are identical. In these embodiments, the binding moiety for the tag conjugate on the docking compound comprises an ALFA-specific single-domain antibody (sdAb), NbALFA-nanobody.
In some embodiments, the docking compound comprises one binding moiety for a tag, e.g., one NbALFA-nanobody. In some embodiments, the tag conjugate comprises two, preferably identical tags, e.g., ALFA-tags, and the docking compound comprises one binding moiety for a tag, e.g., one NbALFA-nanobody. In these embodiments, a binding moiety for a tag, e.g., a NbALFA-nanobody, of two different docking compounds may bind to one of the tags, e.g., ALFA-tags, of a tag conjugate. In some embodiments, a docking compound may be monovalent for binding to a tag, e.g., an ALFA-tag, and optionally monovalent for binding to target antigen.
In some embodiments, the docking compound comprises two binding moieties for a tag, e.g., two NbALFA-nanobodies. In some embodiments, the tag conjugate comprises two, preferably identical tags, e.g., ALFA-tags, and the docking compound comprises two binding moieties for a tag, e.g., two NbALFA-nanobodies. In these embodiments, each binding moiety for a tag, e.g., each NbALFA-nanobody, of a docking compound may bind to one of the tags, e.g., ALFA- tags, of a tag conjugate. In some embodiments, a docking compound may be bivalent for binding to a tag, e.g., an ALFA-tag, and optionally bivalent for binding to target antigen.
In embodiments, wherein a docking compound is bivalent for binding to an ALFA-tag and bivalent for binding to target antigen, the docking compound may comprise a full-length antibody binding to a primary target, wherein each of the heavy chains of the full-length antibody is C-terminally linked to an NbALFA-nanobody. Thus, the docking compound may comprise the structure IgG x NbALFA. Alternatively, the docking compound comprises the Fc domain of an antibody, e.g., IgG or IgA antibody, wherein each of the N-termini is linked to a binding moiety, e.g., a single-domain antibody such as a VHH, binding to a primary target and each of the C-termini is linked to an NbALFA-nanobody. Thus, the docking compound may comprise the structure VHH - Fc x NbALFA.
In some embodiments, an ALFA-tag for use herein comprises a cyclized amino acid sequence selected from the group consisting of Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr- Glu), Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu, and Pro-Ser-Arg-Leu- cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu. A particulary preferred ALFA-tag for use herein comprises the cyclized amino acid sequence Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg- cyclo(Lys-Arg-Leu-Thr-Glu).
In some embodiments, a tag conjugate comprises at least one pAEEA structure.
In some embodiments, a tag comprises a high affinity tag, such as a high affinity ALFA-tag. In some embodiments, a tag comprises a high affinity tag, such as a high affinity ALFA-tag, and the docking compound comprises two binding moieties for a tag, e.g., two NbALFA- nanobodies. In some embodiments, a tag comprises an ALFA-tag selected from the group consisting of:
Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu-NH2,
Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-(cyclo8)Lys-Arg-Arg-Leu-Thr-Glu-NH2, Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-(cyclol3)Glu-NH2, Ac-Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2, and Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Glu-Glu-Leu-Arg-(cyclolO)Lys-Arg-Leu-Thr-Glu-NH2, and the docking compound comprises two binding moieties for the tag, e.g., two NbALFA- nanobodies. In some of these embodiments, a tag conjugate comprises at least two tags, e.g., two tags.
In these embodiments, a pre-formed complex of tag conjugate and docking compound may be administered to a subject.
In some embodiments, a tag comprises a medium/low affinity tag, such as a medium/low affinity ALFA-tag. In some embodiments, a tag comprises a medium/low affinity tag, such as a medium/low affinity ALFA-tag, and the docking compound comprises a single binding moiety for a tag, e.g., a single NbALFA-nanobody. In some embodiments, a tag comprises an ALFA-tag selected from the group consisting of:
- Ac-Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2,
Ac-Pro-Ser-Arg-Leu-Glu-(cyclo6)Lys-Glu-Leu-Arg-(cyclolO)Glu-Arg-Leu-Thr-Glu-NH2, and Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2, and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody. In some of these embodiments, a tag conjugate comprises at least two tags, e.g., two tags.
In these embodiments, a complex of tag conjugate and docking compound may be formed in the body of a subject. For example, following administration of RNA encoding docking compound, docking compound is produced in a subject's body for binding to target antigen and tag conjugate.
In some embodiments, a tag conjugate comprises a tag comprising the ALFA-tag Ser-Arg-Leu- Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu, Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg- Leu-Thr-(cyclol3)Glu or Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr- Glu. In some embodiments, the tag conjugate comprises two of said tags. In some embodiments, the docking compound comprises two binding moieties for a tag, e.g., two NbALFA-nanobodies. In some embodiments, the tag conjugate comprises two of said tags and the docking compound comprises two binding moieties for the tag, e.g., two NbALFA- nanobodies. In some of these embodiments, the payload comprised in the tag conjugate comprises the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety having the Formula (XX-181), (XX-6'), (XX-51'), (XX-53'), (XX-73'a), or (XX-73'b).
In some embodiments, a tag conjugate comprises two tags each tag comprising the ALFA-tag Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu, Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg- (cyclo9)Lys-Arg-Leu-Thr-(cyclol3)Glu or Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys- Arg-Leu-Thr-Glu, and the docking compound comprises two binding moieties for the tag, e.g., two NbALFA-nanobodies. In some of these embodiments, the payload comprised in the tag conjugate comprises the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety having the Formula (XX-181), (XX-6'), (XX-51'), (XX-53'), (XX-73'a), or (XX-73'b).
In some embodiments, a tag conjugate comprises two tags each tag comprising the ALFA-tag Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg-Leu-Thr-Glu, and the docking compound comprises two binding moieties for the tag, e.g., two NbALFA-nanobodies. In some of these embodiments, the payload comprised in the tag conjugate comprises the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety having the Formula (XX- 18'), (XX-61), (XX-51'), (XX-53'), (XX-73'a), or (XX- 73'b).
In some embodiments, a tag conjugate comprises two tags each tag comprising the ALFA-tag Ser-Arg-Leu-Glu-(cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, and the docking compound comprises two binding moieties forthe tag, e.g., two NbALFA-nanobodies. In some of these embodiments, the payload comprised in the tag conjugate comprises the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety having the Formula (XX-181), (XX-61), (XX-51'), (XX-53'), (XX- 73'a ), or (XX-73'b).
In some embodiments, a tag conjugate comprises a tag comprising the ALFA-tag Ser-Arg-Leu- Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, or Pro-Ser-Arg-Leu-(cyclo5)Glu- Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu. In some embodiments, the tag conjugate comprises two of said tags. In some embodiments, the docking compound comprises a single binding moiety for a tag, e.g., a single NbALFA-nanobody. In some embodiments, the tag conjugate comprises two of said tags and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody. In some of these embodiments, the payload comprised in the tag conjugate comprises the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety having the Formula (XX-18'), (XX-6'), (XX-51'), (XX-53'), (XX-73'a), or (XX-73'b).
In some embodiments, a tag conjugate comprises two tags each tag comprising the ALFA-tag Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, or Pro-Ser-Arg-Leu- (cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu, and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody. In some of these embodiments, the payload comprised in the tag conjugate comprises the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety having the Formula (XX-18'), (XX-6’), (XX-511), (XX-53’), (XX-73'a), or (XX-73'b).
In some embodiments, a tag conjugate comprises two tags each tag comprising the ALFA-tag Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu, and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody.
In some embodiments, a tag conjugate comprises two tags each tag comprising the ALFA-tag Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu-(cyclo9)Lys-Arg-Arg-Leu-Thr-Glu, and the docking compound comprises a single binding moiety for the tag, e.g., a single NbALFA-nanobody. In some of these embodiments, the payload comprised in the tag conjugate comprises the STING agonist of Formula (XX), (XXH), or (XXK). In some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK). in some embodiments, the payload moiety comprised in the tag conjugate comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-511), (XX-531), (XX- 73'a), or (XX-73'b).
In some embodiments, a tag conjugate comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19), EX-024 to EX- 035, EX-032 to EX-035, and EX-047 to EX-054. In some embodiments, a tag conjugate comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19), or a tag conjugate is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19). In some preferred embodiments, a tag conjugate comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8), or a tag conjugate is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8). In some particularly preferred embodiments, a tag conjugate comprises a structure of a compound shown herein as SD18321 (PIC7), or a tag conjugate is SD18321 (PIC7). Cells for targeted delivery
According to the disclosure, a payload is delivered specifically to a target cell by targeting a target on target cells, e.g., an antigen on target cells, also referred to herein as "primary target".
In some embodiments, the primary target is a structure such as a protein present on the surface of a target cell such as a cell surface antigen including a cell surface receptor.
Terms such as "expressed on the cell surface", "associated with the cell surface" or "cell surface molecule" mean that a molecule such as a receptor or antigen is associated with and located at the plasma membrane of a cell, wherein at least a part of the molecule faces the extracellular space of said cell and is accessible from the outside of said cell, e.g., by a binding molecule such as an antibody located outside the cell. In this context, a part is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association may be direct or indirect. For example, the association may be by one or more transmembrane domains, one or more lipid anchors, or by the interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of a cell. For example, a molecule associated with the surface of a cell may be a transmembrane protein having an extracellular portion or may be a protein associated with the surface of a cell by interacting with another protein that is a transmembrane protein. "Cell surface" or "surface of a cell" is used in accordance with its normal meaning in the art, and thus includes the outside of the cell which is accessible to binding by proteins and other molecules. An antigen is expressed on the surface of cells if it is located at the surface of said cells and is accessible to binding by e.g. antigen-specific antibodies added to the cells. In one embodiment, an antigen expressed on the surface of cells is an integral membrane protein having an extracellular portion recognized by a binding molecule such as an antibody.
The term "extracellular portion" or "exodomain" in the context of the present invention refers to a part of a molecule such as a protein that is facing the extracellular space of a cell and preferably is accessible from the outside of said cell, e.g., by binding molecules such as antibodies located outside the cell.
In some embodiments, a primary target may be present on a diseased cell. The primary target may be upregulated during a disease, e.g. infection or cancer. In diseased tissues, markers can differ from healthy tissue and offer unique possibilities for therapy, especially targeted therapy.
In some embodiments, the primary target is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen. This allows diseased cells to be targeted by the methods and agents described herein, e.g., for delivering a pharmaceutically active agent.
The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. Disease-associated antigens may be associated with infection by microbes, typically microbial antigens, or associated with cancer, typically tumors.
In some embodiments, the primary target is a tumor antigen. In the context of the present disclosure, the term "tumor antigen" or "tumor-associated antigen" relates to proteins that are under normal conditions specifically expressed in a limited number of tissues and/or organs or in specific developmental stages, for example, the tumor antigen may be under normal conditions specifically expressed in stomach tissue, preferably in the gastric mucosa, in reproductive organs, e.g., in testis, in trophoblastic tissue, e.g., in placenta, or in germ line cells, and are expressed or aberrantly expressed in one or more tumor or cancer tissues. In this context, "a limited number" preferably means not more than 3, more preferably not more than 2. The tumor antigens in the context of the present disclosure include, for example, differentiation antigens, preferably cell type specific differentiation antigens, i.e., proteins that are under normal conditions specifically expressed in a certain cell type at a certain differentiation stage, cancer/testis antigens, i.e., proteins that are under normal conditions specifically expressed in testis and sometimes in placenta, and germ line specific antigens. In the context of the present disclosure, the tumor antigen is preferably associated with the cell surface of a cancer cell and is preferably not or only rarely expressed in normal tissues. Preferably, the tumor antigen or the aberrant expression of the tumor antigen identifies cancer cells. In the context of the present disclosure, the tumor antigen that is expressed by a cancer cell in a subject, e.g., a patient suffering from a cancer disease, is preferably a selfprotein in said subject. In preferred embodiments, the tumor antigen in the context of the present disclosure is expressed under normal conditions specifically in a tissue or organ that is non-essential, i.e., tissues or organs which when damaged by the immune system do not lead to death of the subject, or in organs or structures of the body which are not or only hardly accessible by the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between the tumor antigen which is expressed in normal tissues and the tumor antigen which is expressed in cancer tissues.
Examples for tumor antigens include p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP- 1, CASP-8, CDC27/m, CDK4/m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, GaplOO, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-l/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO- 1, NY-BR-1, pl90 minor BCR-abL, Pml/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT. Particularly preferred tumor antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).
In some embodiments, the primary target is a structure such as a protein present on the surface of a target cell such as a cell surface antigen or cell surface receptor the presence or amount of which is characteristic for certain cell types compared to others. This allows certain cell types characterized by the presence or increased amounts to be targeted by the methods and agents described herein.
In some embodiments, the cells for targeted delivery are immune effector cells and the primary target is a cell surface antigen that is characteristic for immune effector cells.
In some embodiments, a primary target, e.g., CD3, such CD3e, CD4 or CD8, may be present on an immune cell, such as an immune effector cell.
In some embodiments, a primary target may be present on a diseased cell, such as a tumor cells, and the agents and methods described herein may deliver a payload to immune cells close to the diseased cell. For example, if the docking compound comprises Fc sequences, e.g., of the heavy chains of an immunoglobulin, the complex described herein comprising a tag conjugate and a docking compound targeted to diseased cells may by taken up by immune cells located in vicinity to the targeted diseased cells by Fc-mediated internalization.
Immune effector cells
The immune cells used in connection with the methods and agents described herein include, in particular, immune effector cells such as cells with lytic potential, in particular lymphoid cells, and are preferably T cells, in particular cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes triggers the destruction of target cells. For example, cytotoxic T cells trigger the destruction of target cells by either or both of the following means. First, upon activation T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a caspase cascade in the cytoplasm that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between the T cells and target cells. The cells used in connection with the present disclosure will preferably be autologous cells, although heterologous cells or allogenic cells can be used.
The term "effector functions" in the context of the present disclosure includes any functions mediated by components of the immune system that result, for example, in the killing of diseased cells such as tumor cells, or in the inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis. Preferably, the effector functions in the context of the present disclosure are T cell mediated effector functions. Such functions comprise in the case of a helper T cell (CD4+ T cell) the release of cytokines and/or the activation of CD8+ lymphocytes (CTLs) and/or B cells, and in the case of CTL the elimination of cells, i.e., cells characterized by expression of an antigen, for example, via apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-g and TNF-a, and specific cytolytic killing of antigen expressing target cells.
The term "immune effector cell" or "immunoreactive cell" in the context of the present disclosure relates to a cell which exerts effector functions during an immune reaction. An "immune effector cell" in some embodiments is capable of binding an antigen such as an antigen presented by in the context of MHC on a cell or expressed on the surface of a cell and mediating an immune response. For example, immune effector cells comprise T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present disclosure, "immune effector cells" are T cells, preferably CD4+ and/or CD8+ T cells, most preferably CD8+ T cells. The term "immune effector cell" also includes a cell which can mature into an immune cell (such as T cell, in particular T helper cell, or cytolytic T cell) with suitable stimulation. Immune effector cells comprise CD34+ hematopoietic stem cells, immature and mature T cells and immature and mature B cells. The differentiation of T cell precursors into a cytolytic T cell, when exposed to an antigen, is similar to clonal selection of the immune system.
A "lymphoid cell" is a cell which, is capable of producing an immune response such as a cellular immune response, or a precursor cell of such cell, and includes lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. A lymphoid cell may be an immune effector cell as described herein. A preferred lymphoid cell is a T cell which can be modified to express an antigen receptor on the cell surface. In some embodiments, the lymphoid cell lacks endogenous expression of a T cell receptor.
The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) which comprise cytolytic T cells.
T cells belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors (TCR). The thymus is the principal organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with a distinct function.
T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.
Cytotoxic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. "Regulatory T cells" or "Tregs" are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.
As used herein, the term "naive T cell" refers to mature T cells that, unlike activated or memory T cells, have not encountered their cognate antigen within the periphery. Naive T cells are commonly characterized by the surface expression of L-selectin (CD62L), the absence of the activation markers CD25, CD44 or CD69 and the absence of the memory CD45RO isoform.
As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that have previously encountered and responded to their cognate antigen. At a second encounter with the antigen, memory T cells can reproduce to mount a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells may be either CD4+ or CD8+ and usually express CD45RO.
As used herein, the term "T cell" also includes a cell which can mature into a T cell with suitable stimulation.
A majority of T cells have a T cell receptor (TCR) existing as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCRa and TCRP) genes and are called a- and P-TCR chains. y6 T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. However, in y6 T cells, the TCR is made up of one y-chain and one 6-chain. This group of T cells is much less common (2% of total T cells) than the a0 T cells.
All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitors derived from hematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classed as double-negative (CD4 CD8 ) cells. As they progress through their development they become double-positive thymocytes (CD4+CD8+), and finally mature to single-positive (CD4+CD8 or CD4 CD8+) thymocytes that are then released from the thymus to peripheral tissues.
As used herein, the term "NK cell" or "Natural Killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor. As provided herein, the NK cell can also be differentiated from a stem cell or progenitor cell.
Targeted delivery of payloads
The agents and methods described herein find use in a variety of applications in which it is desired to deliver a payload to a target cell. The agents described herein may be administered by in vitro or in vivo protocols. Delivery of payloads using the methods and agents described herein can be used with a variety of target cells such that the payload is delivered to the target cells and optionally introduced into the target cells (or cells in vicinity to the target cells). The present disclosure may provide for in vitro or in vivo delivery of the payload to the target cell, depending on the location of the target cell. For example, where the target cell is an isolated cell, the payload may be delivered directly to the cell under cell culture conditions permissive of viability of the target cell. Alternatively, where the target cell or cells are part of a multicellular organism, the targeting compounds described herein (tag conjugate/docking compound) may be provided to the organism or host in a manner such that the targeting compounds are able to reach and optionally enter the target cell(s) (or cells in vicinity to the target cells). By "in vivo” it is meant that the targeting compounds (or a nucleic acid encoding therefor) are administered to a living body of an animal. By "ex vivo" it is meant that cells are modified outside of the body. Such cells may be returned to a living body. The route of administration of the targeting compounds (or a nucleic acid encoding therefor) to the multicellular organism depends on several parameters, including the nature of the targeting compounds. Of particular interest as systemic routes are vascular routes, by which the targeting compounds (or a nucleic acid encoding therefor) are introduced into the vascular system of the host, e.g., an artery or vein, where intravenous routes of administration are of particular interest in many embodiments. For administration, targeting compounds (or a nucleic acid encoding therefor) typically are present in a pharmaceutical preparation, e.g., comprising a pharmaceutically acceptable carrier, diluent and/or adjuvant, and include an effective amount of the payload. In certain embodiments, the targeting compounds (or a nucleic acid encoding therefor) are administered in an aqueous delivery vehicle, e.g., a saline solution. As such, in many embodiments, the targeting compounds (or a nucleic acid encoding therefor) are administered intravascularly, e.g., intraarterially or intravenously, employing an aqueous based delivery vehicle, e.g., a saline solution.
In many embodiments, the targeting compounds (or a nucleic acid encoding therefor) are administered to a multicellular organism in an in vivo manner such that the payload is introduced into a target cell of the multicellular organism. In the case of nucleic acid, administration is typically under conditions sufficient for expression of the nucleic acid to occur. In some embodiments, the agents and methods described herein result in transient expression of the nucleic acid, as opposed to persistent expression, as indicated above. By transient expression is meant that the expression of nucleic acid at a detectable level does not persist for an extended period of time, following administration of the nucleic acid. By extended period of time is meant at least 1 week, usually at least 2 months and more usually at least 6 months. By detectable level is meant that the expression of the nucleic acid is at a level such that one can detect the encoded protein in the mammal, e.g., in the serum of the mammal, at a therapeutic concentration.
In some embodiments, the above-described transient expression is achieved without integration of the nucleic acid into the target cell genome of the host.
Binding moieties and agents
The present disclosure describes binding moieties or agents such as antibodies or antibody derivatives. Moreover, the disclosure describes bispecific or multispecific binding agents such as bispecific antibodies comprising a first and a second binding domain, wherein the first binding domain is capable of binding to a primary target and the second binding domain is capable of binding to a tag conjugate.
The term "binding agent" as used herein refers to any agent capable of binding to desired antigens. In certain embodiments, the binding agent is or comprises an antibody, antibody fragment, or any other binding protein, or any combination thereof.
The term "binding moiety" as used herein refers to any moiety, group or domain capable of binding to desired antigens. In certain embodiments, the binding moiety is or comprises an antibody, antibody fragment, or any other binding protein, or any combination thereof.
As used herein, the term "antigen" is a molecule capable of being bound by a binding moiety or agent, such as an antibody. An antigen may additionally be capable of inducing a humoral immune response and/or cellular immune response leading to the production of B- and/or T- lymphocytes. An antigen may have one or more epitopes (B-cell and T-cell epitopes).
The term "epitope" refers to a part or fragment of a molecule or antigen that is recognized by a binding agent. For example, the epitope may be recognized by an antibody or any other binding protein. An epitope may include a continuous or discontinuous portion of the antigen and may be between about 5 and about 100, such as between about 5 and about 50, more preferably between about 8 and about 30, most preferably between about 8 and about 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is between about 10 and about 25 amino acids in length. The term "epitope" includes structural epitopes.
The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. The hinge region is the region between the CHI and CH2 domains of the heavy chain and is highly flexible. Disulphide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)).
The term "antibody" (Ab) as used herein refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to bind, preferably specifically bind to an antigen. In some embodiments, binding takes place under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The term "antigen-binding region", "binding region" or "binding domain", as used herein, refers to the region or domain which interacts with the antigen and typically comprises both a VH region and a VL region. The term antibody when used herein comprises not only monospecific antibodies, but also multispecific antibodies which comprise multiple, such as two or more, e.g. three or more, different antigen-binding regions. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. As indicated above, the term antibody as used herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that are antigen-binding fragments, i.e., retain the ability to specifically bind to the antigen, and antibody derivatives, i.e., constructs that are derived from an antibody. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" include (i) a Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in W02007059782 (Genmab); (ii) Ffab'Jz fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341, 544-546 (1989)), which consists essentially of a VH domain and also called domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov;21(ll):484-90); (vi) camelid or Nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan;5(l):lll-24) and (vii) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure, as well as bispecific formats of such fragments, are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.
The phrase "single chain Fv" or "scFv" refers to an antibody in which the variable domains of the heavy chain and of the light chain (VH and VL) of a traditional two chain antibody have been joined to form one chain. Optionally, a linker (usually a peptide) is inserted between the two chains to allow for proper folding and creation of an active binding site.
A single-domain antibody, also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. In some embodiments, a single-domain antibody is a variable domain (VH) of a heavy-chain antibody. These are called VHH fragments. Like a whole antibody, a single-domain antibody is able to bind selectively to a specific antigen. The first single-domain antibodies were engineered from heavy-chain antibodies found in camelids. Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG) from humans or mice into monomers. Although most research into single-domain antibodies is currently based on heavy chain variable domains, nanobodies derived from light chains have also been shown to bind specifically to target epitopes.
An antibody can possess any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (for instance IgGl, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes. When a particular isotype, e.g. IgGl, is mentioned herein, the term is not limited to a specific isotype sequence, e.g. a particular IgGl sequence, but is used to indicate that the antibody is closer in sequence to that isotype, e.g. IgGl, than to other isotypes. Thus, e.g. an IgGl antibody may be a sequence variant of a naturally- occurring IgGl antibody, including variations in the constant regions.
In various embodiments, an antibody is an IgGl antibody, more particularly an IgGl, kappa or IgGl, lambda isotype (i.e. IgGl, K, X), an lgG2a antibody (e.g. lgG2a, K, X), an lgG2b antibody (e.g. lgG2b, K, X), an lgG3 antibody (e.g. lgG3, K, X) or an lgG4 antibody (e.g. lgG4, K, X). The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.
The term "chimeric antibody" as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. The terms "variable region" or "variable domain" as used in the context of chimeric antibodies, refer to a region which comprises the CDRs and framework regions of both the heavy and light chains of the immunoglobulin. Chimeric antibodies may be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. The chimeric antibody may be a genetically or an enzymatically engineered recombinant antibody. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody may be performed by other methods than described herein.
The term "humanized antibody" as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92/22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back- mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.
The term "human antibody" as used herein, refers to antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse or rat, have been grafted onto human framework sequences. Human monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibody can be employed, e.g., viral or oncogenic transformation of B-lymphocytes or phage display techniques using libraries of human antibody genes. A suitable animal system for preparing hybridomas that secrete human monoclonal antibodies is the murine system. Hybridoma production in the mouse is a very well established procedure. Immunization protocols and techniques for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known. Human monoclonal antibodies can thus e.g. be generated using transgenic or transchromosomal mice or rats carrying parts of the human immune system rather than the mouse or rat system. Accordingly, in some embodiments, a human antibody is obtained from a transgenic animal, such as a mouse or a rat, carrying human germline immunoglobulin sequences instead of animal immunoglobulin sequences. In such embodiments, the antibody originates from human germline immunoglobulin sequences introduced in the animal, but the final antibody sequence is the result of said human germline immunoglobulin sequences being further modified by somatic hypermutations and affinity maturation by the endogeneous animal antibody machinery, see e.g. Mendez et al. 1997 Nat Genet. 15(2):146-56. When used herein, unless contradicted by context, the term "Fab-arm", "binding arm" or "arm" includes one heavy chain-light chain pair and is used interchangeably with "halfmolecule" herein.
