DE212016000029U1 - Compositions of antibody construct agonist conjugates - Google Patents

Abstract

A conjugate comprising: a) an immunostimulatory compound; b) an antibody construct comprising an antigen-binding domain and an Fc domain, wherein i) a Kd for binding said antigen binding domain to said first antigen in the presence of said immunostimulatory compound less than about 100 nM and not greater than about 100 times a Kd for the Binding of said antigen-binding domain to said first antigen in the absence of said immunostimulatory compound; ii) a Kd for binding of said Fc domain to an Fc receptor in the presence of said immunostimulatory compound is not greater than about 100 times a Kd for binding of said Fc domain to said Fc receptor in the absence of said immunostimulatory compound; and c) a linker which attaches said antibody construct to said immunostimulatory compound, said linker being covalently linked to said immunostimulatory compound, and wherein a molar ratio of said immunostimulatory compound to said antibody construct is less than 8.

Description

CROSS REFERENCE

This application claims the benefit of US Provisional Patent Application No. 62 / 371,141, filed on Aug. 4, 2016, US Patent Application No. 15 / 173,075, filed on Jun. 3, 2016, and US Provisional Application No. 62 / 264,260, filed December 7, 2015, each of which is incorporated herein by reference in its entirety.

BACKGROUND

One of the leading causes of death in the United States is cancer. Conventional methods of cancer treatment, such as chemotherapy, surgery or radiotherapy, tend to be either highly toxic or nonspecific for a cancer, or both, resulting in limited efficacy and harmful side effects. However, the immune system has the potential to be a powerful, specific tool in the fight against cancer. In many cases, tumors can specifically express genes whose products are required for the induction or maintenance of the malignant state. These proteins can serve as antigenic markers for the development and establishment of a more specific anti-cancer immune response. The enhancement of this specific immune response has the potential to be a potent anti-cancer treatment, which may be more effective than conventional cancer treatment procedures and may have fewer side effects.

INCLUSION BY REFERENCE

All publications, patents and patent applications mentioned in this specification are incorporated herein to the same extent as if each individual publication, patent or patent application were expressly and individually indicated to be incorporated by reference.

SUMMARY

The composition described herein relates to various embodiments of a conjugate. In various embodiments, a conjugate comprises a) an immunostimulatory compound; B) an antibody construct comprising an antigen binding domain and an Fc domain, wherein the antigen binding domain binds to a first antigen, and wherein a K _d for binding the Fc domain to an Fc receptor in the presence of the immunostimulatory compound is not greater than about 100 a K _d for the binding of the Fc domain to that Fc receptor in the absence of the immunostimulatory compound; and c) a linker, wherein the linker attaches the antibody construct to that immunostimulatory compound. In some aspects, that antigen-binding domain binds that first antigen in the presence of that immunostimulatory compound. In some aspects, a K _d for attaching that antigen binding domain to that first antigen in the presence of that immunostimulatory compound is less than about 100 nM and not more than about 100 times a K _d for the binding of that antigen binding domain to that first antigen Absence of that immunostimulatory compound. In some aspects, that K _d for binding that antigen binding domain to that first antigen in the presence of that immunostimulatory compound is less than about 100 nM and not greater than about 10 times that K _{d of} the binding of that antigen binding domain to that first antigen in the absence that immunostimulatory compound; and that K _d for the binding of the Fc domain to that Fc receptor in the presence of that immunostimulatory compound is not greater than about 10 times that K _d for the binding of that Fc domain to that Fc receptor in the absence of the immunostimulatory compound , In some aspects, a molar ratio of immunostimulatory to antibody construct is less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, or less than 2.

In some aspects, the conjugate further comprises a targeting binding domain, wherein that targeting domain is attached to that antibody construct. In some aspects, that targeting binding domain binds a second antigen. In some aspects, that targeting binding domain is bound to the antibody construct at a C-terminal end of that Fc domain.

In some aspects, that antigen-binding domain is an antibody or non-antibody framework.

The conjugate of any one of claims 1 to 9, wherein the antigen-binding domain is at least 80% homologous to an antigen-binding domain from an antibody or non-antibody framework. In some aspects, that non-antibody backbone is a DARPin, Affimer, Avimer, Knottin, Monobody, or an affinity bracket. In some aspects, that antigen binding domain is at least 80% homologous to an antigen binding domain from a DARPin, Affimer, Avimer, Knottin, Monobody, or an affinity bracket.

In some aspects, that antigen-binding domain recognizes a single antigen. In some aspects, that antigen-binding domain recognizes two or more antigens. In some aspects, that first antigen is a tumor antigen. In some aspects, the first antigen is at least 80% homologous to CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC. HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate binding protein, A33, G250, prostate specific membrane antigen, ferritin, GD2, GD3, GM2, Le ^y, CA-125, CA19-9, epidermal growth factor, p185HER2, IL 2-receptor, de2-7 EGFR, fibroblast activating protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2 / neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1, PMSA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SA RT3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, Legumain, Tie3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC1, MUC15, MSLN, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, or Fos-related antigen 1. In some aspects, that first antigen is expressed on an immune cell. In some aspects, that first antigen is expressed on an antigen presenting cell. In some aspects, that first antigen is expressed on a dendritic cell, a macrophage or a B cell. In some aspects, wherein the first antigen is CD40. In some aspects, that antigen binding domain is a CD40 agonist.

In some aspects, that antibody construct is an antibody. In some aspects, that antibody construct is a human antibody or a humanized antibody. In some aspects, that antibody construct comprises a light chain sequence that is at least 80%, 90% or 100% homologous to SEQ ID NO: 4, at least 80%, 90%, or 100% homologous to SEQ ID NO: 26 or at least 80%. , 90% or 100% homologous to SEQ ID NO: 34. In some aspects, said antibody construct comprises a light chain variable domain sequence that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 6. In some aspects, said antibody construct comprises: a) a heavy chain sequence at least 80%, 90% or 100% homologous to SEQ ID NO: 15; b) a heavy chain sequence that is at least 80%, 90% or 100% homologous to SEQ ID NO: 16; b) a heavy chain sequence which is at least 80%, 90% or 100% homologous to SEQ ID NO: 17; d) a heavy chain sequence that is at least 80%, 90% or 100% homologous to SEQ ID NO: 18; e) a heavy chain sequence that is at least 80%, 90% or 100% homologous to SEQ ID NO: 22; or f) a heavy chain sequence which is at least 80%, 90% or 100% homologous to SEQ ID NO: 30. In some aspects, that antibody construct comprises a heavy chain variable domain that is at least 80%, 90%, or 100% homologous to SEQ ID NO: 20. In some aspects, that antibody binding domain comprises at least 80%, 90% or 100% homology to: a) HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 23, HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 24, an HC -CDR3 comprising an amino acid sequence of SEQ ID NO: 25, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 27, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 28 and LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 29; or b) HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 31, HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 32, an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 33, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 35, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 36 and LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 37.

In some aspects, that Fc domain is an antibody. In some aspects, that Fc domain is at least 80% homologous to an Fc domain of an antibody. In some aspects, binding of that Fc domain to that Fc receptor in the presence of that immunostimulatory compound results in Fc receptor-mediated signaling. In some aspects, binding of that Fc domain to that Fc receptor in the presence of that immunostimulatory compound results in increased antigen presentation on an immune cell. In some aspects, that Fc domain is a human Fc domain. In some aspects, that Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, and a human IgG4 Fc domain. In some aspects, where that Fc domain is an Fc domain variant comprising at least one amino acid residue change compared to a wild-type sequence of the Fc domain. In some aspects, that Fc domain binds that Fc receptor with altered affinity compared to a wild-type Fc domain. In some aspects, wherein said Fc receptor is selected from the group consisting of CD16a, CD16b, CD32a, CD32b, and CD64. In some aspects, that Fc receptor is a CD16a-F158 variant or a CD16a-V158 variant. In some aspects, that Fc domain binds that Fc receptor with a higher affinity than a wild-type Fc domain. In some aspects, that Fc receptor is selected from a group consisting of: CD16a, CD16b, CD32a, CD32b or CD64. In some aspects, that Fc receptor is a CD16a-F158 variant or a CD16a-V158 variant. In some aspects, that Fc domain has at least one amino acid residue change as compared to wild-type, wherein that at least one amino acid residue change is a) F243L, R292P, Y300L, L235V and P396L, wherein the numbering of amino acid residues in that Fc domain is according to the EU index such as done in Kabat; b) S239D and I332E, where the numbering of amino acid residues in that Fc domain follows the EU index as in Kabat; or c) S298A, E333A and K334A, where the numbering of amino acid residues in that Fc domain is according to the EU index as in Kabat.