The term "full-length" when used in the context of an antibody indicates that the antibody is not a fragment, but contains all of the domains of the particular isotype normally found for that isotype in nature, e.g. the VH, CHI, CH2, CH3, hinge, VL and CL domains for an IgGl antibody.
When used herein, unless contradicted by context, the term "Fc region" refers to an antibody region consisting of the two Fc sequences of the heavy chains of an immunoglobulin, wherein said Fc sequences comprise at least a hinge region, a CH2 domain, and a CH3 domain.
The term "specificity" as used herein is intended to have the following meaning unless contradicted by context. Two antibodies have the "same specificity" if they bind to the same antigen and the same epitope.
Naturally occurring antibodies are generally monospecific, i.e. they bind to a single antigen. Described herein are binding agents, e.g., docking compounds, binding to different epitopes on e.g. a primary target and a tag conjugate. Such binding agents are at least bispecific or multispecific such as trispecific, tetraspecific and so on. Thus, the binding agent may comprise two or more antibodies as described herein or fragments thereof. In particular, a binding agent described herein may be an artificial protein that is composed of two different antibodies, an antibody and a fragment of a different antibody, and fragments of two different antibodies (said fragments of two different antibodies forming two binding domains).
According to the disclosure, a bispecific binding agent, in particular a bispecific protein, such as a bispecific antibody is a molecule that has two different binding specificities and thus may bind to two epitopes. Particularly, the term "bispecific antibody" as used herein refers to an antibody comprising two antigen-binding sites, a first binding site having affinity for a first epitope and a second binding site having binding affinity for a second epitope distinct from the first.
The term "bispecific" as used herein refers to an agent having two different antigen-binding regions binding to different epitopes.
"Multispecific binding agents" are molecules which have more than two different binding specificities. Many different formats and uses of bispecific antibodies are known in the art, and were reviewed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and; MAbs, 2012 Mar- Apr;4(2):182-97.
A bispecific binding agent according to the present disclosure is not limited to any particular bispecific format or method of producing it.
Examples of bispecific antibody molecules which may be used herein comprise (i) a single antibody that has two arms comprising different antigen-binding regions; (ii) a single chain antibody that has specificity to two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; (iii) a dual-variable-domain antibody (DVD-lg), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD- lg™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) a chemically- linked bispecific (Fab')2 fragment; (v) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (vi) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (vii) a so-called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (viii) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fab-arm; and (ix) a diabody.
The term "bispecific antibody" includes diabodies. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g. , Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444- 6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Bispecific antibodies also include bispecific single chain antibodies. The term "bispecific single chain antibody" denotes a single polypeptide chain comprising two binding domains. In particular, the term "bispecific single chain antibody" or "single chain bispecific antibody" or related terms as used herein preferably mean antibody constructs resulting from joining at least two antibody variable regions in a single polypeptide chain devoid of the constant and/or Fc portion(s) present in full immunoglobulins. For example, a bispecific single chain antibody may be a construct with a total of two antibody variable regions, for example two VH regions, each capable of specifically binding to a separate epitope, and connected with one another through a short polypeptide spacer such that the two antibody variable regions with their interposed spacer exist as a single contiguous polypeptide chain. Another example of a bispecific single chain antibody may be a single polypeptide chain with three antibody variable regions. Here, two antibody variable regions, for example one VH and one VL, may make up an scFv, wherein the two antibody variable regions are connected to one another via a synthetic polypeptide linker, the latter often being genetically engineered so as to be minimally immunogenic while remaining maximally resistant to proteolysis. This scFv is capable of specifically binding to a particular epitope, and is connected to a further antibody variable region, for example a VH region, capable of binding to a different epitope than that bound by the scFv. Yet another example of a bispecific single chain antibody may be a single polypeptide chain with four antibody variable regions. Here, the first two antibody variable regions, for example a VH region and a VL region, may form one scFv capable of binding to one epitope, whereas the second VH region and VL region may form a second scFv capable of binding to another epitope. Within a single contiguous polypeptide chain, individual antibody variable regions of one specificity may advantageously be separated by a synthetic polypeptide linker, whereas the respective scFvs may advantageously be separated by a short polypeptide spacer as described above. According to some embodiments, the first binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. According to some embodiments, the first binding domain of the bispecific antibody comprises two antibody variable domains, preferably a scFv, i.e. VH-VL or VL-VH. According to some embodiments, the second binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. Accordingto some embodiments, the second binding domain of the bispecific antibody comprises two antibody variable domains, preferably a scFv, i.e. VH-VL or VL-VH. In its minimal form, the total number of antibody variable regions in the bispecific antibody is thus only two. For example, such an antibody could comprise two VH or two VHH domains. According to some embodiments, the first binding domain and the second binding domain of the bispecific antibody each comprise one antibody variable domain, preferably a VHH domain. According to some embodiments, the first binding domain and the second binding domain of the bispecific antibody each comprise two antibody variable domains, preferably a scFv, i.e. VH-VL or VL-VH. In this embodiment, the binding agent preferably comprises (i) a heavy chain variable domain (VH) of a first antibody, (ii) a light chain variable domain (VL) of a first antibody, (iii) a heavy chain variable domain (VH) of a second antibody and (iv) a light chain variable domain (VL) of a second antibody.
In some embodiments, the bispecific molecules comprise two Fab regions, each being directed against different epitopes. In some embodiments, the molecule of the disclosure is an antigen binding fragment (Fab)2 complex. The Fab2 complex is composed of two Fab fragments, one Fab fragment comprising a Fv domain, i.e. VH and VL domains, specific for one epitope, and the other Fab fragment comprising a Fv domain specific for another epitope. Each of the Fab fragments may be composed of two single chains, a VL-CL module and a VH-CH module. Alternatively, each of the individual Fab fragments may be arranged in a single chain, preferably, VL-CL-CH-VH, and the individual variable and constant domains may be connected with a peptide linker.
In some embodiments, the binding agent according to the disclosure includes various types of bivalent and trivalent single-chain variable fragments (scFvs), fusion proteins mimicking the variable domains of two antibodies. Divalent (or bivalent) single-chain variable fragments (di- scFvs, bi-scFvs) can be engineered by linking two scFvs. This can be done by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. The disclosure also includes multispecific molecules comprising more than two scFvs binding domains.
Another possibility is the creation of scFvs with linker peptides that are too short for the two variable regions to fold together (about five amino acids), forcing scFvs to dimerize. This type is known as diabodies. Still shorter linkers (one or two amino acids) lead to the formation of trimers, so-called triabodies or tribodies. Tetrabodies have also been produced. They exhibit an even higher affinity to their targets than diabodies.
A particularly preferred example of a bispecific antibody fragment is a diabody (Kipriyanov, Int. J. Cancer 77 (1998), 763-772), which is a small bivalent and bispecific antibody fragment. Diabodies comprise a heavy chain variable domain (VH) and a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain. This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites.
In some embodiments, the bispecific or multispecific molecule according to the disclosure comprises variable (VH, VL) and constant domains (C) of immunoglobulins. In some embodiments the bispecific molecule is a minibody, preferably, a minibody comprising two single VH-VL-C chains that are connected with each other via the constant domains (C) of each chain. According to this aspect, the corresponding variable heavy chain regions (VH), corresponding variable light chain regions (VL) and constant domains (C) are arranged, from N-terminus to C-terminus, in the order VH(Epitope l)-VL(Epitope l)-(C) and VH(Epitope 2)- VL(Epitope 2)-C, wherein C is preferably a CH3 domain, Epitope 1 refers to a first epitope and Epitope 2 refers to a second epitope. Pairing of the constant domains results in formation of the minibody.
According to another aspect, the bispecific binding agent of the disclosure is in the format of a bispecific single chain antibody construct, whereby said construct comprises or consists of at least two binding domains. In some embodiments, each binding domain comprises one variable region from an antibody heavy chain ("VH region"), wherein the VH region of the first binding domain specifically binds to Epitope 1, and the VH region of the second binding domain specifically binds to Epitope 2. The two binding domains are optionally linked to one another by a short polypeptide spacer. Each binding domain may additionally comprise one variable region from an antibody light chain ("VL region"), the VH region and VL region within each of the first and second binding domains being linked to one another via a polypeptide linker long enough to allow the VH region and VL region of the first binding domain and the VH region and VL region of the second binding domain to pair with one another.
In some embodiments, the binding agent described herein comprises an antibody, e.g., a full- length antibody, comprising the first binding domain. In some embodiments, the binding agent described herein comprises an antibody fragment such as scFv or VHH comprising the second binding domain which is covalently linked to the antibody comprising the first binding domain. In some embodiments, the binding agent comprises the antibody fragment such as scFv or VHH covalently linked to the N-terminus or C-terminus of the light chain or heavy chain of the antibody.
In some embodiments, a binding moiety described herein, e.g., a binding moiety comprised in a docking compound binding to a primary target, comprises a DARPin. In some embodiments, the binding moiety directs a payload to target cells, such as cancer cells.
The term "DARPin" refers to designed ankyrin repeat proteins. DARPins are based on naturally occurring ankyrin repeat proteins, yet contain one or more amino acid mutations that can affect, for example, their binding affinity to a target molecule, their cell surface expression, and the like. DARPins preferably include 2 to 3 ankyrin repeat modules flanked by N- and C- capping repeats. Each ankyrin repeat module includes about 33 amino acid residues.
Ankyrin repeat proteins have been identified in 1987 through sequence comparisons between four such proteins in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans. Breeden and Nasmyth reported multiple copies of a repeat unit of approximately 33 residues in the sequences of swi6p, cddOp, notch and lin-12 (Breeden et al., Nature 329, 651- 654 (1987)). The subsequent discovery of 24 copies of this repeat unit in the ankyrin protein led to the naming of this repeat unit as the ankyrin repeat (Lux et al., Nature 344, 36-42 (1990)). Later, this repeat unit has been identified in several hundreds of proteins of different organisms and viruses (Bork, Proteins 17(4), 363-74 (1993)). These proteins are located in the nucleus, the cytoplasm or the extracellular space. This is consistent with the fact that the ankyrin repeat domain of these proteins is independent of disulfide bridges and thus independent of the oxidation state of the environment. The number of repeat units per protein varies from two to more than twenty. Tertiary structures of ankyrin repeat units share a characteristic fold (Sedgwick and Smerdon, Trends Biochem Sci. 24(8), 311-6 (1999)) composed of a (3-hairpin followed by two antiparallel a-helices and ending with a loop connecting the repeat unit with the next one. Domains built of ankyrin repeat units are formed by stacking the repeat units to an extended and curved structure. Proteins containing ankyrin repeat domains often contain additional domains. While the latter domains have variable functions, the function of the ankyrin repeat domain is most often the binding of other proteins. When analysing the repeat units of these proteins, the target interaction residues are mainly found in the P-hairpin and the exposed part of the first a-helix. These target interaction residues are hence forming a large contact surface on the ankyrin repeat domain. This contact surface is exposed on a framework built of stacked units of a-helix 1, a-helix 2 and the loop.
DARPins that bind to specific targets can be identified by screening combinatorial libraries of DARPins and selecting those with desired binding properties for the target. Such screening methods are described in, e.g., Muench et aL, Molecular Therapy, 16(4), 686-693, 2011. For example, ribosomal display or phage display methods can be used to select target-specific DARPins from diverse libraries.
The term "repeat protein" refers to a (polyjpeptide/protein comprising one or more repeat domains. In one embodiment, a repeat protein comprises up to four repeat domains. In one embodiment, a repeat protein comprises up to three repeat domains. In one embodiment, a repeat protein comprises up to two repeat domains. In the most preferred embodiment, a repeat protein comprises one repeat domain.
The individual domains of a repeat protein may be connected to each other directly or via (poly)peptide linkers. The term "(poly)peptide linker" refers to an amino acid sequence which is able to link two protein domains. Such linkers include, for example, glycine-serine-linkers of variable lengths and are known to the person skilled in the relevant art.
The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat units (modules). In one embodiment, said repeat units are structural units having the same or a similar folding structure, and preferably stack tightly to preferably create a superhelical structure having a joint hydrophobic core.
The term "structural unit" refers to a locally ordered part of a (poly)peptide, formed by three- dimensional interactions between two or more segments of secondary structure that are near one another along the (poly)peptide chain. Such a structural unit comprises a structural motif. The term "structural motif" refers to a three-dimensional arrangement of secondary structure elements present in at least one structural unit. Structural motifs are well known to the person skilled in the relevant art. Said structural units may alone not be able to acquire a defined three-dimensional arrangement; however, their consecutive arrangement as repeat modules in a repeat domain leads to a mutual stabilization of neighbouring units which may result in a superhelical structure.
The term "repeat modules" refers to the repeated amino acid sequences of the repeat proteins, which are derived from the repeat units of naturally occurring proteins. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family of naturally occurring repeat proteins, e.g., ankyrin repeat proteins.
The term "set of repeat modules" refers to the total number of repeat modules present in a repeat domain. Such "set of repeat modules" present in a repeat domain comprises two or more consecutive repeat modules, and may comprise just one type of repeat module in two or more copies, or two or more different types of modules, each present in one or more copies. Such set of repeat modules comprising, for example, 3 repeat modules may comprise consecutively, form N- to C-terminus, repeat module 1, repeat module 2, and repeat module
3. Different repeat domains may have an identical number of repeat modules per repeat domain or may differ in the number of repeat modules per repeat domain.
Preferably, the repeat modules comprised in a set are homologous repeat modules. In the context of the present disclosure, the term "homologous repeat modules" refers to repeat modules, wherein more than 70% of the framework residues of said repeat modules are homologous. Preferably, more than 80% of the framework residues of said repeat modules are homologous. Most preferably, more than 90% of the framework residues of said repeat modules are homologous. Computer programs to determine the percentage of homology between polypeptides, such as Fasta, Blast or Gap, are known to the person skilled in the relevant art.
The term "repeat unit" refers to amino acid sequences comprising sequence motifs of one or more naturally occurring proteins, wherein said "repeat units" are found in multiple copies, and which exhibit a defined folding topology common to all said motifs determining the fold of the protein. Such repeat units comprise framework residues and interaction residues.
One example of such repeat units is an ankyrin repeat unit. Naturally occurring proteins containing two or more such repeat units are referred to as "naturally occurring repeat proteins". The amino acid sequences of the individual repeat units of a repeat protein may have a significant number of mutations, substitutions, additions and/or deletions when compared to each other, while still substantially retaining the general pattern, or motif, of the repeat units.
The term "repeat sequence motif" or "repeat consensus sequence" refers to an amino acid sequence, which is deduced from one or more repeat units. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. Said framework residue positions correspond to the positions of framework residues of said repeat units. Said target interaction residue positions correspond to the positions of target interaction residues of said repeat units. Such repeat sequence motifs comprise fixed positions and randomized positions. The term "fixed position" refers to an amino acid position in a repeat sequence motif, wherein said position is set to a particular amino acid. Frequently, such fixed positions correspond to the positions of framework residues.
The term "randomized position" refers to an amino acid position in a repeat sequence motif, wherein two or more amino acids are allowed at said amino acid position. Frequently, such randomized positions correspond to the positions of target target interaction residues. However, some positions of framework residues may also be randomized.
The term "folding topology" refers to the tertiary structure of said repeat units. The folding topology will be determined by stretches of amino acids forming at least parts of a-helices or P-sheets, or amino acid stretches forming linear polypeptides or loops, or any combination of a-helices, P-sheets and/or linear polypeptides/loops.
The term "consecutive" refers to an arrangement, wherein said modules are arranged in tandem.
In repeat proteins, there are at least 2, frequently 6 or more, 10 or more, or 20 or more repeat units, usually about 2 to 6 repeat units. For the most part, the repeat proteins are structural proteins and/or adhesive proteins, being present in prokaryotes and eukaryotes, including vertebrates and non-vertebrates.
In most cases, said repeat units will exhibit a high degree of sequence identity (same amino acid residues at corresponding positions) or sequence similarity (amino acid residues being different, but having similar physicochemical properties), and some of the amino acid residues might be key residues being strongly conserved in the different repeat units found in naturally occurring proteins.
However, a high degree of sequence variability by amino acid insertions and/or deletions, and/or substitutions between the different repeat units found in naturally occurring proteins will be possible as long as the common folding topology is maintained.
The term "framework residues" relates to amino acid residues of the repeat units, or the corresponding amino acid residues of the repeat modules, which contribute to the folding topology, i.e. which contribute to the fold of said repeat unit (or module) or which contribute to the interaction with a neighboring unit (or module). Such contribution might be the interaction with other residues in the repeat unit (module), or the influence on the polypeptide backbone conformation as found in a-helices or P-sheets, or amino acid stretches forming linear polypeptides or loops.
The term "target interaction residues" refers to amino acid residues of the repeat units, or the corresponding amino acid residues of the repeat modules, which contribute to the interaction with target substances. Such contribution might be the direct interaction with the target substances, or the influence on other directly interacting residues, e.g. by stabilising the conformation of the (poly)peptide of said repeat unit (module) to allow or enhance the interaction of said directly interacting residues with said target.
A "target" may be an individual molecule such as a nucleic acid molecule, a (poly)peptide protein, a carbohydrate, or any other naturally occurring molecule, including any part of such individual molecule, or complexes of two or more of such molecules. The target may be, in particular, a molecule on immune effector cells, in particular CD8.
In one embodiment, the repeat modules are directly connected. In the context of the present invention, the term "directly connected" refers to repeat modules, which are arranged as direct repeats in a repeat protein without an intervening amino acid sequence.
In another embodiment, the repeat modules are connected by a (poly)peptide linker. Thus, the repeat modules may be linked indirectly via a (poly)peptide linker as intervening sequence separating the individual modules. An "intervening sequence" may be any amino acid sequence, which allows to connect the individual modules without interfering with the folding topology or the stacking of the modules. Preferentially, said intervening sequences are short (poly)peptide linkers of less than 10, and even more preferably, of less than 5 amino acid residues.
In one embodiment, a repeat protein further comprises an N- and/or a C-terminal capping module having an amino acid sequence different from any one of said repeat modules. The term "capping module" refers to a polypeptide fused to the N- or C- terminal repeat module of a repeat domain, wherein said capping module forms tight tertiary interactions with said repeat module thereby providing a cap that shields the hydrophobic core of said repeat module at the side not in contact with the consecutive repeat module from the solvent.
Said N- and/or C-terminal capping module may be, or may be derived from, a capping unit or other domain found in a naturally occurring repeat protein adjacent to a repeat unit.
The term "capping unit" refers to a naturally occurring folded (poly)peptide, wherein said (poly)peptide defines a particular structural unit which is N- or C-terminally fused to a repeat unit, wherein said (poly)peptide forms tight tertiary interactions with said repeat unit thereby providing a cap that shields the hydrophobic core of said repeat unit at one side from the solvent. Such capping units may have sequence similarities to said repeat sequence motif. Nucleic acids
The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In some embodiments, a nucleic acid is DNA. in some embodiments, a nucleic acid is RNA. In some embodiments, a nucleic acid is a mixture of DNA and RNA. A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis. The term "nucleoside" (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.
The five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine. The five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively. However, thymidine is more commonly written as "dT" ("d" represents "deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.
A modified purine (A or G) or pyrimidine (C, T, or U) base moiety is, in some embodiments, modified by one or more alkyl groups, e.g., one or more C1-4 alkyl groups, e.g., one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N7-CI-4 alkyl-guanine, N6-CI-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uraci I, and N ( 1)- Ci-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5- methyl-uracil, and N(l)-methyl-uracil.
DNA
Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of nonnucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA.
Nucleic acids may be comprised in a vector. The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.
RNA
The term "RNA" relates to a nucleic acid molecule which includes ribonucleotide residues. In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a |3- D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered/modified nucleotides (/.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (/.e., naturally occurring) nucleotide residues or analogs thereof).
"RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), selfamplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA. The term "in vitro transcription" or "IVT" as used herein means that the transcription (i.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).
According to the present disclosure, the term '"RNA" includes "mRNA". According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide or polypeptide. mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.
According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.
In some embodiments, the mRNA which preferably encodes a peptide or polypeptide has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.
As established in the art, mRNA generally contains a 5' untranslated region (5'-UTR), a peptide/polypeptide coding region and a 3' untranslated region (3'-UTR). In some embodiments, the mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, the mRNA is produced by in vitro transcription using a DNA template. The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™). For providing modified mRNA, correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and/or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and/or added to the mRNA after transcription.
In some embodiments, RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.
In some embodiments of the present disclosure, the RNA is "replicon RNA" or simply a "replicon", in particular "self-replicating RNA" or "self-amplifying RNA". In certain embodiments, the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).
Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication (trans-amplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans- replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.
In some embodiments of the present disclosure, the RNA (in particular, mRNA) described herein contains one or more modifications, e.g., in order to increase its stability and/or increase translation efficiency and/or decrease immunogenicity and/or decrease cytotoxicity. For example, in order to increase expression of the RNA (in particular, mRNA), it may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO 2007/036366 and PCT/EP2019/056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and/or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA).
In some embodiments, the RNA (in particular, mRNA) described herein comprises a 5'-cap structure. In some embodiments, the RNA does not have uncapped 5'-triphosphates. In some embodiments, the RNA (in particular, mRNA) may comprise a conventional 5'-cap and/or a 5'- cap analog. The term "conventional 5'-cap" refers to a cap structure found on the 5'-end of an RNA molecule and generally comprises a guanosine 5'-triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the RNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the RNA). The guanosine may be methylated at position N7 (resulting in the cap structure m7Gppp). The term "5'-cap analog" includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2'- or 3'-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphorothioate modified 5'-cap analogs at the ^-phosphate (such as m27,2 OG(5l)ppSp(5')G (referred to as beta-S-ARCA or 0-S-ARCA)), as described in PCT/EP2019/056502. Providing an RNA (in particular, mRNA) with a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5'-cap compound, wherein said 5'-cap structure is co-transcriptionally incorporated into the generated RNA (in particular, mRNA) strand, or the RNA (in particular, mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
In some embodiments, the RNA (in particular, mRNA) comprises a 5'-cap structure selected from the group consisting of m27'2 OG(5')ppSp(5')G (in particular its DI diastereomer), m2 7-3'0G(5')ppp(5,)G, and m27'3 OGppp(mi2' °)ApG.
In some embodiments, the RNA (in particular, mRNA) comprises a capO, capl, or cap2, preferably capl or cap2. According to the present disclosure, the term "capO" means the structure "m7GpppN", wherein N is any nucleoside bearing an OH moiety at position 2'. According to the present disclosure, the term "capl" means the structure "m7GpppNm", wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'. According to the present disclosure, the term "cap2" means the structure "m7GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH3 moiety at position 2'.
The 5'-cap analog beta-S-ARCA (fJ-S-ARCA) has the following structure: The "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)" is the diastereomer of beta-S- ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time. The HPLC preferably is an analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml/min can be applied. In some embodiments, a gradient of methanol in ammonium acetate, for example, a 0-25% linear gradient of methanol in 0.05 M ammonium acetate, pH = 5.9, within 15 min is used. UV-detection (VWD) can be performed at 260 nm and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.
The 5'-cap analog m27'3' °Gppp(mi2L°)ApG (also referred to as m27'30G(5,)ppp(5,)m2L°ApG) which is a building block of a capl has the following structure:
An exemplary capO mRNA comprising p-S-ARCA and mRNA has the following structure:
An exemplary capO mRNA comprising m27'3'0G(5')ppp(5')G and mRNA has the following structure:
An exemplary capl mRNA comprising m27,3 OGppp(mi2' °)ApG and mRNA has the following structure:
As used herein, the term "poly-A tail" or "poly-A sequence" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA (in particular, mRNA) molecule. Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs (in particular, mRNAs) described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical. RNAs (in particular, mRNAs) disclosed herein can have a poly-A tail attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.
It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly-A tail (Holtkamp et ai., 2006, Blood, vol. 108, pp. 4009-4017).
The poly-A tail may be of any length. In some embodiments, a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of" means that most nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.
In some embodiments, a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.
In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. Consequently, in some embodiments, the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. In some embodiments, the poly(A) tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence of 10 nucleotides.
In some embodiments, no nucleotides other than A nucleotides flank a poly-A tail at its 3'-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.
In some embodiments, a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.
In some embodiments, RNA (in particular, mRNA) described in present disclosure comprises a 5'-UTR and/or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). A 5'-UTR, if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. A 3'-UTR, if present, is located at the 3'-end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does generally not include the poly-A sequence. Thus, the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly-A sequence. Incorporation of a 3'-UTR into the 3'- non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency. A synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced. In certain embodiments, the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2-globin, alphal-globin, or beta-globin, e.g., beta-globin, e.g., human beta-globin. For example, the RNA (e.g., mRNA) may be modified by the replacement of the existing 3'-UTR with or the insertion of one or more, e.g., two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alphal- globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.
In some embodiments, a 5'-UTR is or comprises a modified human alpha-globin 5'-UTR. In some embodiments, a 3'-UTR comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.
The RNA (in particular, mRNA) described herein may have modified ribonucleotides in order to increase its stability and/or decrease immunogenicity and/or decrease cytotoxicity. For example, in some embodiments, uridine in the RNA (in particular, mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.
In some embodiments, the modified uridine replacing uridine is selected from the group consisting of pseudouridine (i|>), Nl-methyl-pseudouridine (mlip), 5-methyl-uridine (m5U), and combinations thereof.
In some embodiments, the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5- methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl- uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio- uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5- methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncmSU), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1- propynyl-pseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5-methyl-2- thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls4ip), 4-thio-l-methyl- pseudouridine, 3-methyl-pseudouridine (m3ip), 2-thio-l-methyl-pseudouridine, 1-methyl-l- deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropyljpseudouridine (acp3 ip), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-0-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (cpm), 2-thio-2'-O-methyl- uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O- methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)~ 2'-0-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.
An RNA (preferably mRNA) which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine) is referred to herein as "ip-modified", whereas the term "mlUJ-modified" means that the RNA (preferably mRNA) contains N(l)- methylpseudouridine (replacing partially or completely, preferably completely, uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5- methyluridine (replacing partially or completely, preferably completely, uridine). Such or ml4<- or m5U-modified RNAs usually exhibit decreased immunogenicity compared to their unmodified forms and, thus, are preferred in applications where the induction of an immune response is to be avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(l)-methylpseudouridine replacing completely uridine.
The codons of the RNA (in particular, mRNA) described in the present disclosure may further be optimized, e.g., to increase the GC content of the RNA and/or to replace codons which are rare in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject). In some embodiments, the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence. This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence. In some embodiments, the codonoptimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.
The term "codon-optimized" refers to the alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions may be codon-optimized for optimal expression in a subject to be treated using the RNA (in particular, mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (in particular, mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".
In some embodiments, the guanosine/cytosine (G/C) content of the coding region of the RNA (in particular, mRNA) described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides.
In various embodiments, the G/C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA. A combination of the above described modifications, i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (MJ) or N(l)-methylpseudouridine (mlMJ) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency. Thus, in some embodiments, the RNA (in particular, mRNA) described in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (iii) alteration of the 5'- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5- methylcytidine for cytidine and/or pseudouridine (MJ) or N(l)-methylpseudouridine (mlUJ) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.
Particles
A nucleic acid such as RNA may be administered with one or more delivery vehicles that protect the nucleic acid from degradation, maximize delivery to on-target cells and minimize exposure to off-target cells. Such delivery vehicles may complex or encapsulate the nucleic acid and include a range of materials, including polymers, lipids and mixtures thereof. In some embodiments, such delivery vehicles may form particles with the nucleic acid.
In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolameliar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.
The term "nanoparticle" relates to a nano-sized particle comprising at least one particle forming agent, e.g., at least one cationic or cationically ionizable lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.
In some embodiments, the particles described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, such as in the range of 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.
Particles described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.
A "nucleic acid particle" can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the nucleic acid. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable compound combines together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.
In some embodiments, nucleic acid may be noncovalently associated with a particle. In some embodiments, the nucleic acid may be adhered to the outer surface of the particle (surface nucleic acid) and/or may be contained in the particle (encapsulated nucleic acid). The N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles.
The term "particle forming components" or "particle forming agents" relates to any components which form particles, e.g., by associating with nucleic acid. Nucleic acid delivery vehicles such as particle forming agents useful herein include polymers, polymer derivatives, lipids, and mixtures thereof. Such components include any component which can be part of nucleic acid particles, e.g., cationic or cationically ionizable lipids.
A "polymer," as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties.
If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can be a copolymer. The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
In certain embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
In certain embodiments, polymer may be protamine or polyalkyleneimine. The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.
According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.
In some embodiments, the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75-102 to 107 Da, preferably 1000 to 105 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.
Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).
Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In some embodiments, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
Particles described herein may also comprise polymers other than cationic polymers, i.e., noncationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.
In some embodiments of the present disclosure, the nucleic acid such as RNA described herein may be present in lipoplex particles.
Lipoplexes (LPX) are electrostatic complexes which are generally formed by mixing preformed cationic lipid liposomes with anionic nucleic acid. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid lipoplexes.
Generally, lipoplex particles are obtainable by adding nucleic acid to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic or cationically ionizable lipids and additional lipids, is injected into an aqueous solution under stirring.
In some embodiments, liposomes are self-closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase. A prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.
In certain embodiments, the nucleic acid lipoplex particles include both a cationic lipid and an additional lipid, in an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.
In some embodiments, nucleic acid described herein is present in the form of lipid nanoparticles (LNPs).
In general, lipid nanoparticles are obtainable from direct mixing of nucleic acid, e.g., RNA, in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol. In that case, lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.
LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid such as PEG-lipid. In some embodiments, the LNP comprises from 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid.
In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent.
In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to 45 mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent. In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer-conjugated lipid. In some embodiments, the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer-conjugated lipid; and the nucleic acid, encapsulated within or associated with the lipid nanoparticle.
In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.
In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.
In some embodiments, the steroid is cholesterol.
In some embodiments, the polymer conjugated lipid is a pegylated lipid.
In some embodiments, the cationically ionizable lipid component of the LNPs has one of the following structures:
Pharmaceutical compositions
The agents described herein, e.g., tag conjugates, docking compounds, complexes comprising tag conjugate and docking compound, and nucleic acid encoding docking compound, may be administered in pharmaceutical compositions and may be administered in the form of any suitable pharmaceutical composition. Accordingly, described herein are pharmaceutical compositions comprising the agents described herein, e.g., tag conjugates, docking compounds, complexes comprising tag conjugate and docking compound, and nucleic acid encoding docking compound.
In some embodiments, the agents described herein may be administered in a pharmaceutical composition which may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more stabilizers etc. In some embodiments, the pharmaceutical composition is for therapeutic or prophylactic treatments, e.g., for use in treating or preventing a disease. The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation.
The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".
The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.
The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses and/or agents. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.
Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal. The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.
The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water.
The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline.
Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).
Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.
In some embodiments, pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration.
Treatments
The agents, compositions and methods described herein can be used to treat a subject with a disease, e.g., a disease characterized by the presence of diseased cells expressing an antigen. The agents, compositions and methods described herein may be used in the therapeutic or prophylactic treatment of various diseases. Particularly preferred diseases are cancer diseases. In some embodiments, the agents, compositions and methods described herein are useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen.
For example, if the antigen is derived from a virus, the agents, compositions and methods may be useful in the treatment of a viral disease caused by said virus. If the antigen is a tumor antigen, the agents, compositions and methods may be useful in the treatment of a cancer disease wherein cancer cells express said tumor antigen.
The term "disease" refers to an abnormal condition that affects the body of an individual. A disease is often construed as a medical condition associated with specific symptoms and signs. A disease may be caused by factors originally from an external source, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death to the individual afflicted, or similar problems for those in contact with the individual. In this broader sense, it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes these may be considered distinguishable categories. Diseases usually affect individuals not only physically, but also emotionally, as contracting and living with many diseases can alter one's perspective on life, and one's personality.
In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.
The term "therapeutic treatment" relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.
The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.
The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g. mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer) but may or may not have the disease or disorder. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".
The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.
In some embodiments of the disclosure, the aim is to deliver a pharmaceutically active agent to diseased cells expressing an antigen such as cancer cells expressing a tumor antigen, and to treat a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen.
In some embodiments of the disclosure, the aim is to deliver a payload to immune cells to modulate, e.g., induce or enhance development, priming, expansion, differentiation and/or survival of, immune cells. In some embodiments, immune cells may target diseased cells expressing an antigen such as cancer cells expressing a tumor antigen for treating a disease such as a cancer disease involving cells expressing an antigen such as a tumor antigen. In some embodiments, immune cells exert one or more immune effector functions on diseased cells, e.g., kill diseased cells by means of a cellular immune response.
The term "disease involving an antigen", "disease involving cells expressing an antigen" or similar terms refer to any disease which implicates an antigen, e.g. a disease which is characterized by the presence of an antigen. The disease involving an antigen can be an infectious disease, or a cancer disease or simply cancer. As mentioned above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. In some embodiments, a disease involving an antigen is a disease involving cells expressing an antigen, preferably on the cell surface.
The term "infectious disease" refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent (e.g. common cold). Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, hepatitis, sexually transmitted diseases (e.g. chlamydia or gonorrhea), tuberculosis, HIV/acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), the bird flu, and influenza. The terms "cancer disease" or "cancer" refer to or describe the physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the sexual and reproductive organs, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal cancer, spinal axis tumors, glioma, meningioma, and pituitary adenoma. The term "cancer" according to the disclosure also comprises cancer metastases.
The term "solid tumor" or "solid cancer" as used herein refers to the manifestation of a cancerous mass, as is well known in the art for example in Harrison's Principles of Internal Medicine, 14th edition. Preferably, the term refers to a cancer or carcinoma of body tissues other than blood, preferably other than blood, bone marrow, and lymphoid system. For example, but not by way of limitation, solid tumors include cancers of the prostate, lung cancer, colorectal tissue, bladder, oropharyngeal/laryngeal tissue, kidney, breast, endometrium, ovary, cervix, stomach, pancrease, brain, and central nervous system.
The methods and agents described herein are, in particular, useful for the treatment of cancers, e.g., solid cancers, characterized by diseased cells expressing an antigen a docking compound is directed to.
Citation of documents and studies referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.
The following description is presented to enable a person of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein wiil be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be accorded the scope consistent with the claims.
Abbreviations
Some abbreviations that may appear in this application are defined hereinafter:
The present invention pertains, in particular, to the following:
1. A kit comprising:
(i) a compound comprising a binding moiety binding to a target antigen and a binding moiety for a tag, or a nucleic acid encoding said compound; and
(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist.
2. The kit of item 1, wherein the STING agonist has the following Formula (XX): wherein: Ring A is selected from the group consisting of
G and Gi are independently N, CH, or C-X1-R2; or when G and Gi are each C-X1-R2, the R2 groups are optionally linked to form L2;
G' and G2 are independently N or CH;
X is N-R, O, or S;
X' is N or CH;
Xi is CH2, 0 or S;
R is hydrogen or C1-4 alkyl;
L1 and L2 are each independently C2-4 alkylene or C2-4 alkenylene;
R2 is selected from the group consisting of hydrogen, C2-4 cyclic ether, C1-4 alkylene-(C2-4 cyclic
Ri and R3 are independently selected from the group consisting of ,
Ring B is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 2 heteroatoms independently selected from N, O, and S;
Rs is -OH or -NR9R10;
R9 and Rioare independently selected from hydrogen and C1-6 alkyl; X2 and X3 are independently NH or S;
Yi and Y2 are independently a 5-membered heteroaryl or heterocyclic ring, wherein the 5- membered heteroaryl or heterocyclic ring (i) has 1 to 4 heteroatoms independently selected from N, O, and S, (ii) is attached to the remainder of the STING agonist via a C ring atom of the
5-membered heteroaryl or heterocyclic ring, and (iii) is optionally substituted with 1 to 4 R21, wherein each R21 is independently C1-4 alkyl (such as methyl, ethyl, propyl, or butyl);
Rs, Re, and R7 are independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, , or Rs and Re are optionally connected to form a 5- or 6- membered heterocyclic ring;
Ris is -OH or -NR9R10;
Ring C is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 ring heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 4 ring heteroatoms independently selected from N, O, and S; n, p, q, q', t, and v are independently an integer from 2 to 6; and k, I, m, o, u, and w are independently an integer from 1 to 6, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
3. The kit of item 2, wherein the STING agonist has the following formula (XXH): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH). 3a. The kit of item 2, wherein the STING agonist has the following formula (XXA): wherein G is CH, C-SCH3, C-OCH3, or N, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXA).
3b. The kit of item 2, wherein the STING agonist has the following formula (XXB): wherein G is CH, C-SCH3, C-OCH3, or N; Rg is -OH or -NH2; Yi and Yz are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXB).
The kit of item 2, wherein the STING agonist has the following formula (XXC): wherein Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R?i, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXC).
3d. The kit of item 2, wherein the STING agonist has the following formula (XXD): wherein Rar is hydrogen or C1-4 alkyl, preferably the STING agonist is a STING agonist moiety of the STING agonist of Formula (XXD).
3e. The kit of item 2, wherein the STING agonist has the following formula (XXE): wherein X is S or O, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXE).
3f. The kit of item 2, wherein the STING agonist has the following formula (XXF): wherein G is CH, C-SCHs, C-OCH3, or N, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXF).
The kit of item 2, wherein the STING agonist has the following formula (XXG): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXG).
3h. The kit of item 2, wherein the STING agonist has the following formula (XXJ): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXJ). 3i. The kit of item 2, wherein the STING agonist has the following formula (XXK): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of
Formula (XXK).
4. The kit of any one of items 1 to 3, 3a, 3b, and 3g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-18'): wherein Rz is a bond, -(CH2)3-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably Rz is
-(CH2)3-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, more preferably Rz is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point of Rz to the remainder of the compound under (ii); and # represents the attachment point of Rz to the remainder of the STING agonist moiety.
4a. The kit of any one of items 1 to 3, 3a, 3b, and 3g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-51'): wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
4b. The kit of any one of items 1 to 3, 3a, 3b, and 3g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-73'a): wherein R21 is a bond, -(CFhh-, or #-(CH2)3(piperazin-l,4-diyl)-*, preferably R2' is #- (CH2)3(piperazin-l,4-diyl)-* wherein * represents the attachment point of R2' to the remainder of the compound under (ii); and # represents the attachment point of R2' to the remainder of the STING agonist moiety.
4c. The kit of any one of items 1 to 3, 3a, 3b, and 3g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-73'b): wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Ri- is #-C(O)NHNH-*), wherein * represents the attachment point of Rr to the remainder of the compound under
(ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
4d. The kit of any one of items 1, 2, 3a, 3b, 3h, and 3i, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-6'): wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound under (ii); and # represents the attachment point of Rr to the remainder of the STING agonist moiety.
4e. The kit of any one of items 1, 2, 3a, 3b, 3h, and 3i, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-531):
wherein Ra1 is a bond, -(CHah-, MCHahMCHs)-*, or #-(CH2)3N(CH3)NH-*, preferably R2' is #- (CH2)SN(CH3)NH-*, wherein * represents the attachment point of R2' to the remainder of the compound under (ii); and # represents the attachment point of R2' to the remainder of the
STING agonist moiety.
5. The kit of any one of items 1 to 4e, wherein the compound under (ii) comprises one or more than one tag to which the binding moiety for a tag binds.
6. The kit of any one of items 1 to 5, wherein the tag is a peptide tag.
7. The kit of any one of items 1 to 6, wherein the compound under (ii) comprises a moiety comprising a polymer.
8. The kit of any one of items 1 to 7, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer.
9. The kit of item 7 or 8, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.
10. The kit of any one of items 7 to 9, wherein the polymer is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof. 11. The kit of any one of items 7 to 8, wherein the polymer comprises polyethylene glycol) (PEG), or poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
12. The kit of any one of items 7 to 9, wherein the polymer comprises at least one poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
13. The kit of any one of items 1 to 12, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
14. The kit of any one of items 1 to 13, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
15. The kit of any one of items 1 to 14, wherein the payload moiety and the tag or tags are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
16. The kit of any one of items 1 to 15, wherein the nucleic acid is RNA.
17. The kit of any one of items 1 to 16, wherein the compound under (ii) comprises one tag.
18. The kit of any one of items 1 to 17, wherein the total number of tags in the compound under (ii) is one.
19. The kit of any one of items 1 to 18, wherein the compound under (ii) comprises one payload moiety. 20. The kit of any one items 1 to 19, wherein the total number of payload moieties in the compound under (ii) is one.
21. The kit of any one of items 1 to 20, wherein the compound under (ii) comprises a linking moiety connecting a tag and a payload moiety.
22. The kit of item 21, wherein the linking moiety comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
23. The kit of any one of items 1 to 19, wherein the compound under (ii) comprises the formula:
P-L-T wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-511), (XX-531), (XX-73'a), or (XX-73'b);
T comprises a tag; and
L comprises a linking moiety.
24. The kit of item 23, wherein L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
25. The kit of item 23 or 24, wherein L comprises the formula [AEEA]u-[L'-[AEEA]v]w, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
L' comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.
26. The kit of any one of items 23 to 25, wherein L or L' comprises an amino acid.
27. The kit of any one of items 23 to 26, wherein Lor L' comprises the D-isomerof an amino acid.
28. The kit of any one of items 23 to 27, wherein L or L' comprises cysteine or lysine.
29. The kit of any one of items 23 to 28, wherein L or L' is connected to a side chain.
30. The kit of any one of items 1 to 16, wherein the compound under (ii) comprises at least two of said tags.
31. The kit of any one of items 1 to 16 and 30, wherein the total number of tags in the compound under (ii) is two.
32. The kit of any one of items 1 to 16, 30 and 31, wherein the compound under (ii) comprises one or more payload moieties.
33. The kit of any one of items 1 to 16, and 30 to 32, wherein the total number of payload moieties in the compound under (ii) is one.
34. The kit of any one of items 1 to 16, and 30 to 32, wherein the compound under (ii) comprises two or more payload moieties.
35. The kit of any one of items 1 to 16, 30 to 32 and 34, wherein the total number of payload moieties in the compound under (ii) is two.
36. The kit of any one of items 1 to 16, and 30 to 35, wherein the compound under (ii) comprises payload moieties and tags in an unbranched (linear) configuration. 37. The kit of any one of items 1 to 16, and 30 to 36, wherein the compound under (ii) comprises the formula:
P-LA-T-LB-T or
P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-51'), (XX-531), (XX-73'a), or (XX-73’b);
T comprises a tag;
LA comprises a linking moiety;
LB comprises a linking moiety; and
Lc comprises a linking moiety.
38. The kit of item 37, wherein one or more of LA, LB, and Lc comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
39. The kit of item 37 or 38, wherein LB comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
40. The kit of any one of items 1 to 16, and 30 to 35, wherein the compound under (ii) comprises payload moieties and tags in a branched (non-linear) configuration.
41. The kit of any one of items 1 to 16, 30 to 35 and 40, wherein the compound under (ii) comprises the formula:
[[P]m-Li]n-Bi-[L2-T]o wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-51'), (XX-53*), (XX-73'a), or (XX-73'b);
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety;
1.2 comprises a linking moiety; m is an integer from 1 to 4; n is an integer from 1 to 4; and o is an integer from 2 to 4; wherein the different groups [L2-T] may be identical or different, the different groups P may be identical or different, and the different groups [[P] m-Li] may be identical or different.
42. The kit of item 41, wherein Bi comprises an amino acid or bis-amino acid.
43. The kit of item 41 or 42, wherein Bi comprises the D-isomer of an amino acid.
44. The kit of any one of items 41 to 43, wherein Bi comprises cysteine or lysine.
45. The kit of any one of items 41 to 44, wherein Li comprises a 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
46. The kit of any one of items 41 to 45, wherein L2 comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
47. The kit of any one of items 10 to 46, wherein the number of repeating units of 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30. 48. The kit of any one of items 10 to 47, wherein the number of repeating units of 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
49. The kit of any one of items 10 to 48, wherein the number of repeating units of 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
50. The kit of any one of items 41 to 49, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3.
51. The kit of any one of items 41 to 50, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2.
52. The kit of any one of items 41 to 51, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2, n is
1 and m is 1.
53. The kit of any one of items 41 to 51, wherein in formula [ [P] m-Li]n-Bi-[ Lz-T]0 o is 2, n is
2 and m is 2.
54. The kit of any one of items 1 to 53, wherein the compound under (i) comprises one or more binding moieties for the tag.
55. The kit of any one of items 1 to 54, wherein the compound under (i) comprises at least two binding moieties for the tag.
56. The kit of any one of items 1 to 55, wherein the compound under (i) comprises two binding moieties for the tag.
57. The kit of any one of items 1 to 56, wherein the tag is an ALFA-tag.
58. The kit of any one of items 1 to 57, wherein the tag is a cyclic ALFA-tag. 58a. The kit of item 1, wherein the compound under (ii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX- M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19), EX-024 to EX-035, EX-032 to EX-035, and EX-047 to EX-054.
58b. The kit of item 1, wherein the compound under (ii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD1832O (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19), or the compound under (ii) is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19).
58c. The kit of item 1, wherein the compound under (ii) comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8), or the compound under (ii) is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8).
58d. The kit of item 1, wherein the compound under (ii) comprises a structure of a compound shown herein as SD18321 (PIC7), or the compound under (ii) is SD18321 (PIC7).
59. A compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist.
60. The compound of item 59, wherein the STING agonist has the following Formula (XX):
wherein:
Ring A is selected from the group consisting of
G and Gi are independently N, CH, or C-XI-R2; or when G and Gi are each C-X1-R2, the R2 groups are optionally linked to form L2;
G' and G2 are independently N or CH;
X is N-R, O, or S;
X' is N or CH;
Xi is CH2, O or S;
R is hydrogen or C1-4 alkyl;
L1 and L2 are each independently C2-4 alkylene or C2-4 alkenylene;
R2 is selected from the group consisting of hydrogen, C2-4 cyclic ether, C1-4 alkylene-(C2-4 cyclic o o
Ri and R3 are independently selected from the group consisting of
Ring B is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 2 heteroatoms independently selected from N, O, and S;
Rs is -OH or -NR9R10;
R9 and Rio are independently selected from hydrogen and C1-6 alkyl;
X2 and X3 are independently NH or S;
Yi and Y2 are independently a 5-membered heteroaryl or heterocyclic ring, wherein the 5- membered heteroaryl or heterocyclic ring (i) has 1 to 4 heteroatoms independently selected from N, O, and S, (ii) is attached to the remainder of the STING agonist via a C ring atom of the
5-membered heteroaryl or heterocyclic ring, and (iii) is optionally substituted with 1 to 4 R21, wherein each R21 is independently C1-4 alkyl (such as methyl, ethyl, propyl, or butyl);
Rs, Re, and R7 are independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl,
O , or Rs and Re are optionally connected to form a 5- or 6- membered heterocyclic ring;
Ris is -OH or -NR9R10;
Ring C is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 ring heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 4 ring heteroatoms independently selected from N, O, and
S; n, p, q, q1, t, and v are independently an integer from 2 to 6 (i.e., 2, 3, 4, 5, or 6, such as 2, 3,
4, or 5, e.g., 2, 3, or 4); and k, I, m, o, u, and w are independently an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6, such as 1,
2, 3, 4, or 5, e.g., 1, 2, 3, or 4), preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
61. The compound of item 60, wherein the STING agonist has the following formula (XXH): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH).
61a. The compound of item 60, wherein the STING agonist has the following formula (XXA): wherein G is CH, C-SCH3, C-OCH3, or N, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXA).
61b. The compound of item 60, wherein the STING agonist has the following formula (XXB): wherein G is CH, C-SCH3, C-OCH3, or N; Rs is -OH or -NH2; Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXB).
61c. The compound of item 60, wherein the STING agonist has the following formula (XXC): wherein Yi and Y2 are independently oxazolyl, pyrazolyl, thiazolyl, or imidazolyl, each of which is optionally substituted with 1 or 2 R21, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXC).
61d. The compound of item 60, wherein the STING agonist has the following formula (XXD) : wherein Fbr is hydrogen or CM alkyl, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXD).
61e. The compound of item 60, wherein the STING agonist has the following formula (XXE): wherein X is S or O, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXE).
61f. The compound of item 60, wherein the STING agonist has the following formula (XXF): wherein G is CH, C-SCH3, C-OCH3, or N, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXF).
61g. The compound of item 60, wherein the STING agonist has the following formula (XXG):
preferably the payload moiety comprises a STING agonist moiety of the STING agonist of
Formula (XXG).
61h. The compound of item 60, wherein the STING agonist has the following formula (XXJ): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of
Formula (XXJ).
61i. The compound of item 60, wherein the STING agonist has the following formula (XXK) : preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXK).
62. The compound of any one of items 59 to 61, 61a, 61b, and 61g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-181): wherein R2' is a bond, -(CHzh-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, preferably R2' is -(CH2)3-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, more preferably R2' is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point of R2' to the remainder of the compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist; and # represents the attachment point of R2' to the remainder of the STING agonist moiety.
62a. The compound of any one of items 59 to 61, 61a, 61b, and 61g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-511): wherein Ri' is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Ri1 is #-C(O)NHNH-*, wherein * represents the attachment point of Rr to the remainder of the compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist; and
# represents the attachment point of Rr to the remainder of the STING agonist moiety. 62b. The compound of any one of items 59 to 61, 61a, 61b, and 61g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-73'a): wherein R2' is a bond, -(CHzh-, or #-(CH2)3(piperazin-l,4-diyl)-*, preferably R2’ is #- (CH2)3(piperazin-l,4-diyl)-* wherein * represents the attachment point of R2' to the remainder of the compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist; and # represents the attachment point of R2' to the remainder of the STING agonist moiety.