In some aspects, that immunostimulatory compound is a molecule having a damage-associated molecular pattern or a molecule having a pathogen-associated molecular pattern. In some aspects, that immunostimulatory compound is a Toll-like receptor agonist, STING agonist or RIG I agonist. In some aspects, that immunostimulatory compound is a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 Agonist or a TLR10 agonist. In some aspects, that immunostimulatory compound is selected from a group consisting of: S-27609, CL307, UC-IV150, imiquimod, gardiquimod, resiquimod, motolimod, VTS-1463GS-9620, GSK2245035, TMX-101, TMX-201, TMX- 202, isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240, KU34B and CL663.

In some aspects, that immunostimulatory compound comprises one or more rings selected from carbocyclic and heterocyclic rings. In some aspects, that linker is covalently linked to the antibody construct. In some aspects, that linker is covalently linked to the immunostimulatory compound. In some aspects, that linker is not selected to an amino acid residue of the Fc domain from a group consisting of: 221, 222, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239 , 240, 241, 243, 244, 244, 242, 242, 242, 242, 242, 242, 243, 275, 296, 298, 290, 290, 292, 292, 290, 292, 292, 292, 292, 292, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 396, or 428, wherein the numbering of amino acid residues in the Fc domain according to the EU index as in Kabat. In some aspects, that linker is linked to an amino acid residue of the antibody construct through a THIOMAB linker or a sortase A-catalyzed linker. In some aspects, that linker is to the antibody construct via a sulfhydryl group, a primary amine, a hinge cysteine, a CL lysine, an introduced cysteine in a light chain, an introduced light chain glutamine, or an unnatural one introduced into a light chain or heavy chain Bound amino acid. In some aspects, that linker does not affect the Fc domain that binds to that Fc receptor when that linker is linked to an amino acid residue on that antibody construct. In some aspects, that linker does not interfere with Fc receptor-mediated signaling resulting from binding of that Fc domain to that Fc receptor when that linker is attached to that Fc domain at an amino acid residue. In some aspects, that linker is linked to that immunostimulatory compound via an exocyclic nitrogen or carbon atom of that immunostimulatory compound. In some aspects, that immunostimulatory compound is covalently linked to that linker by attachment to an exocyclic carbon or nitrogen atom on that immunostimulatory compound.

In some aspects, where that linker is a peptide. In some aspects, that linker is a cleavable linker. In some aspects, that linker is selected from a group consisting of: a) a valine citrulline linker; b) a valine citrulline linker containing a pentafluorophenyl group; c) a valine citrulline linker containing a succinimide group; d) a valine citrulline linker containing a para-aminobenzoic acid group; e) a valine citrulline linker containing a para-aminobenzoic acid group and a pentafluorophenyl group; and f) a valine citrulline linker containing a para-aminobenzoic acid group and a succinimide group.

In some aspects, that linker is a non-cleavable linker. In some aspects, that linker is selected from a group consisting of: a) a maleimidocaproyl linker; b) a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules; c) a maleimide-PEG4 linker; d) a maleimidocaproyl linker containing a succinimide group; e) a maleimidocaproyl linker containing a pentafluorophenyl group; f) a combination of a maleimidocaproyl linker having a Succinimide group and one or more polyethylene glycol molecules; and g) a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules.

In some aspects, that conjugate induces secretion of cytokine by an antigen presenting cell. In some aspects, that cytokine is IFN-γ, IL-8, IL-12, IL-2, or a combination thereof. In some aspects, that conjugate induces antigen presentation on an antigen presenting cell.

In some aspects, that conjugate is formulated for the treatment of tumors.

In some aspects, wherein that conjugate is in a pharmaceutical formulation.

In some embodiments, a pharmaceutical composition comprises a conjugate of any of the above embodiments and a pharmaceutically acceptable carrier.

In some embodiments, a method of preparing the conjugate of any of the foregoing embodiments comprises: a) selecting an antibody construct; b) selecting an immunostimulatory compound; and c) attaching said antibody construct to said immunostimulatory compound, wherein said immunostimulatory compound is attached to said antibody construct via a linker and said antibody construct comprises an antigen binding domain and an Fc domain, said antigen binding domain specifically binding an antigen in the presence of said immunostimulatory compound, and those Fc Domain specifically binds an Fc receptor in the presence of that immunostimulatory compound.

In some embodiments, a method of preparing the conjugate of any of the foregoing embodiments comprises: a) selecting an antibody construct; b) selecting an immunostimulatory compound; c) selecting a targeting binding domain; d) attaching that targeting-binding domain to that antibody construct; and e) attaching said antibody construct to said immunostimulatory compound, wherein said immunostimulatory compound is attached to said antibody construct via a linker, said antigen binding domain specifically binding a first antigen in the presence of said immunostimulatory compound and said targeting binding a second antigen in the presence of said immunostimulatory compound specifically binds.

In some embodiments, a method of treating a subject in need thereof, administering a therapeutic dose of the conjugate of any of the above embodiments, or the pharmaceutical composition of any of the above embodiments. In some aspects, the subject has cancer. In some aspects, that composition is administered intravenously, cutaneously, subcutaneously or injected at a site of the disease.

In some embodiments, a kit comprises that conjugate of any of the above embodiments.

A composition comprising a light chain sequence that is at least 80%, 90% or 100% homologous to SEQ ID NO: 4 and a heavy chain sequence that is at least 80%, 90% or 100%

• a) eine Sequenz einer leichten Kette, die mindestens 80%, 90% oder 100% homolog zu SEQ ID NO: 4 ist oder mindestens 80%, 90% oder 100% homolog zu SEQ ID NO: 26 ist; und
• b) eine Sequenz einer schweren Kette, die mindestens 80%, 90% oder 100% homolog zu SEQ ID NO: 16 ist, mindestens 80%, 90% oder 100% homolog zu SEQ ID NO: 17 ist oder mindestens 80%, 90% oder 100% homolog zu SEQ ID NO: 28 ist.

A composition comprising:

• a) a sequence of a light chain which is at least 80%, 90% or 100% homologous to SEQ ID NO: 4 or at least 80%, 90% or 100% homologous to SEQ ID NO: 26; and
• b) a heavy chain sequence that is at least 80%, 90% or 100% homologous to SEQ ID NO: 16, at least 80%, 90% or 100% homologous to SEQ ID NO: 17, or at least 80%, 90 % or 100% homologous to SEQ ID NO: 28.

The composition of claim 75, wherein said composition binds to an Fc receptor having a higher affinity than an antibody comprising a heavy chain sequence of SEQ ID NO: 15 or SEQ ID NO: 22.

oder ein pharmazeutisch annehmbares Salz davon, wobei:
X¹ ausgewählt ist aus -OR² und -SR²;
X² ausgewählt ist aus -OR³ und-SR³;
B¹ und B² unabhängig ausgewählt sind aus optional substituierten stickstoffhaltigen Basen;
Y ausgewählt ist aus -OR⁴, -NR⁴R⁴, und Halogen;
R¹, R², R³ und R⁴ bei jedem Auftreten unabhängig ausgewählt sind aus Wasserstoff, -C(=O)R¹⁰⁰, -C(=O)OR¹⁰⁰ und -C(=O)NR¹⁰⁰; C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, von denen jedes optional bei jedem Auftreten unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus; und C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, wobei jeder C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus in R¹, R², R³ und R⁴ optional unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰ -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_1-6-Alkyl, C_2-6-Alkenyl, und C_2-6-Alkinyl; und
R¹⁰⁰ bei jedem Auftreten unabhängig ausgewählt ist aus Wasserstoff; und C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, von denen jedes optional bei jedem Auftreten unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -CN, -NO₂, =O, =S, und Haloalkyl.In some aspects, the present disclosure provides a compound represented by the structure of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:
X ^{1 is} selected from -OR ² and -SR ² ;
X ^{2 is} selected from -OR ³ and-SR ³ ;
B ¹ and B ^{2 are} independently selected from optionally substituted nitrogenous bases;
Y is selected from -OR ⁴ , -NR ⁴ R ⁴ , and halogen;
R ¹ , R ² , R ³ and R ⁴ at each occurrence are independently selected from hydrogen, -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each of which is optionally independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle, and _3-10 -linked heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carboxylic cycle and 3- to 10-membered heterocycle in R ¹ , R ² , R ³ and R ^{4 is} optionally independently substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ -C (O) R ¹⁰⁰ , -C ( O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl; and
R ^{100 is} independently selected at each occurrence from hydrogen; and C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbo cycle, and _3-10 membered heterocycle, each of which is optionally independently substituted on each occurrence with one or more more substituents selected from halogen, -CN, -NO ₂ , = O, = S, and haloalkyl.

oder pharmazeutisch annehmbaren Salzen davon.In various embodiments, the compound of formula (I) is represented by formula (IA):

or pharmaceutically acceptable salts thereof.

oder pharmazeutisch annehmbaren Salzen davon.In various embodiments, the compound of formula (I) is represented by formula (IB):

or pharmaceutically acceptable salts thereof.

In various embodiments, B ¹ and B ^{2 are} independently selected from optionally substituted purines such as optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid and optionally substituted isoguanine. In a preferred embodiment, B ¹ and B ^{2 are} independently selected from optionally substituted adenine and optionally substituted guanine.

In some embodiments, B ¹ and B ^{2 are} independently substituted with one or more substituents, wherein the independent substituents on B ¹ and B ² on each occurrence are independently selected from halogen, = O, = S, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -OC (O) R ¹⁰⁰ , - NO ₂ , -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ and -CN; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each of which is optionally independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carbocycle and _3-10 -linked heterocycle; and C _3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C _3-10 carbocycle and 3- to 10-membered heterocycle is optionally independently substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , - SR ^100, -N (R ¹⁰⁰⁾ _2, -S (O) R ^100, -S (O) 2 R ¹⁰⁰ -C (O) R ^100, -C (O) OR ^100, -OC (O) R ^100, -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 -alkyl, C _2-6 alkenyl, and C _2-6 alkynyl. In a preferred embodiment, B ¹ and B ^{2 are} optionally independently substituted with one or more substituents, the independent substituents on B ¹ and B ^{2 being} independently selected on each occurrence from halogen, = O, = S, -OR ¹⁰⁰ , -SR ^100, -N (R ¹⁰⁰⁾ _2, -S (O) R ^100, -S (O) 2 R ^100, -C (O) R ^100, -C (O) OR ^100, -OC (O) R ¹⁰⁰ , -NO ₂ , -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN and C _1-10 -alkyl.

In various embodiments, X ^{1 is} selected from -OH and -SH. For example, X ¹ can be -OH. In various embodiments, X ^{2 is} selected from -OH and -SH. For example, X ¹ can be -OH.

In various embodiments, Y is selected from -OH, -OC _1-10 -alkyl, -NH (C _1-10 -alkyl), and -NH ₂ . For example, Y can be -OH.

In various embodiments, R ^{100 is} independently selected from hydrogen and C _1-10 alkyl at each occurrence, optionally substituted on each occurrence with one or more substituents selected from halo, -CN, -NO ₂ , = O, and = S.

oder pharmazeutisch annehmbaren Salzen davon. In einigen Ausführungsformen ist die Verbindung von Formel (IC) dargestellt durch Formel (ID):

oder pharmazeutisch annehmbaren Salzen davon.In various embodiments, the compound of formula (I) is represented by formula (IC):

or pharmaceutically acceptable salts thereof. In some embodiments, the compound of formula (IC) is represented by formula (ID):

or pharmaceutically acceptable salts thereof.

In various embodiments, the compound is a pharmaceutically acceptable salt. The compound or salt may agonize a stimulator of interferon genes (STING).

In some aspects, the present disclosure provides an antibody-drug conjugate comprising a compound or a salt as described above, an antibody and a linker group, wherein the compound or salt is linked to the antibody through the linker group. The linker group may be selected from a cleavable and a non-cleavable linker.

oder ein pharmazeutisch annehmbares Salz davon, wobei:
X¹ ausgewählt ist aus -OR² und -SR²;
X² ausgewählt ist aus -OR³ und -SR³;
B¹ und B² unabhängig ausgewählt sind aus optional substituierten stickstoffhaltigen Basen, wobei jeder optionale Substituent unabhängig ausgewählt ist aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -CN, R⁶ und -X³;
Y ausgewählt ist aus -OR⁴, -SR⁴, -NR⁴R⁴ und Halogen;
Z ausgewählt ist aus -OR⁵ -SR⁵, and -NR⁵R⁵;
R¹, R², R³ und R⁴ bei jedem Auftreten unabhängig ausgewählt sind aus einem -X³; Wasserstoff, -C(O)R¹⁰⁰, -C(=O)OR¹⁰⁰ und -C(O)NR¹⁰⁰; C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, von denen jedes optional bei jedem Auftreten unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -CO)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰ -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus; und C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, wobei jeder C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus in R¹, R², R³, R⁴ und R⁵ optional unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰ -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_1-6-Alkyl, C_2-6-Alkenyl, und C_2-6-Alkinyl;
R⁶ unabhängig ausgewählt ist aus -C(=O)R¹⁰⁰, -C(=O)OR¹⁰⁰ und -C(=O)NR¹⁰⁰; C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, von denen jedes optional bei jedem Auftreten unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus; and C_3-10 carbocycle and 3- to 10-membered heterocycle, und C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, wobei jeder C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus in R⁶ optional unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰ -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂ -OP(O)(OR¹⁰⁰)₂, -CN, C_1-6-Alkyl, C_2-6-Alkenyl, C_2-6-Alkinyl;
R¹⁰⁰ bei jedem Auftreten unabhängig ausgewählt ist aus Wasserstoff; und C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, von denen jedes optional bei jedem Auftreten unabhängig substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -CN, -NO₂, =O, =S, und Haloalkyl; und X³ eine Linkergruppe ist, wobei mindestens eines von R¹, R², R³, R⁴, R⁵, X¹, X², einem B¹-Substituent und einem B²-Substituent -X³ ist.In some aspects, the present disclosure provides a compound represented by the structure of formula (II):

or a pharmaceutically acceptable salt thereof, wherein:
X ^{1 is} selected from -OR ² and -SR ² ;
X ^{2 is} selected from -OR ³ and -SR ³ ;
B ¹ and B ^{2 are} independently selected from optionally substituted nitrogenous bases, wherein each optional substituent is independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN, R ⁶ and -X ³ ;
Y is selected from -OR ⁴ , -SR ⁴ , -NR ⁴ R ⁴ and halogen;
Z is selected from -OR ⁵ -SR ⁵ , and -NR ⁵ R ⁵ ;
R ¹ , R ² , R ³ and R ⁴ are independently selected on each occurrence from a -X ³ ; Hydrogen, -C (O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each of which is optionally independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -CO) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ -NO ₂ , = O , = S, = N (R ^100), -P (O) (OR ¹⁰⁰⁾ _2, -OP (O) (OR ¹⁰⁰⁾ _2, -CN, C _3-10 -Carbozyklus and 3- to 10-membered heterocycle ; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carboxylic cycle and 3- to 10-membered heterocycle are optionally independently substituted into R ¹ , R ² , R ³ , R ⁴ and R ⁵ is one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) ( OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl;
R ^{6 is} independently selected from -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each of which is optionally independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle, and _3-10 -linked heterocycle; and C _3-10 carbocycle and 3- to 10-membered heterocycle, and C _3-10 carbocycle and 3- to 10-membered heterocycle, wherein each C _3-10 carbocycle and 3- to 10-membered heterocycle in R ⁶ optionally independently substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ -OP (O ) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl;
R ^{100 is} independently selected at each occurrence from hydrogen; and C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbo cycle, and _3-10 membered heterocycle, each of which is optionally independently substituted on each occurrence with one or more more substituents selected from halo, -CN, -NO ₂ , = O, = S, and haloalkyl; and X ^{3 is} a linker group, wherein at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , X ¹ , X ² , a B ¹ substituent and a B ² substituent is -X ³ .

oder pharmazeutisch annehmbaren Salzen davon.In various embodiments, the compound of formula (II) is represented by formula (IIA):

or pharmaceutically acceptable salts thereof.

oder pharmazeutisch annehmbaren Salzen davon.In various embodiments, the compound of formula (II) is represented by formula (IIB):

or pharmaceutically acceptable salts thereof.