62c. The compound of any one of items 59 to 61, 61a, 61b, and 61g, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-73'b): wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Ri' to the remainder of the compound comprising a payload moiety and a tag, wherein the payload moiety comprises a STING agonist; and # represents the attachment point of Ri' to the remainder of the STING agonist moiety.
62d. The compound of any one of items 59, 60, 61a, 61b, 61h, and 61i, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-6'): wherein Rr is a bond, -C(O)-, #-C(O)NH-*, or #-C(O)NHNH-*, preferably Rr is #-C(O)NHNH-*, wherein * represents the attachment point of Ri- to the remainder of the compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist; and # represents the attachment point of Ri' to the remainder of the STING agonist moiety.
62e. The compound of any one of items 59, 60, 61a, 61b, 61h, and 61i, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-531): wherein R2' is a bond, -(CFhh-, MCFhhNfCHs)-*, or #-(CH2)3N(CHa)NH-*, preferably R21 is #- (CH2)3N(CH3)NH-*, wherein * represents the attachment point of R21 to the remainder of the compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist; and # represents the attachment point of R2' to the remainder of the STING agonist moiety.
63. The compound of any one of items 59 to 62e, which comprises one or more than one tag.
64. The compound of any one of items 59 to 63, wherein the tag is a peptide tag. 65. The compound of any one of items 59 to 64, which comprises a moiety comprising a polymer.
66. The compound of any one of items 59 to 65, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer.
67. The compound of item 65 or 66, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.
68. The compound of any one of items 65 to 67, wherein the polymer is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
69. The compound of any one of items 65 to 68, wherein the polymer comprises polyethylene glycol) (PEG), or poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
70. The compound of any one of items 65 to 69, wherein the polymer comprises at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
71. The compound of any one of items 59 to 70, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
72. The compound of any one of items 59 to 71, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof. 73. The compound of any one of items 59 to 72, wherein the payload moiety and the tag or tags are coupled through a moiety comprising at least one poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
74. The compound of any one of items 59 to 73, which comprises one tag.
75. The compound of any one of items 59 to 74, wherein the total number of tags in the compound is one.
76. The compound of any one of items 659to 75, which comprises one payload moiety.
77. The compound of any one of items 59 to 76, wherein the total number of payload moieties in the compound is one.
78. The compound of any one of items 59 to 77, which comprises a linking moiety connecting a tag and a payload moiety.
79. The compound of item 78, wherein the linking moiety comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
80. The compound of any one of items 62 to 79, which comprises the formula:
P-L-T wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-511), (XX-53'), (XX-73'a), or (XX-73'b);
T comprises a tag; and
L comprises a linking moiety. 81. The compound of item 80, wherein L comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
82. The compound of item 80 or 81, wherein L comprises the formula [AEEA]U-[L'- [AEEA]v]w, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
L' comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.
83. The compound of any one of items 80 to 82, wherein L or L' comprises an amino acid.
84. The compound of any one of items 80 to 83, wherein L or L' comprises the D-isomer of an amino acid.
85. The compound of any one of items 80 to 84, wherein L or L' comprises cysteine or lysine.
86. The compound of any one of items 80 to 85, wherein L or L' is connected to a side chain.
87. The compound of any one of items 59 to 73, which comprises at least two of said tags.
88. The compound of any one of items 59 to 73 and 87, wherein the total number of tags in the compound is two.
89. The compound of any one of items 62 to 73, 87 and 88, which comprises one or more payload moieties. 90. The compound of any one of items 59 to 73 and 87 to 89, wherein the total number of payload moieties in the compound is one.
91. The compound of any one of items 59 to 73 and 87 to 89, which comprises two or more payload moieties.
92. The compound of any one of items 59 to 73, 87 to 89 and 91, wherein the total number of payload moieties in the compound is two.
93. The compound of any one of items 59 to 73, and 87 to 92, which comprises payload moieties and tags in an unbranched (linear) configuration.
94. The compound of any one of items 59 to 73, and 87 to 93, which comprises the formula:
P-LA-T-LB-T or
P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-61), (XX-511), (XX-531), (XX-73'a), or (XX-73'b);
T comprises a tag;
LA comprises a linking moiety;
LB comprises a linking moiety; and
Lc comprises a linking moiety.
95. The compound of item 94, wherein one or more of LA, LB, and Lc comprises a poly-2-(2- (2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof. 96. The compound of item 94 or 95, wherein LB comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
97. The compound of any one of items 59 to 73, and 87 to 92, which comprises payload moieties and tags in a branched (non-linear) configuration.
98. The compound of any one of items 59 to 73, 87 to 92 and 97, which comprises the formula:
[[P]m-Ll]n-Bi-(L2-T]0 wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-61), (XX-51'), (XX-531), (XX-73'a), or (XX-73'b);
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety;
L2 comprises a linking moiety; m is an integer from 1 to 4; n is an integer from 1 to 4; and o is an integer from 2 to 4; wherein the different groups [L2-T] may be identical or different, the different groups P may be identical or different, and the different groups [[P] m-Li] may be identical or different.
99. The compound of item 98, wherein Bi comprises an amino acid or bis-amino acid.
100. The compound of item 98 or 99, wherein Bi comprises the D-isomer of an amino acid.
101. The compound of any one of items 98 to 100, wherein Bi comprises cysteine or lysine. 102. The compound of any one of items 98 to 101, wherein U comprises a 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
103. The compound of any one of items 98 to 102, wherein L2 comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
103. The compound of any one of items 68 to 103, wherein the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
105. The compound of any one of items 68 to 104, wherein the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
106. The compound of any one of items 68 to 105, wherein the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
107. The compound of any one of items 98 to 106, wherein in formula [[P]m-Li] n-Bi-[L2-T]0 m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3.
108. The compound of any one of items 98 to 107, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2.
109. The compound of any one of items 98 to 108, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2, n is 1 and m is 1.
110. The compound of any one of items 98 to 108, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2, n is 2 and m is 2.
111. The compound of any one of items 59 to 110, wherein the tag is an ALFA-tag. 112. The compound of any one of items 59 to 111, wherein the tag is a cyclic ALFA-tag.
112a. The compound of item 59, which comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19), EX-024 to EX-035, EX-032 to EX-035, and EX-047 to EX-054.
112b. The compound of item 59, which comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19), or which is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19).
112c. The compound of item 59, which comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8), or which is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8).
112d. The compound of item 59, which comprises a structure of a compound shown herein as SD18321 (PIC7), or which is SD18321 (PIC7).
113. A method for treating a subject having a disease, disorder or condition characterized by cells expressing a target antigen, comprising:
(i) providing to the subject a compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag; (ii) allowing the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag to become associated with cells expressing the target antigen; and
(iii) administering to the subject a compound of any one of items 59 to 112 comprising one or more tags to which the binding moiety for a tag binds.
114. The method of item 113, wherein the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is provided to the subject by administering to the subject RNA encoding a polypeptide comprising a binding moiety binding to the target antigen and a binding moiety for a tag; and allowing expression of the polypeptide by cells in the subject.
115. The method of item 114, wherein the cells expressing the polypeptide are transfected with the RNA.
116. The method of item 114 or 115, wherein the RNA is administered as particulate formulation such as formulated as lipid nanoparticles or lipoplex particles.
117. The method of any one of items 114 to 116, wherein the cells expressing the polypeptide secrete the polypeptide.
118. The method of any one of items 114 to 117, wherein the cells expressing the polypeptide express the polypeptide such that it is released into the bloodstream.
119. The method of any one of items 113 to 118, wherein the target antigen is a cell surface antigen.
120. The method of any one of items 113 to 119, wherein the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is a fusion polypeptide comprising the binding moiety binding to the target antigen and the binding moiety for a tag. 121. The method of any one of 113 to 120, wherein the binding moiety binding to the target antigen comprises an antibody or an antibody derivative.
122. The method of any one of items 113 to 121, wherein the binding moiety for a tag comprises an antibody or an antibody derivative.
123. The method of item 121 or 122, wherein the antibody derivative is an antibody fragment.
124. The method of any one of items 113 to 123, wherein the disease, disorder or condition is cancer.
125. The method of any one of items 113 to 124, wherein the cells expressing a target antigen are diseased cells.
126. The method of any one of items 113 to 125, wherein the cells expressing a target antigen are cancer cells.
127. The method of any one of 113 to 126, wherein the target antigen is a tumor antigen.
128. The method of any one of items 113 to 127, wherein the compound under (i) comprises a single binding moiety for the tag.
129. The method of any one of items 113 to 127, wherein the compound under (i) comprises at least two binding moieties for the tag.
130. The method of any one of items 113 to 129, wherein the compound under (i) comprises two binding moieties for the tag.
131. The method of any one of items 113 to 130, wherein the tag is an ALFA-tag.
132. The method of any one of items 113 to 131, wherein the tag is a cyclic ALFA-tag. 132a. The method of item 113, wherein the compound under (iii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC1O), SD18321 (PIC7), SD18322 (PICH), EX-M60 to EX-M121 (in particular, EX- M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15)), EX-B122 to EX-B132 (in particular, EX-B122 (PIC18)), EX-B133 (PIC19), EX-024 to EX-035, EX-032 to EX-035, and EX-047 to EX-054.
132b. The method of item 113, wherein the compound under (iii) comprises a structure of a compound shown herein as any one of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 (PIC10), SD18321 (PIC7), SD18322 (PICll), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19), or the compound under (iii) is selected from the group consisting of SD18317 (PIC8), SD17248, PIC12, PIC13, PIC14, SD17247 (PIC6), SD17553, SD17453 (PIC2), SD17516 (PIC1), SD17537 (PIC3), SD18319 (PIC9), SD18320 ( PIC10), SD18321 (PIC7), SD18322 (PICH), EX-M69 (PIC16), EX-M71 (PIC17), EX-M74 (PIC15), EX-B122 (PIC18), and EX-B133 (PIC19).
132c. The method of item 113, wherein the compound under (iii) comprises a structure of a compound shown herein as any one of SD18321 (PIC7) and SD18317 (PIC8), or the compound under (iii) is selected from the group consisting of SD18321 (PIC7) and SD18317 (PIC8).
132d. The method of item 113, wherein the compound under (iii) comprises a structure of a compound shown herein as SD18321 (PIC7) or the compound under (iii) is SD18321 (PIC7).
Examples
Example 1 - Examples of Bis-ALFA Conjugates and Synthesis
The following describes exemplary bis-ALFA conjugates (tag conjugates) and methods for preparing these conjugates.
Materials and Methods
All anhydrous solvents were provided by commercial suppliers, e.g., Sigma- Aldrich®, in appropriate containers, e.g., Sure/Seal™ bottles, and used without further purification.
Unless otherwise specified, all starting materials were obtained from commercial suppliers and used without further purification. Unless otherwise specified, all temperatures are expressed in °C and all reactions were conducted at RT. Unless otherwise specified, compounds were purified by either flash column chromatography (FCC), preparative HPLC or preparative chiral HPLC. Unless otherwise specified, silica (50 mm average particle size) is the stationary phase used for flash column chromatography purification.
Analytical methods:
UHPLC-MS Instrument and Methods
Instrument: Waters H-Class UPLC with QSM, sample organizer, column heater, PDA UV detector and QDa mass spectrometer.
Instrument: Thermo Vanquish UHPLC with column heater, UV detector, and LTQ. (linear ion trap) mass spectrometer
Column; Waters BEH Cis Column, 100x 2.1 mm, 1.7 pm, 130 A pore size
Lcms ong method: 40°C column temperature. UV absorption wavelength: 214 nm. MS range: 200-1250 Da. Mobile phase A: 0.100% TFA in water. Mobile phase B: 0.085% TFA in acetonitrile. Flowrate: 0.5 mL/min. Gradient:
Column: Waters BEH Cis Column, 50 x 2.1 mm, 1.7 pm, 130 A pore size
Lcms_short method: 40°C column temperature. UV absorption wavelength: 214 nm. MS range: 200-1250 Da. Mobile phase A: 0.1% TFA in water. Mobile phase B: 0.085% TFA in acetonitrile. Flowrate: 0.5 mL/min. Gradient:
Column: ACQUITY UPLC Protein BEH C4 Column, 300 A, 1.7pm, 2.1 mm X 100 mm
Lcms_long_C4 method: 40°C column temperature. UV absorption wavelength: 214 nm. MS range: 200-1250 Da. Mobile phase A: 0.1% TFA in water. Mobile phase B: 0.085% TFA in acetonitrile. Flowrate: 0.5 mL/min. Gradient: Lcms_long_C4_60C method: Same as Lcms_long_C4 method but with 60°C column temperature.
HPLC instrument and methods
Instrument: Waters 2767 Autopure with SQD2 mass spectrometer and UV detector. Mass spectrometer range: 200-3000 Da. UV Detector wavelength: 214 nm.
Column: Waters XBridge Protein BEH C4 OBD Prep Column, 100 x 19 mm, 5 pm particle size, 300 A pore size.
Method: 15 mL/min flowrate. Mobile phase A: 0.05% TFA in water. Mobile phase B: 0.05% TFA in acetonitrile. Sample loading at 5% B, elution performed using linear gradient.
Column: Phenomenex Luna 18 Column, 250x 30 mm, 10 pm particle size, 100 A pore size.
Method: 30 mL/min flowrate. Mobile phase A: 0.05% TFA in water. Mobile phase B: 0.05% TFA in acetonitrile. Sample loading at 5% B, elution performed using linear gradient.
SEC Instrument and Methods
Preparative size-exclusion chromatography was performed on a JAI Preparative Recycling HPLC (LaboACE LC-7080) system equipped with 2.5HR Plus and 2HR Plus columns connected in series (20x600 mm) using 10% MeOH/CHCh as eluent.
General Schematic A: Synthesis of cyclic ALFA intermediates
Step 1: Synthesis of cyclic ALFA peptide intermediate, lnt-A-001:
The cyclic ALFA peptide intermediate is synthesized using Fmoc solid phase peptide synthesis chemistry. Rink amide AM resin is loaded onto an automated peptide synthesizers suspended in 10 mL of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The peptide synthesis is initiated by the deprotection of the N-terminal a Fmoc protecting group using 4 mL of 20% piperidine in DMF heated by microwave for 3 min at 90 °C. After draining, the resin is washed three times with 5mL DMF at five seconds per wash. Then the coupling reaction is performed by the addition of 2.5 mL of the next 0.3 M amino acid (7.5 equiv) in DMF, 1 mL of a 1 M DIC (10 equiv) solution in DMF, and 0.5 mL 1 M Oxyma (5 equiv) with 0.1M diisopropylethylamine (DIEA) solution in DMF to the reaction vessel (RV). The coupling reaction is heated to 90 °C for 2 min. The reaction solution is drained, and the coupling reaction is repeated. After the second coupling, the resin is drained and washed four times with 4 mL of DMF. Then 4 mL of 20% piperidine in DMF is added to the RV to deprotect the Fmoc group and the next amino acid is coupled. This cycle of Fmoc removal and coupling is repeated for every amino acid sequentially. Fmoc-Glu(O-2-PhiPr)-OH and Fmoc-Lys(Mmt)- OH amino acids are incorporated into the peptide intermediate sequence to allow for selective cyclization.
Step 2: Selective deprotection of Lys(Mmt) and Glu(O-2-PhiPr), lnt-A-002
To the resin containing lnt-A-001 in a fritted syringe is added 5 mL of partial deprotection cocktail (2% TFA, 2% TIS in DCM). The reaction is agitated for 5 min, then the deprotection cocktail is removed from the resin via filtration. Another 5 mL of partial deprotection cocktail is added, agitiated for 5 minutes, and removed. This process is repeated six times until the yellow/orange color of the flowthrough disappears (30 minutes total of Mmt and 2-PhiPr removal). The resin is rinsed thoroughly with DCM and DMF to afford lnt-A-002.
Step 3: Lactam cyclization via side chain amide bond formation, lnt-A-003
Cyclization is carried out in the same fritted syringe as the partial deprotection from Step 2. To the syringe is added 5 equiv. of PyAOP and 5 equiv. of HOAt as a solution in DMF. The resin is allowed to swell and then 10 equiv. of DIEA is added. The reaction is agitated for 2-3 hours. The reaction mixture is removed from the resin via filtration and the remaining resin is rinsed thoroughly with DCM and DMF to afford lnt-A-003.
Step 4: Bromoacetamide coupling, lnt-A-004 To the resin containing lnt-A-003 in a fritted syringe was added 7 mL of 20% piperidine in DMF. The reaction is agitated for 10 mins, then rinsed thoroughly with DCM and DMF. Then DIC (3 equiv), Oxyma (3 equiv), and bromoacetic acid (3 equiv) in 7 mL of DMF are added to the resin. After 30 min, the resin is thoroughly washed with DCM and DMF to afford lnt-A-004.
Step 5: Cleavage and global deprotection, lnt-A-005
To the resin containing lnt-A-004 in a fritted syringe is added 10 mL of cleavage cocktail (87.5% TFA, 5% thioanisole, 2.5% water, 2.5% TIS, 2.5% EDT). The reaction is agitated for 2h at room temp followed by 1 h at 40 °C. The resulting peptide intermediate is then precipitated out of solution by adding the reaction solution to ~25 mL cold diethyl ether which is then centrifuged. The supernatant is removed and the resulting pellet is redissolved in 50:50 MeCN:water. The resulting crude peptide intermediated is purified by RP HPLC to afford lnt-A-005.
General Schematic B: Synthesis of bisALFA-DOTA conjugates with general structure 1A
Step 1: Synthesis of DOTA-AEEA-dCys-AEEA-AEEA-cyclic ALFA intermediate, lnt-B-001:
The DOTA-containing cyclic ALFA peptide intermediate is synthesized using an analogous protocol to the synthesis of lnt-A-005 by using Fmoc-AEEA-OH, Fmoc-D-Cys(Trt)-OH, and 1,4,7,10-Tetraazacyclododecane -1,4,7-tris-tert-butyl acetate-10-acetic acid ( DOT A-Tris( tertbutyl ester)). For this intermediate DOTA-Tris(tert-butyl ester) is coupled to the N terminus instead of bromo acetic acid.
Step 2: Synthesis of Bromo-acetamide-AEEA-AEEA-cyclic ALFA intermediate, lnt-B-002:
The Bromoacetamide-containing cyclic ALFA intermediate is synthesized using analogous protocol to the synthesis of lnt-A-005 by using Fmoc-AEEA-OH
Step 3: Thiol conjugation, EX-001:
To a solution of the DOTA-containing intermediate lnt-B-001 (1 equiv) and the cyclic ALFA- containing intermediate lnt-B-002 (2.1 equiv) in 1:1:1 water:DMF:MeCN is added 10 equiv of DIEA. The reaction mixture is stirred at room temperature for 10 min. Then the reaction is purified by RP HPLC and the fractions are lyophilized to afford EX-001 as a white powder.
EX-002:
The title compound was synthesized in an analogous manner to EX-001.
EX-003:
The title compound was synthesized in an analogous manner to EX-001.
EX-024:
The title compound was synthesized in an analogous manner to EX-001 by using Fmoc-
Lys(biotin)-OH and capping the N-terminus with acetic anhydride.
EX-025:
The title compound was synthesized in an analogous manner to EX-001 by using Fmoc-
Lys(biotin)-OH and capping the N-terminus with acetic anhydride.
EX-026:
The title compound was synthesized in an analogous manner to EX-001 by using Fmoc-
Lys(biotin)-OH and capping the N-terminus with acetic anhydride. General Schematic C: Synthesis of DOTA-containing intermediates
Step 1, Synthesis of dCys(Trt)-AEEA-AEEA-Lys(ivDde)-AEEA-AEEA-dCys(Trt) intermediate, lnt-C-001:
The cysteine-containing intermediate is synthesized using Fmoc solid phase peptide synthesis chemistry. Rink amide AM resin is loaded onto an automated peptide synthesizers suspended in 10 mL of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The peptide synthesis is initiated by the deprotection of the N-terminal a Fmoc protecting group using 4 ml of 20% piperidine in DMF heated by microwave for 3 min at 50 °C. After draining, the resin is washed three times with 5mL DMF at five seconds per wash. Then the coupling reaction is performed by the addition of 2.5 mL of the next 0.3 M amino acid (7.5 equiv) in DMF, 1 mL of a 1 M DIC (10 equiv) solution in DMF, and 0.5 mL 1 M Oxyma (5 equiv) with 0.1M diisopropylethylamine (DIEA) solution in DMF to the reaction vessel (RV). The coupling reaction is heated to 60 °C for 10 min. The reaction solution is drained, and the coupling reaction is repeated. After the second coupling, the resin is drained and washed four times with 4 mL of DMF. Then 4 mL of 20% piperidine in DMF is added to the RV to deprotect the Fmoc group and the next amino acid is coupled. This cycle of Fmoc removal and coupling is repeated for every amino acid sequentially.
Step 2, N-terminal acetylation, lnt-C-002:
The cysteine-containing resin lnt-C-001 is transferred to a 20 mL fritted syringe after solid phase peptide synthesis is completed. To the resin is added 7 mL of capping solution (81.25% DMF, 12.5% 2 M DIEA in NMP, 6.25% acetic anhydride) and the reaction is agitated for 15 min. The capping solution is removed, and another 7 mL of capping solution is added and the reaction is agitated for another 15 min. Then the resin is washed thoroughly with DCM and DMF to afford lnt-C-002.
Step 3, Selective deprotection, lnt-C-003:
To the resin containing lnt-C-002 is added 5 mL of 4% anhydrous hydrazine in DMF. The reaction is agitated for 5 mins and then the hydrazine solution is removed via filtration. The process is repeated two more times. Afterwards, the resin is washed thoroughly with DCM and DMF to afford lnt-C-003.
Step 4, Coupling of AEEA and DOTA, lnt-C-004:
The resin containing lnt-C-004 is transferred to an automated peptide synthesized for coupling of AEEA and DOTA. The coupling of AEEA and DOTA is performed in an analogous manner to Step 1 for the synthesis of lnt-C-001 by using Fmoc-AEEA-OH and 1,4,7,10- Tetraazacyclododecane-l,4,7-tris-tert-butyl acetate-10-acetic acid (DOTA-Tris(tert-butyl ester)).
Step 5, Cleavage and global deprotection, lnt-C-005:
To the resin containing lnt-C-004 is added 10 mL of cleavage cocktail (87.5% TFA, 5% thioanisole, 2.5% water, 2.5% TIS, 2.5% EDT). The reaction is agitated for 2 h at room temperature followed by 1 h at 40 °C. The resulting crude material is then precipitated out of solution by addingthe reaction solution to ~25 mL cold diethyl ether which is then centrifuged. The supernatant is removed and the resulting pellet is redissolved in 50:50 MeCN:water. The crude material is purified by RP HPLC and the fractions are lyophilized to afford lnt-C-005.
General Schematic D: Synthesis of bisALFA-DOTA conjugates with general structure 2A and 3A
Synthesis of EX-008:
To a solution of the DOTA-containing intermediate lnt-C-005 (1 equiv) and the cyclic ALFA- containing intermediate lnt-A-005 (2.1 equiv) in 1:1:1 water:DMF:MeCN is added 10 equiv of DIEA. The reaction mixture is stirred at room temperature for 10 min. Then the reaction is purified by RP HPLC and the fractions are lyophilized to afford EX-008 as a white powder.
EX-004:
The title compound is synthesized in an analogous manner to EX-008.
EX-005:
The title compound is synthesized in an analogous manner to EX-008 by using Fmoc-AEPA-OH in the synthesis.
EX-006:
The title compound is synthesized in an analogous manner to EX-008.
EX-007:
The title compound is synthesized in an analogous manner to EX-008,
EX-027:
The title compound is synthesized in an analogous manner to EX-008 by using Fmoc- Lys(biotin)-OH and capping the N-terminus with acetic anhydride.
EX-028:
The title compound is synthesized in an analogous manner to EX-008 by using Fmoc-AEPA-OH, Fmoc-Lys(biotin)-OH and capping the N-terminus with acetic anhydride.
EX-029:
The title compound is synthesized in an analogous manner to EX-008 by using Fmoc- Lys(biotin)-OH and capping the N-terminus with acetic anhydride.
EX-030:
The title compound is synthesized in an analogous manner to EX-008 by using Fmoc- Lys(bioti n)-OH and capping the N-terminus with acetic anhydride.
EX-031:
The title compound is synthesized in an analogous manner to EX-008 by using Fmoc- Lys(biotin)-OH and capping the N-terminus with acetic anhydride.
EX-037: The title compound is synthesized in an analogous manner to EX-008.
EX-038:
The title compound is synthesized in an analogous manner to EX-008.
General Schematic E: Synthesis of bisALFA-Toxin conjugates with general structure IB, 2B,
3B Step 1, Synthesis of azide-containing cyclic ALFA peptide intermediate, lnt-E-001:
The azide-containing cyclic ALFA peptide intermediate is synthesized using an analogous protocol to the synthesis of lnt-A-005 by using Fmoc-AEEA-OH, Fmoc-azidolysine-OH, Fmoc- Lys(Fmoc)-OH, and Fmoc-Cys(Trt)-OH. For this intermediate the N terminus was capped with acetic anhydride.