In various embodiments, B ¹ and B ^{2 are} independently selected from optionally substituted purines. Each of B ¹ and B ² may be independently selected from optionally substituted adenine, guanine and derivatives thereof. Each of B ¹ and B ² may be independently selected from optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid and optionally substituted isoguanine. In a preferred embodiment, B ¹ and B ^{2 are} independently selected from optionally substituted adenine and optionally substituted guanine.

darestellt werden und wobei B¹ optional weiter mit einem oder mehr Substituenten substituiert ist.In various embodiments, B ^{1 is} substituted with X ³ and optionally one or more additional substituents independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O ) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN, and R ⁶ . For example, B ^{1 can} by

and wherein B ^{1 is} optionally further substituted with one or more substituents.

dargestellt werden, und wobei B² optional weiter mit einem oder mehr Substituenten substituiert ist.In various embodiments, B ^{2 is} substituted with X ³ and optionally one or more additional substituents independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O ) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN, and R ⁶ . For example, B ² may be through

and wherein B ^{2 is} optionally further substituted with one or more substituents.

In various embodiments, X ^{1 is} selected from -O-X ³ and -SX ³ . In some embodiments, X ^{1 is} selected from -OH and -SH.

In various embodiments, X ^{2 is} selected from -O-X ³ and -SX ³ . In some embodiments, X ^{2 is} selected from -OH and -SH.

In various embodiments, Y is selected from -NR ⁴ X ³ , -SX ³ , and -O-X ³ . In some embodiments, Y is selected from -OH, -SH, -OC _3-10 -alkyl, -NH (C _3-10 -alkyl), and -NH ₂ .

In various embodiments, Z is selected from -NR ⁴ X ³ , -SX ³ , and -O-X ³ . In some embodiments, Z is selected from -OH, -SH, -OC _1-10 -alkyl, -NH (C _1-10 -alkyl), and -NH ₂ .

In various embodiments, -X ^{3 is} represented by the formula:

wobei RX eine reaktive Gruppe wie Maleimid umfasst.In various embodiments, -X ^{3 is} represented by the formula:

wherein RX comprises a reactive group such as maleimide.

wobei RX* eine reaktive Gruppe ist, die mit einer Gruppe auf einem Antikörper reagiert hat, um ein Antikörper-Medikamenten-Konjugat zu bilden.In various embodiments, -X ^{3 is} represented by the formula:

wherein RX * is a reactive group that has reacted with a group on an antibody to form an antibody-drug conjugate.

wobei RX eine reaktive Gruppe wie Maleimid umfasst.In various embodiments, -X ^{3 is} represented by the formula:

wherein RX comprises a reactive group such as maleimide.

wobei RX* eine reaktive Gruppe ist, die mit einer Gruppe auf einem Antikörper reagiert hat, um ein Antikörper-Medikamenten-Konjugat zu bilden.In various embodiments, -X ^{3 is} represented by the formula:

wherein RX * is a reactive group that has reacted with a group on an antibody to form an antibody-drug conjugate.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein Pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein. Die Verbindung kann durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon dargestellt sein.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the disclosure are set forth in detail in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative aspects in which the principles of the disclosure are used, and the accompanying drawings, in which:

1A Figure 1 illustrates a DNA sequence (SEQ ID NO: 1) of a light chain of a monoclonal human CD40 antibody SBT-040. Furthermore, SEQ ID NO: 1 illustrates a DNA sequence comprising a signal sequence (SEQ ID NO: 2) as in 1B and a variable domain sequence (SEQ ID NO: 3), as shown in FIG 1C shown contains.

1B a DNA sequence of a signal sequence (SEQ ID NO: 2) of a light chain of a monoclonal human CD40 antibody SBT-040 illustrated.

1C a variable domain DNA sequence (SEQ ID NO: 3) in a light chain of a human CD40 monoclonal antibody SBT-040.

2A Figure 5 illustrates an amino acid sequence (SEQ ID NO: 4) of a light chain of a monoclonal human CD40 antibody SBT-040. Furthermore, SEQ ID NO: 4 illustrates an amino acid sequence comprising a signal sequence (SEQ ID NO: 5), as in 1B and a variable domain sequence (SEQ ID NO: 6), as shown in FIG 1C shown contains.

2 B Figure 5 illustrates an amino acid sequence of a signal sequence (SEQ ID NO: 5) of a light chain of a human CD40 monoclonal antibody SBT-040.

2C Figure 4 illustrates a variable domain amino acid sequence (SEQ ID NO: 6) in a light chain of a monoclonal human CD40 antibody SBT-040.

3A FIG. 1 illustrates a wild-type IgG2 isotype IgG2 DNA sequence of a human CD40 monoclonal antibody SBT-040, which SBT-40 antibody heavy chain may also be referred to as SBT-040-G2 , Furthermore, SEQ ID NO: 7 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12), as in 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIG 3G shown contains.

3B exemplifies a wild-type IgG1 heavy chain DNA sequence (SEQ ID NO: 8) of a monoclonal human CD40 antibody SBT-040, which SBT-40 antibody heavy chain can also be referred to as SBT-040-G1WT , Furthermore, SEQ ID NO: 8 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12), as in 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIG 3G shown contains.

3C A DNA sequence (SEQ ID NO: 9) of IgG1 isotype of a monoclonal human CD40 antibody SBT-040 illustrating DNA nucleotide modifications according to amino acid residue modifications L235V, F243L, R292P, Y300L and P396L of a wild-type IgG1 Fc Domain, and this SBT-40 heavy chain heavy chain can also be referred to as SBT-040-G1VLPLL. The modified DNA nucleotides corresponding to the amino acid residue modifications L235V, F243L, R292P, Y300L and P396L are greased. Furthermore, SEQ ID NO: 9 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as in 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIG 3G shown contains.

3D a DNA sequence (SEQ ID NO: 10) of an isotype IgG1 of a monoclonal human CD40 antibody SBT-040 which contains DNA nucleotide modifications corresponding to amino acid residue modifications S239D and 1332E of a wild-type IgG1 Fc domain, said heavy Chain of SBT-40-Anitkörpers can also be referred to as SBT-040-G1DE. The modified DNA nucleotides corresponding to the amino acid residue modifications S239D and I332E are greased. Furthermore, SEQ ID NO: 10 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as described in U.S. Pat 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIG 3G shown contains.

3E a DNA sequence (SEQ ID NO: 11) of an isotype IgG1 of a monoclonal human CD40 antibody SBT-040 which contains DNA nucleotide modifications corresponding to amino acid residue modifications S298A, E333A and K334A of a wild-type IgG1 Fc domain, wherein This heavy chain of the SBT-40 body can also be referred to as SBT-040-G1AAA. The modified DNA nucleotides corresponding to the amino acid residue modifications S298A, E333A and K334A are greased. Furthermore, SEQ ID NO: 11 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as described in U.S. Pat 3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIG 3G shown contains.

3F a DNA sequence of a signal sequence (SEQ ID NO: 12) of a heavy chain of a monoclonal human CD40 antibody SBT-040 is illustrated.

3G a variable domain DNA sequence (SEQ ID NO: 13) in a heavy chain of a human CD40 monoclonal antibody SBT-040.

4A Figure 1 illustrates a wild-type IgG2 isotype IgG2 DNA sequence of a human CD40 monoclonal antibody SBT-040, which SBT-40 antibody heavy chain may also be referred to as SBT-040-G2 , Furthermore, SEQ ID NO: 14 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 19) as described in U.S. Pat 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIG 4G shown contains.

4B a wild-type IgG1 isotype IgG1 human sequence of a human CD40 monoclonal antibody SBT-040, which SBT-40 antibody heavy chain can also be referred to as SBT-040-G1WT , Furthermore, SEQ ID NO: 15 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 19) as described in U.S. Pat 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIG 4G shown contains.

4C A DNA sequence (SEQ ID NO: 16) of IgG1 isotype of a monoclonal human CD40 antibody SBT-040 illustrating DNA nucleotide modifications according to amino acid residue modifications L235V, F243L, R292P, Y300L and P396L of a wild-type IgG1 Fc. Domain, and this SBT-40 heavy chain heavy chain can also be referred to as SBT-040-G1VLPLL. The modified DNA nucleotides corresponding to the amino acid residue modifications L235V, F243L, R292P, Y300L and P396L are greased. Furthermore, SEQ ID NO: 16 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 19) as described in U.S. Pat 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIG 4G shown contains.