Step 2, Synthesis of alkyne-containing cyclic ALFA peptide intermediate, lnt-E-002:
The alkyne-containing cyclic ALFA peptide intermediate is synthesized using an analogous protocol to the synthesis of lnt-A-005 by using Fmoc-AEEA-OH, Fmoc-propargly-Gly-OH, Fmoc- Lys(Fmoc)-OH, and Fmoc-Cys(Trt)-OH. For this intermediate the N terminus was capped with acetic anhydride.
Step 3, Copper click reaction, lnt-E-003:
The azide-containing peptide intermediate lnt-E-001 (6 mg, 2.1 pmol, 1.0 equiv) and the alkyne-containing peptide intermediate lnt-E-002 (6.1 mg, 2.3 pmol, 1.1 equiv) are dissolved in 200 pL of 1:1 water:MeCN.. The resulting solution is bubbled with nitrogen gas for 2 min. In a separate vial, THPTA (10.2 g, 23.5 pmol) and sodium ascorbate (22.8 mg, 115 pmol) are dissolved in 200 pL of water and bubbled with nitrogen gas for 2 min. To the resulting clear solution is added an aqueous solution of copper sulfate pentahydrate (6 mg, 24 pmol). The resulting copper(l) mixture is added to the solution of the two peptide intermediates under nitrogen. After 1-2 h, the reaction is quenched with 50:50 watecMeCN containing 0.05% TFA and purified using RP HPLC to afford lnt-E-003.
Step 4, Thiol conjugation reaction, EX-012:
Conjugation of the toxin payload-linker to the cysteine-containing peptide intermediate was performed using standard cysteine conjugation protocols. If needed, the cysteine-containing peptide intermediate can be treated with TCEP. To a solution of the cysteine-containing peptide intermediate lnt-E-003 (1 equiv) in PBS buffer containing EDTA (pH 7.6, 20 mM EDTA) is added a solution of the toxin payload-linker (alternative toxin payload-linker 1, 4.8 equiv) in DMF. The reaction is stirred under nitrogen at room temperature or 40 °C for 1-2 h. After completion, the reaction is quenched with 50:50 water:MeCN containing 0.05% TFA and purified using RP HPLC to afford EX-012.
EX-009: The title compound is synthesized in an analogous manner to EX-012 by using commercially- available deruxtecan (CAS 1599440-13-7).
EX-010:
The title compound is synthesized in an analogous manner to EX-012.
EX-011:
The title compound is synthesized in an analogous manner to EX-012 by using commercially- available deruxtecan (CAS 1599440-13-7).
EX-013:
The title compound is synthesized in an analogous mannerto EX-012 by using alternative toxin payload-linker 2.
EX-014:
The title compound is synthesized in an analogous mannerto EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-016:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-017:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH.
EX-018:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-019:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-022:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-023:
The title compound is synthesized in an analogous manner to EX-012.
EX-032: The title compound is synthesized in an analogous manner to EX-012 by using an immunomodulator payload-linker.
EX-033:
The title compound is synthesized in an analogous manner to EX-012 by using an immunomodulator payload-linker.
EX-034:
The title compound is synthesized in an analogous manner to EX-012 by using an immunomodulator payload-linker.
EX-036:
The title compound is synthesized in an analogous manner to EX-012.
General Schematic F: Synthesis of bisALFA-Toxin conjugates with general structure 4B Step 1, Synthesis of pAEEA backbone module, lnt-F-001: lnt-F-001 is synthesized at 0.2-mmol scale on Sieber amide resin using standard Fmoc solid phase synthesis protocols. Couplings are performed as 10-minute double couplings with 60 °C coupling and 50 °C Fmoc deprotection. Bromoacetic acid coupling is performed without addition of Oxyma, no final deprotection. The resin is transferred into a fritted syringe and treated with cleavage cocktail (1% TFA/DCM) three times for 30 min each. Cleavage filtrates are combined, volatiles removed on rotavap, and the residue purified on RP HPLC or SEC to afford lnt-F-001 as a white solid.
Step 2, Synthesis of biscALFA peptide intermediate, lnt-F-002: lnt-F-001 (0.5 equiv) and cysteine-containing ALFA (1.1 equiv) are dissolved in a reaction vial in 1:1 MeCNrwater (to ca. 10 mg/mL). BisALFA formation is initiated by addition of DIEA (8 equiv). Upon completion, the reaction mixture is frozen and lyophilized to afford lnt-F-002 as an off-white lyophilization cake. The material is used in subsequent steps without further purification.
Step 3, Trityl deprotection of cysteines and thiol conjugation, EX-015:
To lnt-F-002 is added the deprotection cocktail (5 mg/mL in TFA). After 5 min, trityl is completely removed. The TFA is removed under vacuum or under a nitrogen stream. The resulting residue is dissolved in MeCN/PBS (1:3) and NaOH (0.1 N) is added to adjust the pH of the solution to 7.4. Deruxtecan (CAS 1599440-13-7) is added as solution in DMF (20 mg/mL). Upon completion, the reaction mixture is purified by RP HPLC and lyophilized to afford EX-015 as a yellow solid.
EX-020:
The title compound is synthesized in an analogous manner to EX-015.
EX-021:
The title compound is synthesized in an analogous manner to EX-015.
EX-035:
The title compound is synthesized in an analogous manner to EX-015 by using an immunomodulator payload-linker. General Schematic G: Synthesis of bisALFA-Toxin conjugates with general structure T5A homodimer Step 1, Synthesis of cyclic ALFA peptide intermediate, lnt-G-001:
The cyclic ALFA peptide intermediate is synthesized using an analogous protocol to the synthesis of lnt-A-005 by using solid phase peptide synthesis. Selective cleavage of the peptide from the resin and selective deprotection of the lysine side chain is used to afford lnt-G-001.
Step 2, Crosslinking of cyclic ALFA peptide intermediate, lnt-G-002:
The cyclic ALFA peptide intermediate is coupled together using an activated crosslinking agent such as but not limited to the Bis-N-hydroxysuccinimide ester of 3,6-Dioxaoctanedioic. The resulting material is globally deprotected under acidic conditions using an analogous protocol to Step 5 in the synthesis of lnt-A-005.
Step 3, Thiol conjugation reaction, EX-039:
Conjugation of the toxin payload-linker to the cysteine-containing peptide intermediate is performed using standard cysteine conjugation protocols. The title compound is synthesized in an analogous manner as described in Step 4 for the synthesis of EX-012.
EX-040:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-041:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-042:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH, Fmoc-cysteic acid, and alternative toxin payload-linker 2.
EX-043: The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-044:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-045:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH, Fmoc-cysteic acid, and alternative toxin payload-linker 2.
EX-046:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-047:
The title compound is synthesized in an analogous manner to EX-039 by using an immunomodulator payload-linker.
EX-048:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and immunomodulator payload-linker.
EX-049:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and immunomodulator payload-linker.
EX-050:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH, Fmoc-cysteic acid, and immunomodulator payload-linker. EX-051:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and immunomodulator payload-linker.
EX-052:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and immunomodulator payload-linker.
EX-053:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH, Fmoc-cysteic acid, and immunomodulator payload-linker.
EX-054:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-AEPA-OH and immunomodulator payload-linker.
EX-055:
The title compound is synthesized in an analogous mannerto EX-012 by using Fmoc-N-methyl- AEEA-OH Fmoc-AEPA-OH, and alternative toxin payload-linker 2.
EX-056:
The title compound is synthesized in an analogous manner to EX-012 by using Fmoc-N-methyl- AEEA-OH, Fmoc-AEPA-OH, and alternative toxin payload-linker 2.
EX-057:
The title compound is synthesized in an analogous mannerto EX-012 by using Fmoc-N-methyl- AEEA-OH, Fmoc-AEPA-OH and alternative toxin payload-linker 2.
EX-058: The title compound is synthesized in an analogous manner to EX-039 by coupling via the N terminus.
Example 2 - In Vitro Stability And Solubility Characterization Of Bis-Alfa Conjugates
Plasma Stability Assay
Plasma stability measurements were performed by spiking 5 pL of a 1 mM compound stock solution in DMSO into 495 pL of human or mouse Li-heparin plasma (Innovative Research, Cat. No. IPLALIH5OML (human) and IGMSBC (Balb C mouse). The final mixture was vortexed and incubated at 37°C. Enzymatic reactions were quenched directly after withdrawal by adding 100 pL of acetonitrile/formic acid = 99/1 (v/v) at 0, 1, 3, 6, and 24 hours. After centrifugation, the recovered supernatant was analyzed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). The LC-MS (Liquid Chromatography Mass Spectrometry) system used was a Shimadzu 8060 NX triple quadrupole in positive ion mode. Chromatography separation was achieved on a Waters Cortex T3 50 mm x 3 column x 1.7pm. The gradient program with a flow rate of 0.7 mL/min was started with 5% B followed by a linear gradient to 95% B in 1.4 min. After a linear isocratic hold for 0.2 min at 95% B, it was returned to the starting conditions of 5% B in 0.1 min. Finally, 5% B was kept for 0.6 min before the next injection. Total analysis time was 2.3 min. Plasma stability results for representative molecules are shown in Figure 1.
Solubility Assay
Solubility measurements were performed by spiking 5 pL of a 1 mM solution in dimethyl sulfoxide into a 495 pL media (phosphate or histidine buffer). The phosphate buffer contains 50 mM NazHPCU x 2H2O and 50 mM KH2PO4 (pH 7.4) and histidine buffer consists of 20 mM histidine, 120 mM sodium chloride, 50mM Sucrose and 0.02% polysorbate 20 (pH 6.0). The sample final concentration in each media is 100 pM. The mixtures were incubated at 23 °C under continuous shaking at 1400 rpm. After 24h, the samples were centrifuged, and the supernatant was analyzed by LC-MS (ThermoFisher Scientific Vanquish Liquid Chromatography coupled with ThermoFisher Scientific LTQ. XL) in positive ion mode. The chromatographic column used was an Acquity UPLC Protein BEH C4, 300A, 1.7pm, 2.1mm x 100mm. The gradient program with a flow rate of 0.5 mL/min was started with 0% B followed by a linear gradient to 95% B in 2 min. After a linear isocratic hold for 0.5 min at 95% B, it was returned to the starting conditions of 0% B in 0.1 min. Finally, 5% B was kept for 0.9 min before the next injection. Total analysis time is 3.5 min. The LC autosampler was kept at 232C through the analysis.
Example 3 - Affinity, Avidity and binding Assessment and Complex Formation testing
Methods
Measuring avidity- and affinity-driven interaction of BisALFA and NbALFA-comprising proteins
For affinity and avidity measurements an Octet HTX device from Sartorius was used. The instrument detects molecule:molecule interactions using biolayer interferometry (BLI).
For affinity measurements commercially available SAX2.0 biosensors were soaked for at least 10 min in kinetic buffer (KB) purchased from the instrument manufacturer. Following a 120 sec baseline in KB, a biotinylated derivative of the BisALFA compound with a concentration of 30.99 nM was loaded for 240 sec. Subsequently, a quenching step in a 100 pg/mL biocytin solution was performed, followed by a 120 sec baseline in KB. Association was measured over 900 sec using seven different concentrations of a monovalent ProteinDocker starting at 10 nM in a 1:1 serial dilution series down to 0.15625 nM. As a reference, KB without the NbALFA- containing protein was measured. The dissociation was acquired over 1500 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the second baseline. For interstep correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.
For avidity measurements, the 16-channel mode of the Octet HTX was utilized to allow for simultaneous double referencing. Commercially available SSA biosensors were quenched for 10 min in KB, followed by a 120 sec baseline measurement. Subsequently, eight of the 16 biosensors were loaded with 2.5 pg/mL biotinylated NbALFA for 60 sec while the remaining 8 biosensors were further incubated in KB. All biosensors were quenched for 120 sec in a 100 pg/mL biocytin solution. Association was measured over 900 sec using seven different concentrations per biosensor-column of a BisALFA derivative starting at 2.5 nM in a 1:1 serial dilution series down to 39.0625 pM. As a reference, KB was measured with the eighth biosensor for both biosensor columns. The dissociation was acquired over 1500 sec in KB. The signal of the reference biosensors was subtracted in pairs from the NbALFA-loaded biosensors, followed by subtraction of the reference well signal from all biosensors in the respective column. Afterthis double referencing step, the signal was aligned to the average of the second baseline. For inter-step correction, the data were aligned to the dissociation step and Savitzky- Golay filtering was applied. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.
Binding of BisALFA and bivalent ProteinDocker in solution
To investigate the formation of complexes, bivalent ProteinDocker was mixed in PBS with different concentrations of the BisALFA conjugate. The final concentration was adjusted to 5 pM for the protein, with either 0 pM, 0.5 pM, 5 pM or 50 pM of BisALFA conjugates. After mixing, samples were incubated at room temperature for at least 20 min to allow for the binding for the NbALFA moieties to the ALFA-tags on the BisALFA conjugate.
Nano Differential Scanning Fluorimetry and Dynamic light scattering
Thermal stabilities were investigated using the Prometheus PANTA from NanoTemper. 10 pL of preincubated BisALFA-ProteinDocker complexes or the ProteinDocker alone were loaded into capillaries. After loading the capillaries into the instrument, a heat ramp of rc/min from 25°C to 95°C was applied to all samples. During this process the intrinsic fluorescence of the proteins were measured at 350 nm and 330 nm. The ratio was plotted against the temperature and the first derivative was calculated. Minima and maxima correspond to different TM values that are observed in the process of denaturation of the multidomain protein. TM values of one protein with different concentrations of the BisALFA conjugate were compared. During the heat ramp, DLS measurements were performed in all samples, allowing to assess the turbidity onset in a temperature dependent manner. These values correlate with temperature-induced precipitation of the proteins.
All measurements were performed at least in duplicates.
Size exclusion chromatography to assess large complex formation
Complex formation analysis was performed utilizing an Agilent Infinity II HPLC and a Biozen 1.8 pm dSEC-2, 200 A LC column (300 x 4.6 mm). Flowrates were adjusted to 0.25 mL/min, resulting in approximately 255 bar pressure. As soluble phase 0.2 M potassium phosphate, 250 mM KCI, pH 6.2, 5% acetonitrile was used. Each run took 20 min excluding a 2-3 min wash step between each analysis. 10 pL of the preincubated BisALFA:ProteinDocker complexes or the ProteinDocker alone were applied and detected by absorption at 280 nm.
Binding to Fc receptors to bivalent ProteinDockers in the presence of BisALFA
Binding to CD64 was assessed utilizing the Octet HTX system. After 10 min soaking of Fab2G biosensors, a 60 sec baseline in KB was measured, followed by a 300 sec loading step of the bivalent ProteinDocker or a preincubated BisALFA:ProteinDocker complex at a concentration of 25 nM. After a threshold of 0.7 nm in response was reached, loading was stopped and quenching using a 100 pg/mL biocytin solution was performed over the course of 120 sec followed by a 120 sec baseline step in KB. Association was measured over 600 sec using seven different concentrations of CD64 starting at 5 nM in a 1:1 serial dilution series down to 78.125 pM. As a reference, KB without the CD64 but immobilized ProteinDocker or ProteinDocker- complex, respectively, was measured. The dissociation was acquired over 600 sec in KB. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the second baseline. For interstep correction, the data were aligned to the dissociation step and Savitzky-Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.
To determine binding to FcRn, SAX2.0 biosensors were soaked for 10 min in KB, followed by a 60 sec baseline in KB. Loading of biotinylated human FcRn was done over 300 sec or until a threshold of 1.5 nm in response was reached. After subsequent quenching in a 100 pg/mL biocytin solution, a baseline for 120 sec in 100 mM sodium phosphate, 150 mM NaCI, 0.05% Tween-20, pH6.0 buffer (KB-pH6) was measured. The bivalent ProteinDocker, with or without the prior pre-complexation with BisALFA, was diluted in KB-pH6 in a 1:1 serial dilution from 1600 nM down to 25 nM. Association was measured over 60 sec followed by dissociation in KB-pH6 over 60 sec. The signal of the reference well was subtracted from the signal of all other biosensors and the signals of these processed data were aligned to the average of the second baseline. For inter-step correction, the data were aligned to the dissociation step and Savitzky- Golay filtering was applied to all curves. Association and dissociations were globally fitted using a 1:1 Langmuir binding model. As FcRn interactions show a heterogeneous binding behavior to Fc-parts of antibodies, only the first 5 or 10 sec of the dissociation were fitted.
Results and Discussion
Assessment of affinity and avidity interactions of BisALFA and monovalent ProteinDocker
First, the influence of the bifurcation point and linker length of BisALFA conjugates on binding to the monovalent ProteinDocker was investigated. Therefore, 5 different pairs of BisALFA conjugates were tested, all comprising the same cyclic ALFA moiety (Ac-Ser-Arg-Leu-Glu- (cyclo5)Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-Glu-NH2).
Each pair comprised either a DOT A- or biotin-conjugated derivative of one BisALFA backbone. While affinity measurements gave strong responses due to the size of the antibody, avidity responses were lower but significant. For all tested compounds affinity KDs between 4.986E- 10 and 6.895E-10 M were determined with no significant difference in on- or off rates. In avidity measurements, no off-rate could be calculated for any of the test items as the off-rates were beyond the instrument limits (Table 2).
Table 2: Overview of BisALFA pairs, and their affinity and avidity kinetic binding data to monovalent NbALFA.
A direct comparison of dissociation curves for each pair allows to assess the influence of the structure of the peptides on the avidity effect (Figure 2). There was no major difference between the different pairs, indicating that neither the linker length nor the general structure has an influence on the dynamic range between affinity and avidity.
The difference between the dissociation curve in affinity and avidity measurements is a visualization of the avidity effect. Independent from linker or bifurcation point, this difference was similar between all tested compounds.
In the next step, the influence of the affinity of the ALFA peptide was tested. Therefore, BisALFA conjugates comprising a fixed bifurcation point and fixed linker length were synthesized with different cyclic ALFA moieties displaying different binding affinities to NbALFA. We used Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu-Thr-(cyclol3)Glu- NH2 as high affine ALFA, Ac-Ser-Arg-Leu-Glu-(cyclo5)Asp-Glu-Leu-Arg-(cyclo9)Lys-Arg-Leu- Thr-Glu-NH2 as medium affine ALFA and Ac-Pro-Ser-Arg-Leu-(cyclo5)Glu-Glu-Glu-Leu- (cyclo9)Lys-Arg-Arg-Leu-Thr-Glu-NH2 as low affine ALFA. Again, both DOTA- and biotinderivatives were generated and three pairs of BisALFA conjugates tested for affinity and avidity (Table 3). Biotin-coupled WT linear ALFA (Ac-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-Arg- Leu-Thr-Glu-NHz), which is known to have the highest affinity to NbALFA, was used as a reference in the affinity measurements.
Table 3: Overview of BisALFA pairs, and their affinity and avidity kinetic binding data to monovalent NbALFA.
For affinity measurements, a significant difference in off-rates was observed. Linear ALFA showed the highest off-rates, while the determined affinities of the high, medium and low cyclic ALFA conjugates were lower as expected. However, in avidity measurements, for all of high, medium and low affinity cyclic ALFA conjugates no off-rates could be determined as these surpassed the instrument limits and off-rates exceeded those of WT ALFA. A comparison of the dissociation curves illustrates the dynamic range between the affinity and avidity measurements (Figure 3). This so-called avidity effect can be leveraged in applications where a highly specific binding at a target site is desired. Due to the avidity effect, highly specific and tight binding of a conjugate with comparably low affinity is ensured at the target site with a high density of corresponding interacting binding moieties, while the conjugates will not bind effectively to individual interacting binding moieties at other sites or in circulation. Accordingly, for such applications interacting moieties on the tag conjugate and on the docking compound with affinities in the range of low to medium affine cyclic ALFA are preferred, since these are associated with a larger avidity effect, which is illustrated by the area between the affinity and the avidity curve in the graphs of Figure 3.
While lower affine BisALFA conjugates showed an increased off rate with lower affinities, the off-rate in the avidity measurements stayed unaltered. Therefore, lower affine peptides resulted in a stronger relative avidity effect and are thus more preferred for the intended use as radio diagnostics. Linear ALFA exhibited an off-rate in affinity measurements similar to the off-rates determined in avidity measurement of the its cyclic counterparts as to be seen in Figure 3. The off-rate of the high affine BisALFA conjugates in affinity measurements was also comparable to the off-rate of the linear ALFA, hence underlining that the affinity of linear ALFA is less suitable for a selective avidity effect of a BisALFA conjugate to target-bound monovalent ProteinDocker as compared to medium or low affinity BisALFA compounds.
Based on the observed affinities, it is eminent that off-rates between affinity and avidity should differ as much as possible to allow for a selective avidity effect on target-bound ProteinDocker. However, the calculated off-rates were determined to be 1.0E-07 1/s which is beyond the instrument limitations in the avidity mesurements. As an approximation we therefore also compared the off-rates of the differenc cyclic ALFA conjugates in affinity measurements to the determined affinity of linear ALFA, which can be used as a reference point. The off-rate fold change compared to WT ALFA was found to be of 3.61 for the high affinity cyclic ALFA, 8.11 for the medium affine cyclic ALFA and 20.45 for the low affine cyclic ALFA. Taken together, we concluded that the fold change in off-rate between affinity and avidity should be at least of a factor of >5, preferably >8, better >20, to facilitate a selective avidity effect on target-bound monovalent ProteinDocker.
Assessment of avidity interactions of BisALFA and bivalent ProteinDocker
To investigate the influence of the bifurcation point and the number and the structure of the toxins on BisALFA binding to the bivalent ProteinDocker, avidity measurements were performed. In bivalent proteins, BisALFA always binds with avidity and therefore affinity measurements are not possible to perform. While differences in on- and off-rates were minimal, the overall KD values were mainly identical for all tested compounds, underlining that the BisALFA:NbALFA binding was independent from the nature of bifurcation, and the structure of the toxin conjugated peptides as well as from the number of toxins per BisALFA (Table 4).
Table 4: BisALFA-toxin conjugates and their avidity driven kinetic parameters in binding to bivalent ProteinDocker.
In solution binding of BisALFA to bivalent ProteinDocker
While biolayer interferometric (BLI) binding analysis required that either the BisALFA or the ProteinDocker was immobilized, thermal shift assays using NanoDSF allow for a qualitative analysis of binding in solution. Therefore, a fixed amount of ProteinDocker was preincubated with either no BisALFA or 10-fold lower, equimolar or 10-fold higher molar concentrations of BisALFA. Subsequently, NanoDSF measurements are performed in order to assess the stability of the ProteinDocker in presence of the peptide. While the ProteinDocker alone shows a first TM value at 54.20°C, the TM value rises to above 67.99°C with at least equimolar concentration of BisALFA (Table 5).
Table 5: TM values of the bivalent ProteinDocker in combinations of different concentrations of the BisALFA conjugate EX-036. This increase of stability in the presence of the peptide can be explained by the stabilizing effect of the ALFA:NbALFA interaction on the VHH. As the initial TM value cannot be detected anymore at equimolar concentrations, a quantitative binding of the ProteinDocker and the BisALFA conjugate was confirmed (Figure 4). At 10-fold lower peptide concentrations, the unbound NbALFAs denatures as in the ProteinDocker with no BisALFA as binding was not quantitative.
As the ProteinDocker was a multidomain protein, multiple TM values were observed as different domains denature at different temperatures. The first TM value in the ProteinDocker sample without peptide reflected the stability of the NbALFA VHH. This minimum in the first derivative of the ratio of the fluorescence signals was also present at a 0.1:1 BisALFA:ProteinDocker ratio but completely absent in samples with higher BisALFA contents. In these, the denaturation of NbALFA in complex with the ALFA tag is overlaying with another maximum and therefore not observable.
Influence of BisALFA on the binding of the bivalent ProteinDocker to Fc receptors
The bivalent ProteinDocker comprised an IgGl-like architecture with two NbALFAs fused to the c-terminus of the Fc. The Fc part mediates effector functions by interacting with Fcy- receptors, among them FcyRI/CD64, and prolongs half-life of the antibody by interacting with the neonatal Fc receptor (FcRn). For the envisioned mode of action, effector functions as well as a long half-life are crucial. To investigate if binding of the bivalent ProteinDocker to BisALFA has a negative impact on the interaction with Fc receptors, the ProteinDocker was complexed with a 10-fold molar excess of BisALFA at pH 7.4 and pH 6.0. As a positive control, uncomplexed ProteinDocker was tested in parallel. Using NanoDSF, the successful complexation was verified under neutral and acidified pH conditions by thermal shift (Table 6).
Table 6: TM values of the bivalent ProteinDocker in combinations with a 10-fold excess of EX-
036 at pH 7.4 and pH 6.0
As TM1 was clearly elevated in presence of a 10-fold molar excess of BisALFA, which was also true for the onset of turbidity which reflects the temperature at which the ProteinDocker begins to precipitate, the quantitative formation of the ProteinDocker:BisALFA complex was confirmed.
BLI measurements confirmed that neither FcRn nor CD64 binding affinities are negatively influenced by complexing BisALFA (Table 7). Therefore, full effector functions and an unaltered half-life were to be expected for the ProteinDocker:BisALFA complexes. As the binding of FcRn to the Fc portion of IgG antibodies is pH-dependent, binding studies needed to be performed at pH 6.0.