4D a DNA sequence (SEQ ID NO: 17) of an isotype IgG1 isotype of a monoclonal human CD40 antibody SBT-040 illustrating the DNA nucleotide modifications Amino acid residue modifications S239D and I332E of a wild-type IgG1 Fc domain, which SBT-40 antibody heavy chain can also be referred to as SBT-040-G1DE. The modified DNA nucleotides corresponding to the amino acid residue modifications S239D and I332E are greased. Furthermore, SEQ ID NO: 17 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 19) as described in U.S. Pat 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIG 4G shown contains.

4E a DNA sequence (SEQ ID NO: 18) of an isotype IgG1 of a monoclonal human CD40 antibody SBT-040 which contains DNA nucleotide modifications corresponding to amino acid residue modifications S298A, E333A and K334A of a wild-type IgG1 Fc domain, wherein This heavy chain of the SBT-40 body can also be referred to as SBT-040-G1AAA. The modified DNA nucleotides corresponding to the amino acid residue modifications S298A, E333A and K334A are greased. Furthermore, SEQ ID NO: 18 illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 19) as described in U.S. Pat 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIG 4G shown contains.

4F a DNA sequence of a signal sequence (SEQ ID NO: 19) of a heavy chain of a monoclonal human CD40 antibody SBT-040 is illustrated.

4G a variable domain DNA sequence (SEQ ID NO: 20) in a heavy chain of a monoclonal human CD40 antibody SBT-040.

5A . 5B & 5C a CLUSTAL O (1.2.1) multiple DNA sequence alignment of the DNA sequences of SBT-040-G1VLPLL (SEQ ID NO: 9), SBT-040-G1AAA (SEQ ID NO: 11), SBT-040-G1WT (SEQ ID NO: 8) and SBT-040-G1DE (SEQ ID NO: 10). The SBT-040 G1VLPLL sequence is an IgG1 monoclonal human CD40 antibody heavy chain DNA sequence containing DNA nucleotide modifications corresponding to L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications of a wild-type IgG1 Fc domain , The modified DNA nucleotides corresponding to the amino acid residue modifications L235V, F243L, R292P, Y300L and P396L are greased. The SBT-040-G1AAA sequence is an IgG1 isotype heavy chain DNA sequence of a monoclonal human CD40 antibody containing DNA nucleotide modifications corresponding to amino acid residue modifications S298A, E333A and K334A of a wild-type IgG1 Fc domain. The modified DNA nucleotides corresponding to the amino acid residue modifications S298A, E333A and K334A are outlined. The SBT-040 G1WT sequence is an IgG1 isotype heavy chain DNA sequence of a monoclonal human CD40 antibody. The SBT-040-G1AAA sequence is an IgG1 isotype heavy chain DNA sequence of a monoclonal human CD40 antibody containing DNA nucleotide modifications corresponding to amino acid residue modifications S239D and I332E of a wild-type IgG1 Fc domain. The modified DNA nucleotides corresponding to the amino acid residue modifications S239D and I332E are in italics. 5A shows the beginning of the sequence alignment. 5B shows the middle of the sequence alignment as a continuation of 5A , 5c shows the end of the sequence alignment as a continuation of 5C ,

6 a CLUSTAL O (1.2.1) multiple DNA sequence alignment of the DNA sequences of SBT-040-G1VLPLL (SEQ ID NO: 16), SBT-040-G1AAA (SEQ ID NO: 18), SBT-040-G1WT (SEQ ID NO: 15) and SBT-040-G1DE (SEQ ID NO: 17). The SBT-040 G1VLPLL sequence is an IgG1 monoclonal human CD40 antibody heavy chain DNA sequence containing DNA nucleotide modifications corresponding to L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications of a wild-type IgG1 Fc domain , The modified DNA nucleotides corresponding to the amino acid residue modifications L235V, F243L, R292P, Y300L and P396L are greased. The SBT-040-G1AAA sequence is an IgG1 isotype heavy chain DNA sequence of a monoclonal human CD40 antibody containing DNA nucleotide modifications corresponding to amino acid residue modifications S298A, E333A and K334A of a wild-type IgG1 Fc domain. The modified DNA nucleotides corresponding to the amino acid residue modifications S298A, E333A and K334A are in italics. The SBT-040 G1WT sequence is an IgG1 isotype heavy chain DNA sequence of a monoclonal human CD40 antibody. The SBT-040-G1AAA sequence is an IgG1 isotype heavy chain DNA sequence of a monoclonal human CD40 antibody containing DNA nucleotide modifications corresponding to amino acid residue modifications S239D and I332E of a wild-type IgG1 Fc domain. The modified DNA nucleotides corresponding to the amino acid residue modifications S239D and I332E are in italics. In addition, the hinge region of each amino acid sequence is separated from the other regions of the amino acid sequence by parentheses. The left bracket indicates the top of the hinge region (UH). The four residues between the staples are the middle region of the hinge region. The left bracket indicates the lower portion of the hinge region (LH).

7 a scheme of an antibody is illustrated. The antibody includes two heavy chains, gelled in gray, and two light chains, shown in light gray. A region of heavy chains contains Fc domains ( 705 and 720 ). An antibody contains two antigen-binding sites ( 710 and 715 ).

8th a scheme of an exemplary conjugate is illustrated. An antibody construct is an antibody containing two heavy chains as shown in gray and two light chains as shown in light gray. The antibody contains two antigen-binding sites ( 810 and 815 ), and a region of heavy chains contains Fc domains ( 805 and 820 ). The immunostimulatory compounds ( 830 and 840 ) are linked to the antibody by linker ( 860 and 870 ) conjugated.

9 a scheme of an exemplary conjugate is illustrated. An antibody construct is an antibody containing two heavy chains as shown in gray and two light chains as shown in light gray. The antibody contains two antigen-binding sites ( 910 and 915 ), and a region of heavy chains contains Fc domains ( 905 and 920 ). The immunostimulatory compounds ( 930 and 940 ) are linked to the antibody by linker ( 960 and 970 ) conjugated. Targeting binding domains are conjugated to the antibody ( 980 and 985 ).

10 a scheme of an exemplary conjugate is illustrated. An antibody construct is an antibody containing two heavy chains as shown in gray and two scaffolds as shown in light gray. The antibody construct contains two antigen-binding sites ( 1010 and 1015 ), and a region of heavy chains contains Fc domains ( 1005 and 1020 ). The immunostimulatory compounds ( 1030 and 1040 ) are linked to the antibody construct by linkers ( 1060 and 1070 ) conjugated.

11 a scheme of an exemplary conjugate is illustrated. An antibody construct is an antibody containing two heavy chains as shown in gray and two scaffolds as shown in light gray. The antibody construct contains two antigen-binding sites ( 1110 and 1115 ), and a region of heavy chains contains Fc domains ( 1105 and 1120 ). The immunostimulatory compounds ( 1130 and 1140 ) are linked to the antibody construct by linkers ( 1,160 and 1170 ) conjugated. Targeting binding domains are conjugated to the antibody construct ( 1180 and 1185 ).

12 a scheme of an exemplary conjugate is illustrated. An antibody construct contains the F (ab ') ₂ region of an antibody with heavy chains shown in dark gray and light chains shown in light gray. The antibody construct contains two antigen-binding sites ( 1210 and 1215 ) and a region of two scaffolds contains Fc domains ( 1205 and 1220 ). The immunostimulatory compounds ( 1230 and 1240 ) are linked to the antibody construct by linkers ( 1260 and 1270 ) conjugated.

13 a scheme of an exemplary conjugate is illustrated. An antibody construct contains the F (ab ') ₂ region of an antibody with heavy chains shown in dark gray and light chains shown in light gray. The antibody construct contains two antigen-binding sites ( 1310 and 1315 ) and a region of two scaffolds contains Fc domains ( 1305 and 1320 ). The immunostimulatory compounds ( 1330 and 1340 ) are linked to the antibody construct by linkers ( 1360 and 1370 ) conjugated. Targeting binding domains are conjugated to the antibody construct ( 1380 and 1385 ).

14 a scheme of an exemplary conjugate is illustrated. An antibody construct contains two scaffolds, as shown in light gray, and two scaffolds, as shown in dark gray. The antibody construct contains two antigen-binding sites ( 1410 and 1415 ), and a region of the two dark gray frameworks contains Fc domains ( 1405 and 1420 ). The immunostimulatory compounds ( 1430 and 1440 ) are linked to the antibody construct by linkers ( 1460 and 1470 ) conjugated.

15 a scheme of an exemplary conjugate is illustrated. An antibody construct contains two scaffolds, as shown in light gray, and two scaffolds, as shown in dark gray. The antibody construct contains two antigen-binding sites ( 1510 and 1515 ), and a region of the two dark gray frameworks contains Fc domains ( 1505 and 1520 ). The immunostimulatory compounds ( 1530 and 1540 ) are linked to the antibody construct by linkers ( 1560 and 1570 ) conjugated. Targeting binding domains are conjugated to the antibody construct ( 1580 and 1585 ).

16 is the two-dimensional structure of the heavy chain of Dacetuzumab.

17 is the two-dimensional structure of the light chain of Dacetuzumab.

18 is the two-dimensional structure of bleselumab heavy chain.

19 is the two-dimensional structure of Bleselumab light chain.

20 is the two-dimensional structure of the lucatumumab heavy chain.

21 is the two-dimensional structure of the lucatumumab light chain.

22 is the two-dimensional structure of ADC-1013 heavy chain.

23 is the two-dimensional structure of the light chain of ADC-1013.

24 is the two-dimensional structure of the humanized rabbit antibody heavy chain APX005.

25 is the two-dimensional structure of the humanized rabbit antibody light chain APX005.

26 the two-dimensional structure of the heavy chain of Chi praise is 7/4.

27 the two-dimensional structure of the light chain of Chi is praise 7/4.

28 HPLC analysis of SBT-040-G1WT conjugated to a Cys-directed drug linker compound shows.

29 HPLC analysis of SBT-040-G1WT conjugated to ATAC-2 shows.

30 HPLC analysis of SBT-040-G2WT conjugated to ATAC-2 shows.

31A shows the concentration of IL-12p70 produced by dendritic cells (DCs) from donor 358 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 compared to SBT-050-WT.

31B shows the concentration of IL-12p70 produced by DCs from donor 363 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 compared to SBT-050-WT.

31C shows the concentration of TNFα produced by DCs from donor 358 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 compared to SBT-050-WT.

31D shows the concentration of TNFα produced by DCs from donor 363 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 compared to SBT-050-WT.

32A the concentration of DC-produced IL-12p70 after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-040-AAA- ATAC22, SBT-040 VLPLL ATAC22, SBT040 WT ATAC1, SBT040 G2 ATAC1, SBT040 WT ATAC12, SBT040 G2 ATAC12, SBT040 WT ATAC30, SBT-040-G1AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC23, and SBT-040-AAA-ATAC23 compared to SBT-050-G2 or CD40 ligand shows.

32B the concentration of IL-6 produced by DCs from donor 2 after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-040 AAA ATAC22, SBT040 VLPLL ATAC22, SBT040 WT ATAC1, SBT040 G2 ATAC1, SBT040 WT ATAC12, SBT040 G2 ATAC12, SBT040 WT -ATAC30, SBT-040-G1AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC23, and SBT-040-AAA- ATAC23 compared to SBT-050-G2 or CD40 ligand shows. Results are given for the immunostimulatory cytokines IL-12p70 and IL-6.

33A a dose-dependent increase in CD86 expression on dendritic cells after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040-VLPLL-ATAC22, SBT-040-AAA-ATAC23 compared to treatment SBT-050-WT or staining with isotype control shows.

33B a dose-dependent increase in CD83 expression on dendritic cells after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040-VLPLL-ATAC22, SBT-040-AAA-ATAC23 compared to treatment SBT-050-WT or staining with isotype control shows.

33C demonstrated a dose-dependent increase in MHC class II expression on dendritic cells after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040-VLPLL-ATAC22, SBT-040-AAA-ATAC23 compared to treatment Control SBT-050-WT or isotype control staining shows.

DETAILED DESCRIPTION

Additional aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, which illustrates and describes illustrative aspects of the present disclosure. As will be appreciated, the present disclosure is capable of other and various aspects, and its various details are capable of modifications in various points, all without departing from the disclosure. Accordingly, the drawings and description are to be considered as illustrative in nature and not as restrictive.

Cancer is one of the leading causes of death in the United States. Conventional methods of cancer treatment such as chemotherapy, surgery or radiotherapy may be limited in their effectiveness as they are often non-specific to the cancer. In many cases, however, tumors can specifically express genes whose products are required for the induction or maintenance of the malignant condition. These proteins can serve as antigenic markers for the development and establishment of efficient anti-cancer treatments.

As used herein, "homologous" or "homology" may refer to the similarity between one DNA, RNA, nucleotide, amino acid or protein sequence to another DNA, RNA, nucleotide, amino acid or protein sequence. The homology can be expressed in terms of a percentage of the sequence identity of a first sequence to a second sequence. The percent (%) sequence identity relative to a reference DNA sequence may be the percentage of DNA nucleotides in a candidate sequence that are identical to the DNA nucleotides in the reference DNA sequence after the sequences have been aligned. Percent (%) sequence identity to a reference amino acid sequence can be the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference amino acid sequence, after aligning the sequences and optionally introducing gaps to achieve the maximum percent sequence identity. and no conservative substitutions were considered as part of sequence identity.

As used herein, the term "antibody" may refer to an immunoglobulin molecule that specifically binds to a specific antigen or is immunologically reactive. Antibodies may, for. Polyclonal, monoclonal, genetically engineered and antigen binding fragments thereof. An antibody may be, for example, murine, chimeric, humanized, heteroconjugated, bispecific, diabody, tri-body or tetrabody. The antigen-binding fragment may include, for example, Fab ', F (ab') ₂ , Fab, Fv, rIgG and scFv.

As used herein, "recognize" may refer to association or binding between an antigen-binding domain and an antigen.

As used herein, a "tumor antigen" can be an antigenic substance associated with a tumor or cancer cell and elicit an immune response in a host.

As used herein, an "antibody construct" may refer to a construct containing an antigen-binding domain and an Fc domain.

As used herein, an "antigen binding domain" can refer to an antigen binding domain from a non-antibody capable of binding the antigen.

As used herein, an "Fc domain" may be an Fc domain of an antibody or a non-antibody capable of binding to an Fc receptor.

As used herein, a "target binding domain" may refer to a construct comprising an antigen binding domain from an antibody or from a non-antibody capable of binding to the antigen.

The term "salt" or "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counterions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Organic acids from which salts can be derived are, for. Acetic, propionic, glycolic, pyruvic, oxalic, maleic, malonic, succinic, fumaric, tartaric, citric, benzoic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, salicylic and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins and the like, especially such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine , In some embodiments, the pharmaceutically acceptable base addition salt is selected from ammonium, potassium, sodium, calcium, and magnesium salts.

The term "C _xy ", when used in conjunction with a chemical group such as alkyl, alkenyl or alkynyl, is intended to include groups containing from x to y carbons in the chain. For example, the term "C _xy alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight chain alkyl and branched chain alkyl groups containing from x to y carbons in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc ,

The terms "C-alkenyl" and "C _xy alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups, which are analogous to the above-described alkyl groups in length and possible substitution, but which contain at least one double or triple bond.

The term "carbocycle" as used herein refers to a saturated, unsaturated or aromatic ring in which each atom of the ring is carbon. Carbocycle includes 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g. Phenyl, to a saturated or unsaturated ring, e.g. As cyclohexane, cyclopentane or cyclohexene, be condensed. Any combination of saturated, unsaturated, and aromatic bicyclic rings, as far as valence permits, is included in the definition of carbocyclic. Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl and naphthyl.

The term "heterocycle" as used herein refers to a saturated, unsaturated or aromatic ring comprising one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic heterocycle may be selected from saturated, unsaturated and aromatic rings wherein at least one of the rings contains a heteroatom. In an exemplary embodiment, an aromatic ring, e.g. Pyridyl, to a saturated or unsaturated ring, e.g. As cyclohexane, cyclopentane, morpholine, piperidine or cyclohexene, be condensed. The term "heteroaryl" includes single aromatic ring structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures comprise at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, wherein at least one of the rings is heteroaromatic, e.g. For example, the other cyclic rings may be aromatic or non-aromatic, carbocyclic or heterocyclic. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like.