Table 7: Kinetic binding parameters of ProteinDocker to CD64 and FcRn in the presence or in the absence of BisALFA.
Complex formation assessment
For bivalent ProteinDocker and BisALFA conjugates a 1:1 interaction was envisioned. However, if one ProteinDocker binds to two independent BisALFA conjugates in a 2:1 fashion, two ALFA tags remain free in this complex and can be engaged by further ProteinDockers. As a result, large macromolecular clusters could form that would potentially have a negative impact on PK and immunogenicity of the antibody. To quantify the risk of the presence of these large complexes, the bivalent ProteinDocker was incubated at 10-fold lower, equimolar or 10-fold higher molar concentrations with the BisALFA conjugate EX-036 and SEC analysis was performed. As a control, uncomplexed ProteinDocker was tested along (Figure 5).
The formation of higher molecular weight complexes is expected to be elevated with higher concentration of ProteinDocker. In highly concentrated samples, different ProteinDocker molecules are in close proximity. Upon binding of one ALFA moiety of the BisALFA conjugate by one NbALFA on the bivalent ProteinDocker, the second ALFA tag could be engaged by a nearby second ProteinDocker. In low concentrated samples, this second ProteinDocker molecule is not as close in proximity, favoring the binding of the second ALFA tag of the BisALFA conjugate by the second NbALFA moeity on the first ProteinDocker.
As in these experiments high concentrated samples are used, the formation of complexes is favored. Therefore, the here observed higher molecular complexes exceed what is expected to be observed in vivo where protein titers will be lower.
Between the 10-fold lower and the uncomplexed ProteinDocker, no difference in retention time was observed. At equimolar or at 10-higher concentration of the peptide a slight shift of the main peak was observed indicating that the intended 1:1 complex has formed. At equimolar concentrations a slight formation of a higher molecular weight species was observed (~3%), which was significantly diminished at 10-fold molecular excess of the peptide (<1%). As the totai amount of higher molecular species at an excess of the BisALFA conjugate was below 1% and this assay was performed at concentrations significantly higher compared to what will be expected in vivo, the risk of large complex formations in patients is considered low.
Example 4 - Radiolabeling of Bis-ALFA-conjugates
Ex-007 and Ex-008 are refered to as Peptide 29, and Peptide 31 respectively in the context of
Example 4.
Ga-68 labeling
Peptide 29 and Peptide 31 were labelled with a decay-corrected molar activity to achieve the desired activity (7-10MBq/nmol) at the time of administration. Baseline purity of the peptides was determined by HPLC prior to labelling; Figure 6 for Peptide 29 and Figure 9 for Peptide 31. The labelling was performed according to the protocol stated below:
• The generator was eluted using a fractionated method in 0.1 M HCL
• The 7-15 MBq/nmol was be transferred to an Eppendorf tube, and the activity will be measured.
• Labelling buffer of 1 M NH4OAc was added in ratio 1:2. Maximum reaction reaction volume 1.5 mL.
• pH was measured between 3.5-4.0
• Abs. EtOH was added to the reaction mixture to a final ethanol concentration of 5.0%
• Portion wise addition of the peptide 29 or peptide to the reaction mixture to reach the desired specific activity. o A decay-corrected specific activity was used to achieve the desired activity ((7- lOMBq/nmol) at the time of administration.
• The Reaction mixture was stirred at 95 °C at 600 RPM for 30 min. o The incorporation was be monitored after 15 min by radio-TLC and radio-HPLC. o an incorporation >92% was obtained after 15 min reaction for both peptides
• After end reaction, the reaction mixture was cooled to room temperature and a sample from the crude was withdrawn for determination of incorporation by radio- HPLC and radio-iTLC.
• The product may be purified depending on the degree of incorporation, according to the purification criteria stated in Table 8.
• The reaction was quenched with 4 mM DTPA in ratio 1:10 of total reaction volume. • The final product was be formulated in an aqueous formulation buffer to an activity concentration of 100 MBq/mL (10 MBq in 100 pL injection volume) and a compound concentration of 10 nmol/mL (1 nmol/100 pL).
• The radiochemical purity (RCP) of the final product (EOS) were analyzed by radio-HPLC; Figure 7 for Peptide 29 and Figure 10 for Peptide 31, and radio-TLC, Figure 8 for Peptide 29 and Figure 11 for Peptide 31.
• Buffer stability (RCP) for each compound was evaluated 1 and 4 hours after EOS by radio-HPLC; Figure 12 for Peptide 29 and Figure 13 for Peptide 31.
Discussion of analytical results.
• The optimal reaction time was shown to be 15 min for both compounds.
• An incorporation >92% was obtained.
• A RCP >92% was obtained in final product.
• No signs of instability was observed over the 7 hours monitored
An RCP >94% was obtained for both compounds 7 hours after EOS
In-111 labeling
Peptide 29 and Peptide 31 were labelled with a decay-corrected molar activity to achieve the desired activity (28-30 MBq/nmol) at the time of administration. Figure 6 for Peptide 29 and Figure 9 for Peptide 31. The labelling was performed according to the protocol stated below:
• In-111 (0.02 M HCI) was transferred to an Eppendorf tube, and the activity was measured.
• Labelling buffer 2.5 M NaOAc was added ((volume compound + volume ln-lll)/9= buffer volume).
• pH was measured between 5.0-5.5
• Portion wise addition of the peptide 29 or peptide to the reaction mixture to reach the desired specific activity.
• The Reaction mixture was stirred at 95 °C at 600 RPM for 30 min. o The incorporation was be monitored after 15 min by radio-TLC and radio-HPLC. o an incorporation >98% was obtained after 15 min reaction. • After end reaction, the reaction mixture was cooled to room temperature and a sample from the crude was withdrawn for determination of incorporation by radio- HPLC and radio-iTLC.
• The product may be purified depending on the degree of incorporation, according to the purification criteria stated in Table 8.
• The reaction was quenched with 4 mM DTPA in ratio 1:10 of total reaction volume.
• The final product was formulated in aqueous formulation buffer to an activity concentration of 300 MBq/mL (30 MBq in 100 pL injection volume) and a compound concentration of 10 nmol/mL (1 nmol/100 pL).
• The radiochemical purity (RCP) of the final product (EOS) were analyzed by radio-HPLC; Figure 14 for peptide 29 and Figure 16 for Peptide 31, and radio-TLC; Figure 15 for Peptide 29 and Figure 17 for Peptide 31.
• Buffer stability (RCP) for each compound will be evaluated 1, 4, and 24 hours after EOS by radio-HPLC. Figure 18 for peptide 29 and Figure 19 for Peptide 31.
Discussion of results:
• The optimal reaction time showed to be 15 min for both compounds.
• An incorporation >98% was obtained.
A Radiochemical purity (RCP) >97% was obtained in final product.
The percentage of free In-111 increases over the 24 hours monitored.
An RCP >90% was obtained for both compounds 24 hours after EOS.
Table 8: Example purification criteria Example 5 - Cell death assays using Bis-ALFA-Toxin conjugates
Experiment description
A congenic pair of ES-2 and a variant in which the bivalent ProteinDocker targeting antigen (CLDN6/Claudin-6; antigen+) is overexpressed (ES-2-antigen+), were used to assess potency and specificity of the invention. Cells were seeded at a confluency of 10-20% in a flat-bottom well plate in McCoy's 5A medium supplemented with 10% heat inactivated FCS (5xl03 cells in 96-well format) a day before the treatment. Bivalent ProteinDocker and BisALFA were complexed in an equimolar ratio for 30 min at room temperature prior to preparing a serial dilution of all analyzed compounds. Diluted compounds were mixed with three IncuCyte® dyes that allow measurement of cell death (IncuCyte® Cytotox NIR Dye) and apoptosis induction (IncuCyte® Caspase-3/7 Green Dye and IncuCyte® Annexin V Orange Dye) and added to the cells according to the manufacturer's instructions. The test items were analyzed in 8 concentrations to determine dose-dependent cell death induction. Cells were monitored via IncuCyte® SX5 Live-cell analysis system for 96 hours. Images were acquired every 1-3 hours. Spectral overlap was corrected and thresholds for segmentation were adjusted to the cell line and dye, followed by automated quantification of fluorescent signals by the IncuCyte® Livecell analysis system. The kinetic analysis avoids establishment of optimal readout time point for each cell line and avoids misleading conclusions based on inappropriate time of readout. Multiple redundant cell death readouts (cell confluence, DNA binding dye, Caspase 3/7 activity reporter and Annexin V staining) substantiate correct interpretation of cell death induction, type of cell death induced (necrosis vs. apoptosis) and clear distinguishment of cytotoxic from cytostatic effects or transient cell death unrelated activation of single processes (Caspase activation, Phosphoserine exposure) that are known to mislead interpretation of cell death assays (Kepp et al, Nature Reviews Drug Discovery 2011). Data analyzation, visualization and EC50 value calculation was performed using Graphpad Prims 9. The results are shown in Figure 20.
In future experiments, bystander killing activity will be analyzed in a coculture setting. Therefore, antigen-positive and antigen-negative cells are labeled in different fluorescent colors using proliferation dyes or IncuCyte® CytoLight dyes in order to attribute IncuCyte® Cytotox signals to the specific cell. This allows for the detailed and kinetic analysis of direct antibody-mediated uptake of the invention and the subsequent payload release and cell death induction in antigen-negative surrounding cells.
Additionally, the above-mentioned experiments will be performed using RiboDocker mRNA as starting material. mRNA will be transfected into HEK293 cells that subsequently produce and secrete the bivalent ProteinDockers. 24 hours after transfected supernatant is harvested. Dilutions of supernatant are mixed with fresh medium and incubated with BisALFA before or after they have been applied to the target cells.
Example 6 - Experiments involving Bis-ALFA-lmmunomodulator conjugates
Short description:
RiboDocker immunomodulators consist of two components: 1) The RiboDocker mRNA, which encodes for an Fc-competent, full length IgG (in protein form also called ProteinDocker) binding bivalently to the tumor antigen on one site and having two NanobodyALFAs on the other site; 2) a conjugate consisting of two NanobodyALFA-binding ALFA peptides to which immunomodulatory payloads are attached via a linker, which optionally is cleavable; see Figure 21. Different amounts of payloads are attached to the conjugate creating different DARs (aimed for 2-4). The immunomodulatory payload are agonistic compounds like STING agonists, which, upon cell-entry, induce immune activation.
The following section describes the in vitro methods utilized for selection of conjugates and RiboDocker-conjugate pairs.
Analysis of non-target activity: THP-1 and PBMC assays
The THP-1 and PBMC assays aim at describing the background activity of the immunomodulator conjugates, which are free or complexed with the ProteinDocker, in absence of the tumor antigen. THP-1 Dual Cells, purchased from Invivogen (Cat. Thpd-nfis), are reporter cells, which are utilized to easily monitor pathway activation downstream to PRR activation. PBMCs are additionally used to represent a more biologically realistic scenario. As these are primary human cells, at least two donors are compared. THP-1 cells are seeded at a density of lxlO5 cells/ well and PBMCs are seeded at 5xl05 cells/ well in a 96 well plate. The test items are added at a serial dilution and THP-1 cells are followingly cultivated for 24 h. PBMCs are washed after 1 h stimulation and cultivated for further 23 h. For both cell lines, the assay ends after 24 h, when cells are centrifuged and supernatants are taken. In THP-1 cell supernatant SEAP (secreted alkaline phosphatase), which indicates NFkB activation, and lucia luciferase, which indicates IRF activation, are measured according to manufacturer's instructions. PBMC supernatant is analyzed for secreted cytokines, induced upon immune activation, using Mesoscale Multiplex Assay Kits according to manufacturer's instructions. Based on the data EC50 values are calculated and compared to immune activation induced by the free payload.
Analysis of target-mediated activity: stimulation of antigen+ cells
Depending on the analyzed payload different antigen+ cells are used to analyze the antigen- mediated activity. Tumor cells endogenously expressing the target antigen are utilized if possible. Therefore, 0.1-5xl05 cells/well in a 96 well plate are seeded and a serial dilution of conjugates, which are free or pre-complexed with the antigen-binding ProteinDocker is added. Stimulation is performed for 24 h, after which cells are centrifuged and supernatants are taken to analyze secreted cytokines using Mesoscale Multiplex Assay Kits according to manufacturer's instructions. Alternatively, antigen-mediated activity can be analyzed by a cell line stably expressing the antigen as well as pathway activation reporter elements. For example, antigen-positive HEK reporter cells are seeded at 0.1-5xl05 cells/well in a 96 well plate and rested over night for attachment. The next day, a serial dilution of conjugates, which are free or pre-complexed with the antigen-binding ProteinDocker, is added. Cell supernatants are analyzed for SEAP and lucia luciferase reporter activity 24 h later (see THP-1 assay). EC50 values are calculated and compared between pre-complexed and free conjugates to describe antigen-mediated activity.
Analysis of Fc-mediated activity: co-culture assay
The ProteinDocker is a full length IgG with competent Fc function. Hence, not only activity in antigen+ cells is expected, but also Fc-mediated internalization and activity in FcR+ immune cells. To analyze this effect a customized immune assay was set up (see Figure 22). Antigen+ cells, which themselves do not respond to the payload, are co-cultured with either THP-1 cells or PBMC responder cells, which are FcgR-competent and respond readily to the payload. As Fc-mediated uptake is potentiated by antigen-dependent FcgR clustering, efficient stimulation of a response is dependent on the antigen present on non-responder cells (e.g. tumor cells) as well as the pathway activation in the responder cells. The antigen+ cells are seeded at 0.1- 5xl05 cells/well in a 96 well plate and rested over night for attachment. The next day, the immune cells are added: THP-1 cells at lxlO5 cells or PBMCs at 5xl05 cells. Additionally, the test items are added and left on the cells for 24 h. Conjugates pre-complexed with the ProteinDocker binding to the antigen+ cell line are compared to pre-complexes with a nonbinder to describe the importance of antigen-binding in Fc-mediated activity. Additionally, activity is compared to free-payload to analyze whether Fc-mediated uptake and signaling increases activity over the free payload, which only passively gets into the cell. Activity is analyzed by THP-1 reporter read outs (see THP-1 assay) to describe pathway activation or by analysis of PBMC supernatants for secreted cytokines using the Mesoscale Multiplex Assay Kits according to manufacturer's instructions. EC50 values are calculated and compared between groups to understand the Fc-mediated activity in comparison to the control groups.
Example 7 - Examples of ALFA STING conjugates
Synthesis of monoALFA intermediates Synthesis of SD17248-A
SD17248-A1 SD17248-A
Step 1: Synthesis of cyclic ALFA peptide intermediate, SD17248-A3
The cyclic ALFA peptide intermediate is synthesized using Fmoc solid phase peptide synthesis chemistry. Rink amide AM resin is loaded onto an automated peptide synthesizer suspended in 10 mL of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The peptide synthesis is initiated by the deprotection of the N-terminal a Fmoc protecting group using 4 mL of 20% piperidine in DMF heated by microwave for 3 min at 50 °C. After draining, the resin is washed three times with 5 mL DMF at five seconds per wash. Then the coupling reaction is performed by the addition of 2.5 mL of the next 0.3 M amino acid (7.5 eq.) in DMF, 1 mL of a 1 M DIC (10 eq.) solution in DMF, and 0.5 mL 1 M Oxyma (5 eq.) with 0.1M DIEA solution in DMF to the RV. The coupling reaction is heated to 60 °C for 10 min. The reaction solution is drained, and the coupling reaction is repeated. After the second coupling, the resin is drained and washed four times with 4 mL of DMF. Then 4 mL of 20% piperidine in DMF is added to the RV to deprotect the Fmoc group and the next amino acid is coupled. This cycle of Fmoc removal and coupling is repeated for every amino acid sequentially. Fmoc-Glu(O-2-PhiPr)-OH and Fmoc-Lys(Mmt)-OH amino acids are incorporated into the peptide intermediate sequence to allow for selective cyclization. The peptide- containing resin is transferred to a 20 mL fritted syringe after solid phase peptide synthesis is completed. To the resin is added 7 mL of capping solution (81.25% DMF, 12.5% 2 M DIEA in NMP, 6.25% acetic anhydride) and the reaction is agitated for 15 min. The capping solution is removed, and another 7 mL of capping solution is added and the reaction is agitated for another 15 min. Then the resin is washed thoroughly with DCM and DMF.
Step 2: Selective deprotection of Lys(Mmt) and Glu(O-2-PhiPr), SD17248-A2
To the resin containing SD17248-A3 in a fritted syringe is added 5 mL of partial deprotection cocktail (2% TFA, 2% TIS in DCM). The reaction is agitated for 5 min, then the deprotection cocktail is removed from the resin via filtration. Another 5 mL of partial deprotection cocktail is added, agitated for 5 minutes, and removed. This process is repeated six times until the yellow/orange color of the flowthrough disappears (30 minutes total of Mmt and 2-PhiPr removal). The resin is rinsed thoroughly with DCM and DMF to afford SD17248-A2.
Step 3: Lactam cyclization via side chain amide bond formation, SD17248-A1 Cyclization is carried out in the same fritted syringe as the partial deprotection from Step 2. To the syringe is added 5 eq. of PyAOP and 5 eq. of HOAt as a solution in DMF. The resin is allowed to swell and then 10 eq. of DIEA is added. The reaction is agitated for 2-3 hours. The reaction mixture is removed from the resin via filtration and the remaining resin is rinsed thoroughly with DCM and DMF to afford SD17248-A1.
Step 4: Cleavage and global deprotection, SD17248-A
To the resin containing SD17248-A1 in a fritted syringe is added 10 mL of cleavage cocktail (87.5% TFA, 5% thioanisole, 2.5% water, 2.5% TIS, 2.5% EDT). The reaction is agitated for 2 h at room temp followed by 1 h at 40 °C. The resulting peptide intermediate is then precipitated out of solution by adding the reaction solution to ~25 mL cold diethyl ether which is then centrifuged. The supernatant is removed and the resulting pellet is redissolved in 50:50 MeCN:water. The resulting crude peptide intermediated is purified by RP HPLC to afford SD17248-A. Isolated 159.2 mg from 0.3-mmol scale synthesis (25% yield, >95% pure). [M+2H]2+ = 1038.99
Synthesis of SD18317-A/PIC8-A
PIC8A
The title compound was prepared following analogous protocol to the synthesis of SD17248-A.
[M+2H]2+ = 1184.23
Synthesis of PIC12-A
PIC12A
The title compound was prepared following analogous protocol to the synthesis of SD17248-A using Fmoc-L-Cysteic acid. [M+2H]2+ = 1114.18
Synthesis of PIC13-A
PIC13A
The title compound was prepared following analogous protocol to the synthesis of SD17248-A using Fmoc-AEPA-OH. [M+2H]2+ = 1102.18
Synthesis of PIC14-A
The title compound was prepared following analogous protocol to the synthesis of SD17248-A. [M+3H]3+ = 963.54
Synthesis of bisALFA intermediates
Synthesis of SD17453-B
Step 1: Synthesis of cyclic ALFA peptide intermediate, SD17453-B3:
The cyclic ALFA peptide intermediate is synthesized using Fmoc solid phase peptide synthesis chemistry. Rink amide AM resin is loaded onto an automated peptide synthesizer suspended in 10 mL of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The peptide synthesis is initiated by the deprotection of the N-terminal a Fmoc protecting group using 4 mL of 20% piperidine in DMF heated by microwave for 3 min at 50 °C. After draining, the resin is washed three times with 5mL DMF at five seconds per wash. Then the coupling reaction is performed by the addition of 2.5 mL of the next 0.3 M amino acid (7.5 eq.) in DMF, 1 mL of a 1 M DIC (10 eq.) solution in DMF, and 0.5 mL 1 M Oxyma (5 eq.) with 0.1M DIEA solution in DMF to the RV. The coupling reaction is heated to 60 °C for 10 min. The reaction solution is drained, and the coupling reaction is repeated. After the second coupling, the resin is drained and washed four times with 4 mL of DMF. Then 4 mL of 20% piperidine in DMF is added to the RV to deprotect the Fmoc group and the next amino acid is coupled. This cycle of Fmoc removal and coupling is repeated for every amino acid sequentially. Fmoc-Lys(Fmoc)-OH is used for the first residue coupled to the resin to allow for growth of 2 ALFA peptides simultaneously. Fmoc-Glu(O-2-PhiPr)-OH and Fmoc-Lys(Mmt)-OH amino acids are incorporated into the peptide intermediate sequence to allow for selective cyclization. The peptide-containing resin is transferred to a 20 mL fritted syringe after solid phase peptide synthesis is completed. To the resin is added 7 mL of capping solution (81.25% DMF, 12.5% 2 M DIEA in NMP, 6.25% acetic anhydride) and the reaction is agitated for 15 min. The capping solution is removed, and another 7 mL of capping solution is added and the reaction is agitated for another 15 min. Then the resin is washed thoroughly with DCM and DMF.
Step 2: Selective deprotection of Lys(Mmt) and Glu(O-2-PhiPr), SD17453-B2
To the resin containing SD17453-B3 in a fritted syringe is added 5 mL of partial deprotection cocktail (2% TFA, 2% TIS in DCM). The reaction is agitated for 5 min, then the deprotection cocktail is removed from the resin via filtration. Another 5 mL of partial deprotection cocktail is added, agitated for 5 minutes, and removed. This process is repeated six times until the yellow/orange color of the flowthrough disappears (30 minutes total of Mmt and 2-PhiPr removal). The resin is rinsed thoroughly with DCM and DMF to afford SD17453-B2. Step 3: Lactam cyclization via side chain amide bond formation, SD17453-B1
Cyclization is carried out in the same fritted syringe as the partial deprotection from Step 2. To the syringe is added 10 eq. of PyAOP and 10 eq. of HOAt as a solution in DMF. The resin is allowed to swell and then 20 eq. of DIEA is added. The reaction is agitated for 2-3 hours. The reaction mixture is removed from the resin via filtration and the remaining resin is rinsed thoroughly with DCM and DMF to afford SD17453-B1.
Step 4: Cleavage and global deprotection, SD17453-B
To the resin containing SD17453-B1 in a fritted syringe is added 10 mL of cleavage cocktail (87.5% TFA, 5% thioanisole, 2.5% water, 2.5% TIS, 2.5% EDT). The reaction is agitated for 2 h at room temp followed by 1 h at 40 °C. The resulting peptide intermediate is then precipitated out of solution by adding the reaction solution to ~25 mL cold diethyl ether which is then centrifuged. The supernatant is removed and the resulting pellet is redissolved in 50:50 MeCN:water. The resulting crude peptide intermediated is purified by RP HPLCto afford SD17453-B. Isolated 42 mg from 0.3-mmol scale synthesis (5% yield, >95% pure).
[M+4H]4+ = 1066.52
Synthesis of SD17537-B
The title compound was prepared following analogous protocol to the synthesis of SD17453-B. [M+4H]4+ = 1211.63
Synthesis of SD18319-B
The title compound was prepared following analogous protocol to the synthesis of SD17453-B using Fmoc-AEPA-OH.
[M+5H]5+ = 1225.49
Synthesis of SD18320-B
The title compound was prepared following analogous protocol to the synthesis of SD17453-B.
[M+5H]5+ = 1201.66
Synthesis of a SD18321-B/PIC7-B
The title compound was prepared following analogous protocol to the synthesis of lnt-E-003.
[M+5H]5+ = 1164.25
Synthesis of SD18322-B
The title compound was prepared following analogous protocol to the synthesis of SD17453-B.
[M+4H]4+ = 1113.28
Synthesis of PIC19-B
Step 1: Synthesis of cyclic ALFA homodimer peptide intermediate, PIC19-B1:
The cyclic ALFA peptide intermediate PIC19-B2 was prepared following analogous protocol to the synthesis of SD17248-A using Fmoc-Cys(SIT)- OH. PIC19-B2 (13.8 mg, 0.0048 mmol) was dissolved in DMF (312.5 pL). To this solution was added DIEA (5 pL, 0.028 mmol, 3 eq.) followed by bis-PEGl-NHS ester (CAS 65869-64-9) (0.85 mg, 0.0024 mmol, 0.5 eq.) solution in DMF (43 pL) and DIEA (3 eq. of DIEA to 1 eq. bis-PEGl-NHS ester) as three batches over 15 min. After 60 min, reaction was judged complete by UPLC-MS analysis. The reaction mixture was crashed into diethyl ether (40 mL) and centrifuged to isolate crude PIC19-B1 as off-white precipitate that was used in the next step without further purification. [M+5H]5+ = 1180.74
Step 2: Synthesis of PIC19-B via deprotection of Cys(SIT) on cyclic ALFA homodimer peptide
PIC19-B1 (ca. 0.0048 mmol) was dissolved in DMF (200 pL) containing 5% water and 2.5% DIEA. To the solution was then added dithiothreitol as solid (3.7 mg, 0.024 mmol, 5 eq.). After 3 hours, reaction was judged complete by UPLC-MS analysis. The reaction mixture was crashed into diethyl ether (40 mL) and centrifuged. Supernatant was decanted and the pellet was redissolved in minimal volume of DMF and crashed again in diethyl ether and centrifuged to isolate crude PIC19-B as off-white precipitate that was used in the next step without further purification. [M+5H]5+ = 1139.83
Synthesis of STING drug-linkers
Synthesis of BrAc-AEEA2-16871 drug-linker
NH2-AEEA2-16871 BrAc-AEEA2-16871
BrAc-AEEA2-16871 was prepared over 3 steps beginning with SD16871. SD16871 (45 mg, 0.0576 mmol, 1 eq.) was dissolved in DMF (2.5 mL). To this solution was added Boc-AEEA2-OH (CAS 1069067-08-8) (28.2 mg, 0.0691 mmol, 1.2 equiv.), HATU (65.6 mg, 0.173 mmol, 3 eq.), and DIEA (30 uL, 0.173 mmol, 3 equiv.). After 40 min, reaction was judged complete by UPLC- MS analysis. DMF was removed in vacuo, product was redissolved in 10% MeOH in CHCh and purified by SEC.