The term "substituted" refers to radicals having substituents which contain a hydrogen on one or more carbons or substitutable heteroatoms, e.g. NH, replace the structure. It goes without saying that "substitution" or "substituted with" implies that such substitution matches the allowable valency of the substituted atom and substituent and that the substitution results in a stable compound, ie, a compound that does not undergo spontaneous conversion, such as z. In certain embodiments, substituted on radicals having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group, for example, by rearrangement, cyclization, elimination, etc. As used herein, it is contemplated that the term "substituted" includes all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents for suitable organic compounds may be one or more and the same or different. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valencies of the heteroatoms.

In some embodiments, substituents may include any substituents described herein, for example: halo, hydroxy, oxo (= O), thioxo (= S), cyano (-CN), nitro (-NO ₂ ), imino (= NH), oximo (= N-OH), hydrazino (= N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC (O) -R ^a , -R ^b -OC (O) -OR ^a , -R ^b -OC (O) -N (R ^a ) ₂ , -R ^b -N (R ^a ) ₂ , -R ^b -C (O) R ^a , -R ^b -C (O) OR ^a , -R ^b - C (O) N (R ^a ) ₂ , -R ^b -OR ^c -C (O) N (R ^a ) ₂ , -R ^b -N (R ^a ) C (O) OR ^a , -R ^b -N (R ^a ) C (O) R ^a , -R ^b -N (R ^a ) S (O) _t R ^a (where t is 1 or 2), -R ^b -S (O) _t R ^a (where t 1 or 2), -R ^b -S (O) _t OR ^a (where t is 1 or 2), and -R ^b -S (O) _t N (R ^a ) ₂ (where t is 1 or 2) ; and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl, each of which may optionally be substituted with alkyl, alkenyl, alkynyl, halo, haloalkyl, haloalkenyl, haloalkynyl, Oxo (= O), thioxo (= S), cyano (-CN), nitro (-NO ₂ ), imino (= NH), oximo (= N-OH), hydrazine (= N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC (O) -R ^a , -R ^b -OC (O) -OR ^a , -R ^b -OC (O) -N (R ^a ) ₂ , -R ^b -N (R ^a ) ₂ , -R ^b -C (O) R ^a , -R ^b -C (O) OR ^a , -R ^b -C (O) N (R ^a ) ₂ , -R ^b -OR ^c - C (O) N (R ^a ) ₂ , -R ^b -N (R ^a ) C (O) OR ^a , -R ^b -N (R ^a ) C (O) R ^a , -R ^b -N (R ^a ) S (O) _t R ^a (wherein t is 1 or 2), -R ^b -S (O) _t R ^a (wherein t is 1 or 2), -R ^b -S (O) _t OR ^a ( where t is 1 or 2) and -R ^b -S (O) _t N (R ^a ) ₂ (where t is 1 or 2); wherein each R ^{a is} independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, where each R ^a , as far as the valence allows, may be optionally substituted with alkyl, alkenyl, alkynyl, Halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (= O), thioxo (= S), cyano (-CN), nitro (-NO ₂ ), imino (= NH), oximo (= N-OH), hydrazine (= N-NH ₂ ), -R ^b -OR ^a , -R ^b -OC (O) -R ^a , -R ^b -OC (O) -OR ^a , -R ^b -OC (O) -N (R ^a ) _2, -R ^b N (R ^a) _2, -R ^b -C (O) R ^a, R ^b -C (O) OR ^a, -R ^b -C (O) N (R ^a) ₂ , -R ^b -OR ^c -C (O) N (R ^a ) ₂ , -R ^b -N (R ^a ) C (O) OR ^a , -R ^b -N (R ^a ) C (O) R ^a , -R ^b -N (R ^a ) S (O) _t R ^a (wherein t is 1 or 2), -R ^b -S (O) _t R ^a (wherein t is 1 or 2), -R ^b - S (O) _t OR ^a (where t is 1 or 2) and -R ^b -S (O) _t N (R ^a ) ₂ (where t is 1 or 2); and wherein each R ^{b is} independently selected from a direct bond or a straight or branched alkylene, alkenylene or alkynylene chain, and each R ^{c is} a straight or branched alkylene, alkenylene or alkynylene chain.

It will be understood by those skilled in the art that substituents themselves may optionally be substituted. Unless expressly designated "unsubstituted", references to chemical entities herein refer to substituted variants. For example, reference to a "heteroaryl" group or moiety implicitly includes both substituted and unsubstituted variants.

Chemical units with carbon-carbon double bonds or carbon-nitrogen double bonds may be in Z- or E-form (or cis or trans form). In addition, some chemical entities may exist in different tautomeric forms. Unless otherwise indicated, chemical entities described herein are also intended to include all Z, E, and tautomeric forms.

A "tautomer" refers to a molecule in which a proton shift from one atom of one molecule to another atom of the same molecule is possible. The compounds shown herein are in certain embodiments as tautomers. In circumstances where tautomerization is possible, there will be a chemical equilibrium of the tautomers. The exact ratio of tautomers depends on several factors, including physical state, temperature, solvent and pH. Some examples of tautomeric equilibrium include:

The compounds disclosed herein are used in various enriched isotopic forms in some embodiments, e.g. B. enriched in the content of ² H, ³ H, ¹¹ C, ¹³ C and / or ¹⁴ C. In a particular embodiment, the compound is deuterated in at least one position. Such deuterated forms can be prepared by the method described in U.S. Pat U.S. Patent No. 5,846,514 and 6,334,997 is described. As in the U.S. Patent No. 5,846,514 and 6,334,997 deuteration may improve metabolic stability and efficacy, thereby increasing the duration of action of drugs.

Unless otherwise indicated, the structures illustrated herein are meant to include compounds that differ only in the presence of one or more isotopically-enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by deuterium or tritium or the replacement of a carbon with ¹³ C- or ¹⁴ C-enriched carbon are within the scope of the present disclosure.

The compounds of the present disclosure optionally contain unnatural levels of atomic isotopes on one or more atoms making up such compounds. For example, the compounds with isotopes such as deuterium ⁽² H), tritium ⁽³ H), iodine-125 ^(125I) or carbon-14 ⁽¹⁴ C) be selected. Isotopic substitution with ² H, ¹¹ C, ¹³ C, ¹⁴ C, ¹⁵ C, ¹² N, ¹³ N, ¹⁵ N, ¹⁶ N, ¹⁶ O, ¹⁷ O, ¹⁴ F, ¹⁵ F, ¹⁶ F, ¹⁷ F, ¹⁸ F, ³³ S, ³⁴ S, ³⁵ S, ³⁶ S, ³⁵ Cl, ³⁷ Cl, ⁷⁹ Br, ⁸¹ Br, ¹²⁵ I are all considered. All isotopic variations of the compounds of the present invention, whether radioactive or otherwise, are included within the scope of the present invention.

In certain embodiments, in the compounds disclosed herein, some or all of the ¹ H atoms are replaced by ² H atoms. Synthetic methods for deuterium-containing compounds are known in the art and include, by way of non-limiting example only, the following synthetic methods. Deuterium-substituted compounds are synthesized using various methods as described in:
Dean, Dennis C .; Editor. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [In: Curr. Pharm. Des., 2000; 6 (10)] 2000, 110 ff ; George W .; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45 (21), 6601-21 ; and Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64 (1-2), 9-32 ,

Deuterated starting materials are readily available and are subjected to the synthetic procedures described herein to provide the synthesis of deuterium containing compounds. A large number of deuterium containing reagents and building blocks are commercially available from chemical manufacturers such as Aldrich Chemical Co.

Compounds of the present invention also include crystalline and amorphous forms of these compounds, pharmaceutically acceptable salts and active metabolites of these compounds with the same type of activity including, for example, polymorphs, pseudopolymorphs, solvates, hydrates, non-solvated polymorphs (including anhydrates), conformational polymorphs and amorphous forms the compounds and mixtures thereof.

The terms "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The term "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without undue toxicity, as part of a well-established medical judgment, Irritation, allergic reaction or other problems or complications, correspond to a reasonable benefit / risk ratio.

The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material , Each carrier must be "acceptable" in terms of compatibility with the other ingredients of the formulation and not harmful to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

"Antibody-drug conjugate" ("ADC") may refer to a conjugate of antibody construct and immunostimulating compound. An ADC may include any embodiment as described herein for a conjugate of antibody construct and immunostimulating compound. Therefore, ADC and composite antibody construct-immunostimulant compound conjugate can be used interchangeably herein.