Pure Boc-AEEA2-16871 (22.27 mg, 0.019 mmol) was dissolved in DCM (2.4 mL). To this solution was added 600 uLTFA. Boc removal was complete after 5 min as judged by UPLC-MS. DCM was removed in vacuo and product was suspended in 5 mL water and lyophilized.
Pure H2N-AEEA2-16871 (20.4 mg, 0.019 mmol) was dissolved in 1.5 mL DMF. To this solution was added bromoacetic acid N-hydroxysuccinimide ester (9 mg, 0.038 mmol, 2 eq.) and DIEA (10 uL, 0.057 mmol, 3 eq.). Reaction was complete after 5 min as judged by UPLC-MS. DMF was removed in vacuo, product was redissolved in 10% MeOH in CHCh and purified by SEC. Isolated 16.25 mg (24% over 3 steps).
[M+2H]2+ = 598.31
Synthesis of BrAc-AEEA2-16803
The title compound was prepared following analogous protocol to the synthesis of BrAc-
AEEA2-16871.
[M+2H]2+ = 611.29
Synthesis of BrAc-AEEA2-15573
BrAc-AEEA2-15573
The title compound was prepared following analogous protocol to the synthesis of BrAc-
AEEA2-16871.
[M+2H]2+ = 639.9 Synthesis of ClAc-GGFG-AM-16803
NH2-GGFG-AM-16803 CIAc-GGFG-AM-16803
The title compound was prepared following analogous protocol to the synthesis of BrAc- AEEA2-16871 using Fmoc-GGFG-AM-OH (CAS 2264011-98-3) and chloroacetic acid. In place of Boc deprotection with TFA, Fmoc was removed by bringing solution to 20% piperidine in DMF. After 10 minutes of reaction time, Fmoc removal was complete. Solution was crashed in MTBE and centrifuged. Supernatant was decanted and the pellet was resuspended in MeOH and crashed again in MTBE to isolate H2N-GGFG-AM-16803. Chloroacetic acid coupling was performed in analogous manner to bromoacetic acid coupling during synthesis of BrAc- AEEA2-16871.
[M+2H]2+ = 646.22
Synthesis of ClAc-GGFG-AM-16871
CIAc-GGFG-AM-16871
The title compound was prepared following analogous protocol to the synthesis of CIAc-
GGFG-AM-16803.
[M+2H]2+ = 632.8
Synthesis of ALFA-STING conjugates via thiol conjugation reaction
Synthesis of SD18317/PIC8
SD18317-A/PIC8-A (22 mg, 0.0093 mmol) and BrAc-AEEA2-16871 (12.2 mg, 0.0102 mmol, 1.2 eq.) were dissolved in DMF (250 uL). To this solution was added DIEA (9 uL, 0.0558 mmol, 6 eq.). Reaction mixture was stirred at room temperature. Reaction was complete after 10 min as judged by UPLC-MS monitoring. Purified by RP-HPLC to isolate 5.5 mg of PIC8 (17% yield, >95% pure).
[M+3H]3+ = 1160.68 Synthesis of SD17248
The title compound was prepared following analogous protocol to the synthesis of PIC8 using BrAc-AEEA2-16803. [M+3H]3+ = 1072.49
Synthesis of PIC12
The title compound was prepared following analogous protocol to the synthesis of PIC8.
[M+3H]3+ = 1160.68
Synthesis of PIC13
The title compound was prepared following analogous protocol to the synthesis of PIC8. Fmoc was removed after conjugation was complete by addition of piperidine to the reaction mixture to 20% v/v.The resulting mixture was then purified by HPLC.
[M+3H]3+ = 1105.91
Synthesis of PIC14
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the BrAc-AEEA-16871 drug-linker. [M+4H]4+ = 1279.46
Synthesis of EX-M74/PIC15
The title compound was prepared following analogous protocol to the synthesis of PIC8 using BrAc-AEEA-15573 drug-linker.
[M+3H]3+ = 1188.69
Synthesis of EX-M69/PIC16
The title compound was prepared following analogous protocol to the synthesis of PIC8 using ClAc-GGFG-AM-18761 drug-linker.
[M+3H]3+ = 1199.21
Synthesis of EX-M71/PIC17
The title compound was prepared following analogous protocol to the synthesis of PIC8 using CIAc-GGFG-AM-18761 drug-linker.
[M+3H]3+ = 1152.89
Synthesis of SD17247
The title compound was prepared following analogous protocol to the synthesis of EX-015 using 2.2 eq. of the BrAc-AEEA-16803 drug-linker. [M+7H]7+ = 1105.59
Synthesis of SD17553
The title compound was prepared following analogous protocol to the synthesis of EX-015 using 4.4 eq. of the CIAc-GGFG-AM-16803 drug-linker.
[M+10H]10+ = 1155.96
Synthesis of SD17453
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the BrAc-AEEA-16803 drug-linker. [M+6H]6+ = 1091.21
Synthesis of SD17516
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the CIAc-GGFG-AM-16803 drug-linker. [M+6H]6+ = 1129.55
Synthesis of SD17537
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the CIAc-GGFG-AM-16803 drug-linker.
[M+6H]6+ = 1226.29
Synthesis of SD18319
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the BrAc-AEEA2-16871 drug-linker.
[M+7H]7+ = 1129.9
Synthesis of SD18320
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the BrAc-AEEA2-16871 drug-linker.
[M+8H]8+ = 1029.44
Synthesis of SD18321/PIC7
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the BrAc-AEEA2-16871 drug-linker. Fmoc was removed after conjugation was complete by addition of piperidine to the reaction mixture to 20% v/v. The resulting mixture was then purified by HPLC.
[M+7H]7+ = 1149.67
Synthesis of EX-B122/PIC18
The title compound was prepared following analogous protocol to the synthesis of PICS using 2.2 eq. of the ClAc-GGFG-AM-16871 drug-linker. Fmoc was removed after conjugation was complete by addition of piperidine to the reaction mixture to 20% v/v. The resulting mixture was then purified by HPLC.
[M+5H]5+ = 1654.95
Synthesis of PIC19
The title compound was prepared following analogous protocol to the synthesis of PIC8 using 2.2 eq. of the BrAc-AEEA2-16871 drug-linker.
[M+5H]5+ = 1585.7
Synthesis of SD18322
The title compound was prepared following analogous protocol to the synthesis of PIC8. [M+5H]5+ = 1113.00
The following MonoALFA STING conjugates can be prepared using protocols as outlined for the examples specified above:
The following BisALFA STING conjugates can be prepared using protocols as outlined for the examples specified above:
Example 8 - Affinity, Avidity and binding Assessment and Complex Formation testing
Material and Methods
Measuring avidity-driven interaction of BisALFA and NbALFA-comprising proteins
For avidity measurements, the 16-channel mode of the Octet HTX was utilized to allow for simultaneous double referencing. Commercially available SSA biosensors were quenched for 10 min in KB, followed by a 120 sec baseline measurement. Subsequently, eight of the 16 biosensors were loaded with 2.5 pg/ml biotinylated NbALFA for 60 sec while the remaining 8 biosensors were further incubated in KB. All biosensors were quenched for 120 sec in a 100 pg/ml biocytin solution. Association was measured over 900 sec using seven different concentrations per biosensor-column of a BisALFA derivative starting at 2.5 nM in a 1:1 serial dilution series down to 39.0625 pM. As a reference, KB was measured with the eighth biosensor for both biosensor columns. The dissociation was acquired over 1500 sec in KB. The signal of the reference biosensors was subtracted in pairs from the NbALFA-loaded biosensors, followed by subtraction of the reference well signal from all biosensors in the respective column. After this double referencing step, the signal was aligned to the average of the second baseline. For inter-step correction, the data were aligned to the dissociation step and Savitzky- Golay filtering was applied. Association and dissociations were globally fitted using a 1:1 Langmuir binding model.
Binding of BisALFA and bivalent ProteinDocker in solution
To investigate the formation of complexes, bivalent ProteinDocker was mixed in PBS with different concentrations of the BisALFA conjugate. The final concentration was adjusted to 3 pM for the protein, with 30 pM of BisALFA conjugates. After mixing, samples were incubated at room temperature for at least 20 min to allow for the binding for the NbALFA moieties to the ALFA-tags on the BisALFA conjugate.
Dynamic light scattering for complex detection Dynamic light scattering measurements were performed using the Prometheus PANTA from NanoTemper. 10 pL of preincubated BisALFA-ProteinDocker complexes or the ProteinDocker alone were loaded into capillaries. The intensity distribution was plotted against the radius of the test items (proteins), allowing for assessing the presence of larger aggregated complexes in the sample.
All measurements were performed at least in duplicates.
Results
Assessment of avidity-driven interaction of Bis-ALFA STING agonists with NbALFA
First, we strived to investigate whether both ALFA tags of the BisALFA conjugate can bind simultaneously with two NbALFA VHHs and if the presence of the STING agonist interferes with this interaction. In biolayer interferometric measurements using NbALFA as ligand and BisALFA as analyte revealed a high affine interaction with an off-rate surpassing instrument limit (Fig. 23). This extremely slow off-rate is a clear indication of an avidity-driven interaction showing that both ALFA-tags can simultaneously be engaged by two NbALFA VHHs and that the STING agonist does not interfere with this interaction.
Complex formation assessment
For bivalent ProteinDocker and BisALFA conjugates a 1:1 interaction was envisioned. However, if one ProteinDocker binds to two independent BisALFA conjugates in a 2:1 fashion, two ALFA tags remain free in this complex and can be engaged by further ProteinDockers. As a result, large macromolecular clusters could form that would potentially have a negative impact on PK and immunogenicity of the antibody.
To investigate complex formation, a ProteinDocker comprising two NbALFA VHH moeties was investigated in DLS. The resulting intensity distribution yielded a low cumulant radius indicating that no aggregates are present in the protein solution. Next, the ProteinDocker was incubated with a 10-fold excess of BisALFA STING agonist and the DLS measurement was repeated (Fig. 24). Slight peaks at high radii values were observed, however, DLS overrepresents larger molecules as they scatter light more strongly. As the monomer peak is the most prominent one, the larger complexes must be present in very low abundance.
Taken together, we demonstrated that BisALFA STING agonists are effectively bound by NbALFA with a high avidity via both ALFA tags and do not lead to undesirably high molecular complex upon encountering a ProteinDocker exhibiting two NbALFA moieties.
Example 9 - Pretrageting approach using monovalent RiboDocker and radiolabeled bivalent ALFA (bisALFA) peptide
Biodistribution of Ga68-DOTA-bisALFA with and without RiboDocker (Figure 25)
Athymic nude mice bearing an OV-90 xenograft were administered 30 pg of RiboDocker directed against a tumor antigen and comprising an anti-ALFA VHH. Subsequently, approximately a few hours later (a timeframe that has been demonstrated in previous studies to be sufficient for the elimination of RiboDocker from the circulation), Ga68-radiolabeled DOTA-bisALFA (comprising two cyclized ALFA peptides, each binding with a low affinity to the anti-ALFA VHH) was administered intravenously at a dose of 0.1 nmol (10 MBq). At 3.5 hours post-administration of Ga68-DOTA-bisALFA, the mice were euthanized, and blood samples were collected via cardiac puncture. Subsequently, the tumor and relevant internal organs were harvested, weighed, and counted in a gamma counter with energy windows optimized for Ga-68. The percentage injected dose per gram of tissue (%ID/g) was calculated.
Following the administration of RiboDocker, a notable accumulation of bisALFA peptide was identified within the tumor tissue (10.3% I D/g), with negligible levels retained in normal tissues except the kidneys. The radiolabeled bisALFA is excreted via the kidneys due to its low molecular weight (~5 kDa). The peptide appears to be reabsorbed, resulting in the generation of a pronounced signal in this region.
It is notable that tumors that were not pretargeted with RiboDocker exhibited a low uptake of Ga-68-DOTA-bisALFA (0.3 % ID/g) in the tumor, indicating that the specific accumulation of bisALFA in the tumor was derived from the pre-localization of the RiboDocker. In conclusion, the findings of this study indicate that pretargeting with a RiboDocker with only one binding site to a low-affinity ALFA peptide and a bivalent Ga68-labeled bisALFA peptide has the potential to be a highly sensitive tumor diagnostic modality.
Due to the monovalent binding of the RiboDocker to the bivalent ALFA peptide utilized in this study, the AES (Affinity Enhancement System Effect) was employed. This resulted in a low affinity between the RiboDocker and ALFA peptide in the circulation, while a strong avidity effect was observed in the tumor.
Potential of the RiboDocker and bisALFA approach for therapy (Figure 26)
The objective of this study is to ascertain whether an alternative isotope (in addition to Ga-68) can be employed in conjunction with the RiboDocker bisALFA pretargeting approach. Moreover, the aim is to ascertain the possibility of monitoring the tumor signal beyond the 3.5-hour bisALFA postinjection time point. A longer residence time in the tumor would be beneficial from a therapeutic perspective.
Nude mice bearing OV-90 were first treated with 30 pg of RiboDocker, as previously described. Subsequently, following the clearance of the majority of RiboDocker from the bloodstream, 0.1 nmol radiolabeled DOTA-bisALFA was administered. Instead of using Ga-68 to label the bisALFA peptide, as was done in the previously described experiment, Cu-64 was employed. Given that the half-life of Cu64 is longer than that of Ga68 (12.5 hours vs. 68 minutes, respectively), this isotope is more suitable for analyzing data from later post-injection measurement times.
The data provides substantial insight. It is feasible to attain analogous outcomes by modifying the isotope. Furthermore, the tumor signal is detectable for up to 24 hours after peptide injection (8.8 %l D/g), which is indicative of an effective therapeutic approach. The longer the conjugated bisALFA remains at the tumor site, the greater the possibility of an effective treatment. In this way, we can exchange the Cu64 isotope with a therapeutic isotope or exchange the isotope with a toxin or immunomodulator. Example 10 - Pretrageting approach using monovalent RiboDocker and fluorophore-labeled monovalent ALFA (monoALFA) conjugate
The experiments were conducted using a mouse model with established OV-90 tumors in NSG female mice. A RiboDocker (LNP-formulated RNA encoding a construct directed against a tumor antigen, which is fused to one anti-ALFA VHH) or a ProteinDocker (recombinant and purified protein with the same design as the RiboDocker) was administered as a reference by intravenous injection. Subsequently, a monovalent cyclized ALFA peptide conjugated with an Alexa Fluor 680 fluorophore (monALFA-AF680, Sequence: Ac(C-
Alexa680)(PEG6)PSRLEEELRRRLTE-NH2) was administered to the mice at specified time points following the initial injection. At the designated time points, the mice were anesthetized via inhalation of isoflurane. In vivo imaging was conducted using a Xenogen I VIS Spectrum Imaging System. The maximum near-infrared signals were quantified using Living Image image analysis software.
While a distribution in the body and a weak tumor accumulation can be detected at the early imaging time points, the tumor signal increases with time, and the signal in other parts of the body decreases (Figure 27). Therefore, monALFA-AF680 demonstrates effective tumor targeting and minimal accumulation in other organs at later time points.
Example 11 - In vitro activity of pre-complexes using a co-culture of an antigen+ tumor cell line, which was transfected to express the antigen, and THP-1 Dual reporter cells indicating IRF and NFB induction
THP-1 co-culture assay protocol
TM
THP-1 Dual Cells , were purchased from Invivogen (Cat. Thpd-nfis) and used to determine the biological activity of the test items on a molecular level and to determine downstream activation of STING signaling (IRF and NFKB-pathway activation) with and without antigen presence. Briefly, cultured antigen+ tumor cells were seeded one day prior to experiment start (96 well format in cell-type recommended medium + 10 % heat inactivated FBS). 24 hours after seeding, THP-1 cells were added at 1 x 10s cells per well (cell-type recommended medium without FBS). A serial dilution of the test items was subsequently added. These test items consisted of the binding domain (the ProteinDocker) and the immune-conjugate. These two components were pre-complexed for 30 minutes at room temperature and followingly diluted and added to the antigen+ cells. A total of 8 concentrations was used to determine a dosedependent activation of the NFKB and IRF pathway in THP-1 cells. In presence of the antigen, the pre-complexes were taken up into the THP-1 cells via FcgR-engagement, leading to STING- mediated immune activation. Immune activation was detected 24 hours after stimulation by centrifugation of cells and harvest of supernatants for the analysis of secreted alkaline
TM phosphatase (measured by QUANTI-Blue detection solution, Cat. Rep-qbs Invivogen) and TM lucia luciferase (measured by QUANTI-Luc reagent, Cat. Rep-qlcl Invivogen). The read outs were performed according to the manufacturer's instructions. Graphical depiction was created using GraphPad Prism 9.
BisALFA in vitro co-culture
Figures 28 and 29 depict activity data for pre-complexes consisting of the ProteinDocker and different immune-conjugates, which were added for 24 hours to co-seeded antigen+ tumor cells and THP-1 Dual reporter cells. Compared are conjugates complexed with the binding ProteinDocker and a non-binding control. Analyzed was the STING-mediated signal in the THP- 1 Dual reporter cells. Complexes of ProteinDocker and immune-conjugate were presumed to bind to the antigen on the tumor cells and furthermore to FcyRs on myeloid cells, leading to uptake and activation of the bystander, antigen- immune cells. The analyzed activity signal refers to the second way of uptake only, as tumor cell stimulation was not read out. All immune-conjugates exhibited greater activity in conjunction with the binding ProteinDocker compared to the non-binder, as seen for co-culture with two different antigen+ tumor cell lines. Pre-complexes with the conjugates with the SD16871 payload exhibited activity onset at a higher concentration compared to conjugates with the SD16803 payload, which is more hydrophobic. This difference in activity was expected, as the activity between the payloads alone differed by 20x (Figure 30), which was also seen in the pre-complex activity assay.
Residual activity unrelated to antigen expression was seen, as non-binder pre-complexes induced signal at high concentrations. The shift between binder and non-binder was seen at different extent but all tested conjugates exhibited elevated activity in presence of antigen compared to the non-binder. The background for the non-binder was reduced for conjugates containing the SD16871 payload (corresponding to Formula XX-18) compared to the SD16803- based compounds (SD16803 corresponds to Formula XX-53). In general, pre-complexes with strong antigen-specific activity but low background activity in an antigen-negative setting are preferred. High activity in an antigen-negative environment might indicate off-target activity. Hence, despite slightly lower activity in an antigen-specific context, the SD16871-based conjugates would be preferred based on the low activity of the non-binder.
MonoALFA in vitro co-culture
Figure 31 depicts activity data for pre-complexes consisting of the ProteinDocker and different immune-conjugates, which were added for 24 hours to co-seeded antigen+ tumor cells and THP-1 Dual reporter cells. Compared are conjugates complexed with the binding ProteinDocker and a non-binding control. Analyzed was the STING-mediated signal in the THP- 1 Dual reporter cells. Complexes of ProteinDocker and immune-conjugate were presumed to bind to the antigen on the tumor cells and furthermore to FcyRs on myeloid cells, leading to uptake and activation of the bystander, antigen- immune cells. The analyzed activity signal refers to the second way of uptake only, as tumor cell stimulation was not read out.
Both immune-conjugates (Figure 31(A) and (B)) exhibited greater activity in conjunction with the binding ProteinDocker compared to the non-binder, as seen for co-culture with two different antigen+ tumor cell lines. Pre-complexes with SD18317 exhibited activity onset at a higher concentration compared to SD17248 for the first cell line. The two compounds differ in the attached payload. SD18317 contains the SD16871 payload while SD17248 contains the SD16803 payload, which is more hydrophobic. This difference in activity was expected, as the activity between the payloads alone differed by 20x (Figure 30). For the cell line, which endogenously expressed the antigen (B), no clear activity differences were seen.
Residual activity unrelated to antigen expression was seen, as non-binder pre-complexes induced signal at high concentrations. Both tested conjugates exhibited elevated activity in presence of antigen compared to the non-binder. For the first cell line (A), the background of the non-binder pre-complex was reduced for SD18317 compared to SD17248. Again, this effect was less prominent in the second cell line (B). In general, pre-complexes with strong antigen-specific activity but low background activity in an antigen-negative setting are preferred. High activity in an antigen-negative environment might indicate off-target activity. As SD18317 exhibited a trend towards lower background activity compared to SD17248, conjugates containing the SD16871 would be preferred.
All immune-conjugates tested in Figure 31(C) exhibited greater activity in conjunction with the binding ProteinDocker compared to the non-binder (solid lines vs. dashed lines, respectively). Only for SD18317 (PIC8), the non-binder showed no activity at all. The greatest shift between binder and non-binder was seen for this conjugate. The shift between binder and non-binder was seen at different extent for the different conjugates. However, all tested conjugates exhibited elevated activity in presence of antigen compared to the non-binder. In general, precomplexes with strong antigen-specific activity but low background activity in an antigennegative setting are preferred. High activity in an antigen-negative environment might indicate off-target activity. Hence, PIC8 exhibited the best profile but all tested conjugates showed a good binder to non-binder activity profile.
Example 12 - In vivo testing of pre-complex efficacy in syngraft
Protocol for in vivo testing of pre-complex efficacy in syngraft
Anti-tumoral efficacy of the pre-complexes of ProteinDocker and immune-conjugate were tested in the murine B16F10 melanoma model, which was stably transfected to overexpress the model antigen. C57Bl/6JRjx mice were injected subcutaneously with 3 x IQ5 B16F10 cells. Two pre-complexes with an antigen binding ProteinDocker and a non-binder were injected intravenously on day 13 and day 19 after tumor inoculation. The test items were formulated in PBS containing 10 % 5x HEPES, 10 % Glycerol, and 0.1 % DMSO. Tumor size was monitored using caliper measurement ((mm)3=(widthxwidthx|ength)/2) until mice reached exclusion criteria.
Anti-tumoral effect of immune conjugates in syngraft To describe the anti-tumoral effect of two immune conjugates in conjunction with the ProteinDocker, an in vivo experiment was carried out using SD17453, which has no specified cleavage site, and SD17516, which contains a GGFG cleavage motif. Significantly reduced tumor growth was observed for both pre-complexes upon two intravenous injections (Figure 32). The observed effect was stronger for the antigen-specific ProteinDocker than for a nonbinder, which however also exhibited tumor control greater than the untreated group. This observation is presumably due to the EPR effect (enhanced permeability and retention), by which macromolecules of a certain size (>40 kDa) passively accumulate within the tumor. Nevertheless, usage of an antigen-directed ProteinDocker increased the efficacy compared to this passive accumulation effect. This benefit over the non-binding control was slightly greater for SD17453 than for SD17516.
Anti-tumoral effect of additional immune conjugates in syngraft
The above in vivo experiment was repeated using SD18321 (PIC7) and SD18317 (PIC8). Favorable immune-conjugates should exhibit a balance of good systemic tolerability and anti- tumoral efficacy. Tolerability was seen highest for the bisALFA conjugate PIC7, while activity in vitro was observed to be strongest for the monoALFA conjugate PIC8 (Figure 33). In this study both immune-conjugates were directly compared for anti-tumoral efficacy in a syngeneic model. Anti-tumoral efficacy beyond the level of control groups was seen for both compounds. A non-binding pre-complex exhibited no anti-tumoral effect, similar to a pre-complex with an immune-conjugate carrying no functional payload. Both PIC7 and PIC8 exhibited effects beyond these controls. The anti-tumoral efficacy of PIC8 was stronger than for PIC7, confirming in vitro activity data.
Example 13 - In vitro activity of pre-complexes in whole blood to estimate unspecific systemic activity
Whole blood was taken from three donors using tubes containing sodium citrate as anticoagulant. 175 pl blood and 25 pl test item were added per well (96 well format). The test items consisted of the binding domain (the ProteinDocker) and the immune-conjugate. These two components were pre-complexed for 30 minutes at room temperature and followingly diluted. As controls, free conjugates and free payload were used. A total of 8 concentrations were added to the whole blood to determine dose-dependent immune activation. Immune activation was detected 24 hours after stimulation by centrifugation of cells and harvest of supernatants for the analysis of cytokines (custom U-Plex, Meso Scale Diagnostics LLC). The read outs were performed according to manufacturer's instructions.
The following Table 9 depicts the strength of immune activation in whole blood. For this summarized depiction the AUC of the log(10) test item concentrations was taken. Higher numbers indicate stronger immune activation, while lower numbers indicate weaker immune activation in whole blood. For reference, payload AUC is given. Compared were pre-complexes of ProteinDocker and immune-conjugates, as well as uncomplexed immune-conjugates. Weakest immune activation in whole blood was observed for SD18321 (PIC7) followed by PIC19. None of the tested compounds exhibited unexpected high activity in whole blood.
Table 9: Unspecific immune activation (based on IFNfJ level (left panel) or IL-6 level (right panel)) by pre-complexes in whole blood Example 14- In vivo tolerability of pre-complexes plus different immune-conjugates
In vivo tolerability of the pre-complexes of ProteinDocker and immune-conjugate were tested in the murine B16F10 melanoma model, which was stably transfected to overexpress the model antigen. C57Bl/6JRjx mice were injected subcutaneously with 3 x 105 B16F10 cells. 3 mg/kg of the different pre-complexes with antigen binding ProteinDocker and one precomplex with a non-binder were injected intravenously on day 16 after tumor inoculations. The test items were formulated in PBS containing 10% 5x HEPES, 10% glycerol, and 0.1% DMSO. Plasma was taken 4 and 24 hours after i.v. injection to analyze systemic cytokine induction.
Figures 34 and 35 depict systemically induced IFNa, IFNp, IL-6 and IP-10. Lowest cytokine induction was observed for pre-complexes with SD18321 (PIC7) followed by PIC19. Conjugates with cleavable linkers and monoALFA conjugates exhibited higher systemic cytokine induction than bisALFA uncleavable conjugates. High systemic cytokines were analyzed as a marker of tolerability, as strong systemic cytokine induction indicates systemic immune activation and potentially reduced tolerability compared to compounds with low systemic cytokine induction. The in vivo cytokine response for the different conjugates matched with the in vitro whole blood data, where similarly PIC7 exhibited lowest background activity.
Example 15 - in vivo testing of mRNA LIMP + immune-conjugate efficacy in xenograft
Anti-tumoral efficacy of the RiboDocker mRNA LNP and immune-conjugate were tested in two models: the human ovarian cancer model OV90 (Figure 36) and the teratocarcinoma model PA-1 (Figure 37), which both inherently express the target antigen. Athymic Nude-Foxnlnu mice were injected subcutaneously with the respective tumor cell line. RiboDocker mRNA LNPs were injected intraveneously on days 11, 18, and 25 (OV90) or 16, 23, and 30 (PA-1) after tumor inoculation. SD18321 (PIC7) was administered intraveneously 24 hours after injection of the mRNA LNP. The immune-conjugates were formulated in PBS containing 10% 5x HEPES, 10% glycerol, and 0.1% DMSO. Tumor size was monitored using caliper measurement until mice reached exclusion criteria.
This study aimed at describing the anti-tumoral effect of the RiboDocker mRNA LNP and the bisALFA conjugate PIC7, which has no specified cleavage site. Significantly reduced tumor growth was seen in both models OV90 and PA-1, beyond the effect of the control groups. The efficacy was compared to a group receiving the RiboDocker only or PIC7 only. In OV90 longterm anti-tumoral effects were observed leading to a significantly increased number of survivors. Both studies demonstrated the feasibility of the mRNA LNP pre-targeting followed by immune-conjugate injection and thus confirmed efficacy observed for pre-complexes in the previous studies.
Example 16 - Solubility and Stability Results
Different peptide-immunomodulator conjugates have been tested with respect to (i) their solubility in two different buffers (PBS and HEPES) and (ii) their stability in human plasma. The results are presented in Table 10.
Table 10: Solubility and stabibilty of different peptide-immunomodulator conjugates
Payload: 803 = SD16803, 871 = SD16871; n.t.: not tested
Different solubility and stability trends resulting from structural differences can be seen from the results presented in Table 10. For example, conjugates with SD16871 as the payload appear to be somewhat more soluble in HEPES buffer and significantly more soluble in PBS than conjugates with SD16803 as the payload. Both can be formulated for administration but if a PBS buffer is desired, using SD16871 may be beneficial. All compounds show suitable stability in plasma, although there appears to be a difference in stability using SD16803 vs. SD16871. We also observe a difference in solubility depending on "complexed DAR" (i.e., in pre-complexes of (i) peptide-immunomodulator conjugate and (ii) ProteinDocker) and whether solubility enhancer is charged. Generally, a lower DAR seems to yield higher solubility, and charged solubilizers also appear to increase solubility, as seen when comparing PIC10 and PICH to other conjugates with similar structures aside from these factors.

Claims

Claims
1. A kit comprising:
(i) a compound comprising a binding moiety binding to a target antigen and a binding moiety for a tag, or a nucleic acid encoding said compound; and
(ii) a compound comprising a payload moiety and a tag to which the binding moiety for a tag binds, wherein the payload comprises a STING agonist.
2. The kit of claim 1, wherein the STING agonist has the following Formula (XX): wherein:
Ring A is selected from the group consisting of
G and Gi are independently N, CH, or C-X1-R2; or when G and Gi are each C-X1-R2, the R2 groups are optionally linked to form L2;
G' and G2 are independently N or CH;
X is N-R, O, or S;
X' is N or CH;
Xi is CH2, 0 or S;
R is hydrogen or C1-4 alkyl;
L1 and L2 are each independently C2-4 alkylene or C2-4 alkenylene;
Rz is selected from the group consisting of hydrogen, C24 cyclic ether, Ci-4 alkylene-(C2-4 cyclic ether), C3-4 cycloalkyl. CM alkylene-(C3-4 cycloalkyl), C1-4 alkyl,
o o
Ri and R3 are independently selected from the group consisting of H2N
Ring B is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 2 heteroatoms independently selected from N, O, and S; Rs is -OH or -NR9R10;
R9 and Rio are independently selected from hydrogen and C1-6 alkyl;
X2 and X3 are independently NH or S;
Yi and Y2 are independently a 5-membered heteroaryl or heterocyclic ring, wherein the 5- membered heteroaryl or heterocyclic ring (i) has 1 to 4 heteroatoms independently selected from N, O, and S, (ii) is attached to the remainder of the STING agonist via a C ring atom of the 5-membered heteroaryl or heterocyclic ring, and (iii) is optionally substituted with 1 to 4
R21, wherein each R21 is independently C1-4 alkyl (such as methyl, ethyl, propyl, or butyl);
R15< A
Rs, Rs, and R7 are independently selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, \ /w , or R5 and Rs are optionally connected to form a 5- or 6- membered heterocyclic ring;
Ris is -OH or -NR9R10; Ring C is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 ring heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 4 ring heteroatoms independently selected from N, O, and S; n, p, q, q t, and v are independently an integer from 2 to 6; and k, I, m, o, u, and w are independently an integer from 1 to 6, preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
3. The kit of claim 2, wherein the STING agonist has the following formula (XXH): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of
Formula (XXH).
4. The kit of any one of claims 1 to 3, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-18'): wherein R2' is a bond, -(CFhh-, #-(CH2)3N(CH3)-*, or #-(CH2)sN(CH3)NH-*, preferably R2' is - (CH2)3-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, more preferably R2' is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point of R2' to the remainder of the compound under (ii); and # represents the attachment point of R2' to the remainder of the STING agonist moiety.
5. The kit of any one of claims 1 to 4, wherein the compound under (ii) comprises one or more than one tag to which the binding moiety for a tag binds.
6. The kit of any one of claims 1 to 5, wherein the tag is a peptide tag.
7. The kit of any one of claims 1 to 6, wherein the compound under (ii) comprises a moiety comprising a polymer.
8. The kit of any one of claims 1 to 7 , wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer.
9. The kit of claim 7 or 8, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.
10. The kit of any one of claims 7 to 9, wherein the polymer is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
11. The kit of any one of claims 7 to 8, wherein the polymer comprises polyethylene glycol) (PEG), or poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
12. The kit of any one of claims 7 to 9, wherein the polymer comprises at least one poly- 2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
13. The kit of any one of claims 1 to 12, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
14. The kit of any one of claims 1 to 13, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glyco!) (PEG), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
15. The kit of any one of claims 1 to 14, wherein the payload moiety and the tag or tags are coupled through a moiety comprising at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
16. The kit of any one of claims 1 to 15, wherein the nucleic acid is RNA.
17. The kit of any one of claims 1 to 16, wherein the compound under (ii) comprises one tag.
18. The kit of any one of claims 1 to 17, wherein the total number of tags in the compound under (ii) is one.
19. The kit of any one of claims 1 to 18, wherein the compound under (ii) comprises one payload moiety.
20. The kit of any one claims 1 to 19, wherein the total number of payload moieties in the compound under (ii) is one.
21. The kit of any one of claims 1 to 20, wherein the compound under (ii) comprises a linking moiety connecting a tag and a payload moiety.
22. The kit of claim 21, wherein the linking moiety comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
23. The kit of any one of claims 1 to 19, wherein the compound under (ii) comprises the formula:
P-L-T wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-511), (XX-53'), (XX-73'a), or (XX-73'b);
T comprises a tag; and
L comprises a linking moiety.
24. The kit of claim 23, wherein L comprises a poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
25. The kit of claim 23 or 24, wherein L comprises the formula [AEEA]u-[L'-[AEEA]v]w, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
L' comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.
26. The kit of any one of claims 23 to 25, wherein L or L' comprises an amino acid.
27. The kit of any one of claims 23 to 26, wherein L or L' comprises the D-isomer of an amino acid.
28. The kit of any one of claims 23 to 27, wherein L or L' comprises cysteine or lysine.
29. The kit of any one of claims 23 to 28, wherein L or L' is connected to a side chain.
30. The kit of any one of claims 1 to 16, wherein the compound under (ii) comprises at least two of said tags.
31. The kit of any one of claims 1 to 16 and 30, wherein the total number of tags in the compound under (ii) is two.
32. The kit of any one of claims 1 to 16, 30 and 31, wherein the compound under (ii) comprises one or more payload moieties.
33. The kit of any one of claims 1 to 16, and 30 to 32, wherein the total number of payload moieties in the compound under (ii) is one.
34. The kit of any one of claims 1 to 16, and 30 to 32, wherein the compound under (ii) comprises two or more payload moieties.
35. The kit of any one of claims 1 to 16, 30 to 32 and 34, wherein the total number of payload moieties in the compound under (ii) is two.
36. The kit of any one of claims 1 to 16, and 30 to 35, wherein the compound under (ii) comprises payload moieties and tags in an unbranched (linear) configuration.
37. The kit of any one of claims 1 to 16, and 30 to 36, wherein the compound under (ii) comprises the formula:
P-LA-T-LB-T or P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18' j, (XX-6'), (XX-51’), (XX-53'), (XX-73'a), or (XX-73'b);
T comprises a tag;
LA comprises a linking moiety;
LB comprises a linking moiety; and
Lc comprises a linking moiety.
38. The kit of claim 37, wherein one or more of LA, LB, and Lc comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
39. The kit of claim 37 or 38, wherein LB comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
40. The kit of any one of claims 1 to 16, and 30 to 35, wherein the compound under (ii) comprises payload moieties and tags in a branched (non-linear) configuration.
41. The kit of any one of claims 1 to 16, 30 to 35 and 40, wherein the compound under (ii) comprises the formula:
[[P]m-Li]n-Bi-[L2-T]o wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-51'), (XX-53'), (XX-73'a), or (XX-73'b);
Bi comprises a branching moiety;
T comprises a tag; Li comprises a linking moiety;
L2 comprises a linking moiety; m is an integer from 1 to 4; n is an integer from 1 to 4; and o is an integer from 2 to 4; wherein the different groups [L2-T] may be identical or different, the different groups P may be identical or different, and the different groups [[P]m-Li] may be identical or different.
42. The kit of claim 41, wherein Bi comprises an amino acid or bis-amino acid.
43. The kit of claim 41 or 42, wherein Bi comprises the D-isomer of an amino acid.
44. The kit of any one of claims 41 to 43, wherein Bi comprises cysteine or lysine.
45. The kit of any one of claims 41 to 44, wherein Li comprises a 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
46. The kit of any one of claims 41 to 45, wherein L2 comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
47. The kit of any one of claims 10 to 46, wherein the number of repeating units of 2-(2- (2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
48. The kit of any one of claims 10 to 47, wherein the number of repeating units of 2-(2- (2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
49. The kit of any one of claims 10 to 48, wherein the number of repeating units of 2-(2- (2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
50. The kit of any one of claims 41 to 49, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3.
51. The kit of any one of claims 41 to 50, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2.
52. The kit of any one of claims 41 to 51, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2, n is
1 and m is 1.
53. The kit of any one of claims 41 to 51, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2, n is
2 and m is 2.
54. The kit of any one of claims 1 to 53, wherein the compound under (i) comprises one or more binding moieties for the tag.
55. The kit of any one of claims 1 to 54, wherein the compound under (i) comprises at least two binding moieties for the tag.
56. The kit of any one of claims 1 to 55, wherein the compound under (i) comprises two binding moieties for the tag.
57. The kit of any one of claims 1 to 56, wherein the tag is an ALFA-tag.
58. The kit of any one of claims 1 to 57, wherein the tag is a cyclic ALFA-tag.
59. A compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist.
60. The compound of claim 59, wherein the STING agonist has the following Formula (XX):
wherein:
Ring A is selected from the group consisting of
G and Gi are independently N, CH, or C-X1-R2; or when G and Gi are each C-X1-R2, the R2 groups are optionally linked to form L2;
G' and G2 are independently N or CH;
X is N-R, O, or S;
X' is N or CH;
Xi is CH2, O or S;
R is hydrogen or C1-4 alkyl;
L1 and L2 are each independently C2-4 alkylene or C2-4 alkenylene;
R2 is selected from the group consisting of hydrogen, C2-4 cyclic ether, C1-4 alkylene-(C2-4 cyclic ether), C3-4 cycloalkyl, CM alkylene-(C34 cycloalkyl), C1-4 alkyl. o o
Ri and R3 are independently selected from the group consisting of
Ring B is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 2 heteroatoms independently selected from N, O, and S;
Rs is -OH or -NR9R10;
Rg and Rio are independently selected from hydrogen and Ci-6 alkyl;
X2 and X3 are independently NH or S;
Yi and Y2 are independently a 5-membered heteroaryl or heterocyclic ring, wherein the 5- membered heteroaryl or heterocyclic ring (i) has 1 to 4 heteroatoms independently selected from N, O, and S, (ii) is attached to the remainder of the STING agonist via a C ring atom of the 5-membered heteroaryl or heterocyclic ring, and (iii) is optionally substituted with 1 to 4
R21, wherein each R21 is independently C1-4 alkyl (such as methyl, ethyl, propyl, or butyl);
Rs, Re, and R7 are independently selected from hydrogen, Ci-6 alkyl, C2-6 alkenyl, , , or Rs and Re are optionally connected to form a 5- or 6- membered heterocyclic ring;
Ris is -OH or -NR9R10;
Ring C is 6-membered arylene, 5- or 6-membered heteroarylene comprising 1 to 2 ring heteroatoms independently selected from N, O, and S, or 5- or 6-membered divalent heterocyclic ring comprising 1 to 4 ring heteroatoms independently selected from N, O, and
S; n, p, q, q', t, and v are independently an integer from 2 to 6 (i.e., 2, 3, 4, 5, or 6, such as 2, 3,
4, or 5, e.g., 2, 3, or 4); and k, I, m, o, u, and w are independently an integer from 1 to 6 (i.e., 1, 2, 3, 4, 5, or 6, such as 1,
2, 3, 4, or 5, e.g., 1, 2, 3, or 4), preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX).
61. The compound of claim 60, wherein the STING agonist has the following formula
(XXH): preferably the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XXH).
62. The compound of any one of claims 59 to 61, wherein the payload moiety comprises a STING agonist moiety having the following Formula (XX-18'): wherein Rz is a bond, -(CH2)3-, #-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-\ preferably R2' is - (CH2)3-, *-(CH2)3N(CH3)-*, or #-(CH2)3N(CH3)NH-*, more preferably Rz is #-(CH2)3N(CH3)NH-*, wherein * represents the attachment point of R2' to the remainder of the compound comprising a payload moiety and a tag, wherein the payload comprises a STING agonist; and * represents the attachment point of R2' to the remainder of the STING agonist moiety.
63. The compound of any one of claims 59 to 62, which comprises one or more than one tag-
64. The compound of any one of claims 59 to 63, wherein the tag is a peptide tag.
65. The compound of any one of claims 59 to 64, which comprises a moiety comprising a polymer.
66. The compound of any one of claims 59 to 65, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer.
67. The compound of claim 65 or 66, wherein the polymer is not a polymer of proteinogenic amino acids or their D-isomers.
68. The compound of any one of claims 65 to 67, wherein the polymer is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N- methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
69. The compound of any one of claims 65 to 68, wherein the polymer comprises poly(ethylene glycol) (PEG), or poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
70. The compound of any one of claims 65 to 69, wherein the polymer comprises at least one poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
71. The compound of any one of claims 59 to 70, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine)), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.
72. The compound of any one of claims 59 to 71, wherein the payload moiety and the tag or tags are coupled through a moiety comprising a polymer selected from the group consisting of polyethylene glycol) (PEG), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or a derivative thereof.
73. The compound of any one of claims 59 to 72, wherein the payload moiety and the tag or tags are coupled through a moiety comprising at least one poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
74. The compound of any one of claims 59 to 73, which comprises one tag.
75. The compound of any one of claims 59 to 74, wherein the total number of tags in the compound is one.
76. The compound of any one of claims 659to 75, which comprises one payload moiety.
77. The compound of any one of claims 59 to 76, wherein the total number of payload moieties in the compound is one.
78. The compound of any one of claims 59 to 77, which comprises a linking moiety connecting a tag and a payload moiety.
79. The compound of claim 78, wherein the linking moiety comprises a continuous or non- continuous poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
80. The compound of any one of claims 62 to 79, which comprises the formula:
P-L-T wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-51'), (XX-531), (XX-73'a), or (XX-73'b);
T comprises a tag; and
L comprises a linking moiety.
81. The compound of claim 80, wherein L comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
82. The compound of claim 80 or 81, wherein L comprises the formula [AEEA]U-[L'- [AEEA]v]w, wherein
AEEA is 2-(2-(2-aminoethoxy)ethoxy)acetic acid or a derivative thereof;
L' comprises a linking moiety; u is an integer of 2 or more; each v is an integer of 2 or more; and w is an integer from 1 to 4; wherein the different groups [L'-[AEEA]V] may be identical or different.
83. The compound of any one of claims 80 to 82, wherein L or L' comprises an amino acid.
84. The compound of any one of claims 80 to 83, wherein L or L' comprises the D-isomer of an amino acid.
85. The compound of any one of claims 80 to 84, wherein L or L' comprises cysteine or lysine.
86. The compound of any one of claims 80 to 85, wherein L or L' is connected to a side chain.
87. The compound of any one of claims 59 to 73, which comprises at least two of said tags.
88. The compound of any one of claims 59 to 73 and 87, wherein the total number of tags in the compound is two.
89. The compound of any one of claims 62 to 73, 87 and 88, which comprises one or more payload moieties.
90. The compound of any one of claims 59 to 73 and 87 to 89, wherein the total number of payload moieties in the compound is one.
91. The compound of any one of claims 59 to 73 and 87 to 89, which comprises two or more payload moieties.
92. The compound of any one of claims 59 to 73, 87 to 89 and 91, wherein the total number of payload moieties in the compound is two.
93. The compound of any one of claims 59 to 73, and 87 to 92, which comprises payload moieties and tags in an unbranched (linear) configuration.
94. The compound of any one of claims 59 to 73, and 87 to 93, which comprises the formula:
P-LA-T-LB-T or
P-LA-T-LB-T-LC-P wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-61), (XX-511), (XX-531), (XX-73'a), or (XX-73'b);
T comprises a tag;
LA comprises a linking moiety;
LB comprises a linking moiety; and Lc comprises a linking moiety.
95. The compound of claim 94, wherein one or more of LA, LB, and Lc comprises a poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
96. The compound of claim 94 or 95, wherein LB comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
97. The compound of any one of claims 59 to 73, and 87 to 92, which comprises payload moieties and tags in a branched (non-linear) configuration.
98. The compound of any one of claims 59 to 73, 87 to 92 and 97, which comprises the formula:
[[P]m-Li]n-Br[L2-T]0 wherein
P comprises a payload moiety, wherein the payload comprises a STING agonist, wherein preferably: the payload comprises the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety of the STING agonist of Formula (XX), (XXH), or (XXK), or the payload moiety comprises a STING agonist moiety having the Formula (XX- 18'), (XX-6'), (XX-511), (XX-531), (XX-73'a), or (XX-73'b);
Bi comprises a branching moiety;
T comprises a tag;
Li comprises a linking moiety;
L2 comprises a linking moiety; m is an integer from 1 to 4; n is an integer from 1 to 4; and o is an integer from 2 to 4; wherein the different groups [L2-T] may be identical or different, the different groups P may be identical or different, and the different groups [[P]m-Li] may be identical or different.
99. The compound of claim 98, wherein Bi comprises an amino acid or bis-amino acid.
100. The compound of claim 98 or 99, wherein Bi comprises the D-isomer of an amino acid.
101. The compound of any one of claims 98 to 100, wherein Bi comprises cysteine or lysine.
102. The compound of any one of claims 98 to 101, wherein Li comprises a 2-(2-(2- aminoethoxy)ethoxy)acetic acid (AEEA) moiety or a derivative thereof, or a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
103. The compound of any one of claims 98 to 102, wherein Lz comprises a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof.
103. The compound of any one of claims 68 to 103, wherein the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 30.
105. The compound of any one of claims 68 to 104, wherein the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is between 2 and 10.
106. The compound of any one of claims 68 to 105, wherein the number of repeating units of 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA) or a derivative thereof of a poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) moiety or a derivative thereof is 2, 4 or 6.
107. The compound of any one of claims 98 to 106, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 m is an integer from 1 to 3, n is an integer from 1 to 3, and o is 2 or 3.
108. The compound of any one of claims 98 to 107, wherein in formula [[P]m-Li]n-Bi-[l-2-T]0 o is 2.
109. The compound of any one of claims 98 to 108, wherein in formula [[P]m-Li]n-Bi-[L2-T]0 o is 2, n is 1 and m is 1.
110. The compound of any one of claims 98 to 108, wherein in formula [[P]m-Li]n-Bi-[L.2-T]0 o is 2, n is 2 and m is 2.
111. The compound of any one of claims 59 to 110, wherein the tag is an ALFA-tag.
112. The compound of any one of claims 59 to 111, wherein the tag is a cyclic ALFA-tag.
113. A method for treating a subject having a disease, disorder or condition characterized by cells expressing a target antigen, comprising:
(i) providing to the subject a compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag;
(ii) allowing the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag to become associated with cells expressing the target antigen; and
(iii) administering to the subject a compound of any one of claims 59 to 112 comprising one or more tags to which the binding moiety for a tag binds.
114. The method of claim 113, wherein the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is provided to the subject by administering to the subject RNA encoding a polypeptide comprising a binding moiety binding to the target antigen and a binding moiety for a tag; and allowing expression of the polypeptide by cells in the subject.
115. The method of claim 114, wherein the cells expressing the polypeptide are transfected with the RNA.
116. The method of claim 114 or 115, wherein the RNA is administered as particulate formulation such as formulated as lipid nanoparticles or lipoplex particles.
117. The method of any one of claims 114 to 116, wherein the cells expressing the polypeptide secrete the polypeptide.
118. The method of any one of claims 114 to 117, wherein the cells expressing the polypeptide express the polypeptide such that it is released into the bloodstream.
119. The method of any one of claims 113 to 118, wherein the target antigen is a cell surface antigen.
120. The method of any one of claims 113 to 119, wherein the compound comprising a binding moiety binding to the target antigen and a binding moiety for a tag is a fusion polypeptide comprising the binding moiety binding to the target antigen and the binding moiety for a tag.
121. The method of any one of 113 to 120, wherein the binding moiety binding to the target antigen comprises an antibody or an antibody derivative.
122. The method of any one of claims 113 to 121, wherein the binding moiety for a tag comprises an antibody or an antibody derivative.
123. The method of claim 121 or 122, wherein the antibody derivative is an antibody fragment.
124. The method of any one of claims 113 to 123, wherein the disease, disorder or condition is cancer.
125. The method of any one of claims 113 to 124, wherein the cells expressing a target antigen are diseased cells.
126. The method of any one of claims 113 to 125, wherein the cells expressing a target antigen are cancer cells.
127. The method of any one of 113 to 126, wherein the target antigen is a tumor antigen.
128. The method of any one of claims 113 to 127, wherein the compound under (i) comprises a single binding moiety for the tag.
129. The method of any one of claims 113 to 127, wherein the compound under (i) comprises at least two binding moieties for the tag.
130. The method of any one of claims 113 to 129, wherein the compound under (i) comprises two binding moieties for the tag.
131. The method of any one of claims 113 to 130, wherein the tag is an ALFA-tag.
132. The method of any one of claims 113 to 131, wherein the tag is a cyclic ALFA-tag.
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