An antigen can cause an immune response. An antigen can be a protein, polysaccharide, lipid or glycolipid that can be recognized by an immune cell such as a T cell or a B cell. Exposure of immune cells to one or more of these antigens can cause a rapid cell division and differentiation reaction leading to the formation of clones of the exposed T cells and B cells. B cells can differentiate into plasma cells, which in turn can produce antibodies that selectively bind to the antigens.

In cancer, there are four general groups of tumor antigens: (i) viral tumor antigens that may be identical for each virus tumor of this type, (ii) carcinogenic tumor antigens that may be specific to patients and tumors, (iii) transplant type isoantigens or tumor-specific transplantation antigens that may differ in all individual tumor types but may be the same for different tumors caused by the same virus; and (iv) embryonic antigens.

As a result of the discovery of tumor antigens, tumor antigens have become important in the development of new cancer treatments that can specifically target the cancer. This has led to the development of antibodies directed against these tumor antigens.

In addition to the development of antibodies to tumor antigens for the treatment of cancer, antibodies directed to immune cells have also been developed to enhance the immune response. For example, an anti-CD40 antibody, which is a CD40 agonist, can be used to activate dendritic cells to enhance the immune response.

Cluster of differentiation 40 (CD40) is a member of the Tumor Necrosis Factor Receptor (TNF-R) family. CD40 can be a 50 kDa cell surface glycoprotein found in normal cells such as monocytes, macrophages, B lymphocytes, dendritic cells, endothelial cells, smooth muscle cells, fibroblasts and epithelium, and in tumor cells, including B cell lymphomas and many species of solid tumors, constitutively expressed. The expression of CD40 can be increased in antigen-presenting cells in response to IL-1βp, IFN-γ, GM-CSF, and LPS-induced signaling events.

Humoral and cellular immune responses can be partially regulated by CD40. For example, in the absence of CD40 activation by its corresponding binding partner CD40 ligand (CD40L), antigen presentation may lead to tolerance. However, CD40 activation can improve tolerance. In addition, CD40 activation may positively affect the immune response by improving antigen presentation by antigen presenting cells (APC), increasing cytokine and chemokine secretion, stimulating expression and signaling by costimulatory molecules, and activating the cytolytic activity of various immune cell types. Accordingly, the interaction between CD40 and CD40L may be essential to maintain correct humoral and cellular immune responses.

The intracellular effects of the CD40 and CD40L interaction may involve the association of the cytoplasmic domain of Cd40 with TRAFs (TNF-R-associated factors), which may lead to the activation of NFκB and Jun / AP1 signaling pathways. While the response to activation of NFκB and Jun / AP1 signaling pathways may be cell type specific, such activation often results in increased production and secretion of cytokines, including IL-6, IL-8, IL-12, IL-15; increased production and secretion of chemokines, including MIP1α and β and RANTES; and increased expression of cellular adhesion molecules, including ICAM. While the effects of cytokines, chemokines, and cellular adhesion molecules may be widespread, such effects may include enhanced survival and activation of T cells.

In addition to the enhanced immune responses induced by CD40 activation, CD40 activation may also be involved in chemokine and cytokine-mediated cellular migration and differentiation; Activation of immune cells, including monocytes; Activation and increased cytolytic activity of immune cells, including cytolytic T lymphocytes and natural killer cells; Induction of CD40-positive tumor cell apoptosis and increased immunogenicity of CD40-positive tumors may be involved. In addition, CD40 can initiate and enhance immune responses through many different mechanisms, including induction of maturation of antigen-presenting cells and increased expression of costimulatory molecules, enhancement of cytokine production and secretion, and enhancement of effector functions.

CD40 activation may be effective in inducing immune mediated anti-tumor responses. For example, CD40 activation reverses host immune tolerance to tumor-specific antigens, resulting in improved antitumor responses by T cells. Such antitumor activity may also occur in the absence of immune cells. Similarly, anti-tumor effects may occur in response to anti-CD40 antibody-mediated activation of CD40 and may be independent of antibody-dependent cellular cytotoxicity. In addition to other CD40-mediated mechanisms of anti-tumor effects, CD40L stimulation may cause the maturation and stimulation of dendritic cells. CD40L-stimulated dendritic cells can contribute to the anti-tumor response. In addition, vaccination strategies that include CD40 may lead to regression of CD40 + and CD40 + + tumors.

CD40-activating antibodies (eg, activating monoclonal anti-CD40 antibodies) may be useful in the treatment of tumors. This can occur by one or more mechanisms, including cell activation, antigen presentation, production of cytokines and chemokines, among others. For example, CD40 antibodies activate dendritic cells, resulting in processing and presentation of tumor antigens as well as improved immunogenicity of CD40-positive tumor cells. Improved immunogenicity may not only result in the activation of CD40-positive tumor-specific CD4 ⁺ and CD8 ⁺ T cells, but further antitumor activity may also include monocyte recruitment and activation, enhanced cytolytic cytotoxic lymphocyte and natural killer cell cytolytic activity, and induction of Apoptosis or by stimulation of a humoral response, so that it is aimed directly at tumor cells. In addition, tumor cell debris, including tumor-specific antigens, can be presented to other cells of the immune system by CD40-activated antigen-presenting cells.

Since CD40 may be important in an immune response, there is a need for improved CD40-mediated signaling events to provide reliable and rapid treatment options for patients suffering from diseases that can be improved by treatment with CD40-directed therapeutic strategies.

The HER2 / neu (human epidermal growth factor receptor 2 / receptor tyrosine protein kinase erbB-2) is part of the human epidermal growth factor family. It has been shown that overexpression of this protein plays an important role in the progression of cancer, for example breast cancer. The HER2 / neu protein acts as a receptor tyrosine kinase and autophosphorylates upon dimerization with binding partners. HER2 / neu can enable multiple signal paths, including z. Mitogen-activated protein kinase, phosphoinositide-3-kinase, phospholipase Cγ, protein kinase C, and signal transporter and activator of transcription (STAT). Several compounds have been developed to inhibit HER2 / neu, including the monoclonal antibody trastuzumab and the monoclonal antibody pertuzumab.

Immunostimulatory molecular motifs such as pathogen-associated molecular pattern molecules (PAMPs) can be recognized by receptors of the innate immune system such as Toll-like receptors (TLRs), Nod-like receptors, C-type lectins and RIG-I-like receptors. These receptors can be transmembrane and intraendosomal proteins that can activate the immune system in response to infectious agents such as pathogens. Similar to other protein families, TLRs can have many isoforms, including TLR4, TLR7 and TLR8. Several agonists directed to the activation of various TLRs can be used in various immunotherapies, including vaccine adjuvants and cancer immunotherapies. TLR agonists can range from simple molecules to complex macromolecules. Similarly, the sizes of TLR agonists can range from small to large. TLR agonists may be synthetic or biosynthetic agonists. TLR agonists may also be PAMPs. Additional immunostimulatory compounds such as cytosolic DNA and unique bacterial nucleic acids termed cyclic dinucleotides can be recognized by interferon regulatory factor (IRF) or stimulator of interferon genes (STING), which can affect a cytosolic DNA sensor. Compounds recognized by interferon regulatory factor (IRF) may play a role in immune regulation by TLRs and other pattern recognition receptors.

Imiquimod, a synthetic TLR7 agonist, is currently approved for human therapeutic applications. Contained in a cream and marketed under the brand name Aldara, imiquimod is used as a topical treatment for a variety of indications with immune components such as actinic keratosis, genital warts and basal cell carcinomas. In addition, imiquimod is indicated as an adjuvant candidate for improving the adaptive immune response when applied topically to an immunization site.

Another type of immunostimulatory molecular motif, damage-associated molecular pattern molecules (DAMPs), can initiate and sustain an immune response that occurs as part of the non-infectious inflammatory response. DAMPs may be specifically localized proteins that, when recognized by the immune system at a location other than where DAMPs should be located, activate the immune system. Often, DAMPs can be nuclear or cytosolic proteins, and upon release from the nucleus or cytosol, DAMP proteins can be denatured by oxidation. Examples of DAMP proteins may include chromatin-associated protein high mobility group box 1 (HMGB1), S100 molecules of the calcium-modulated family of proteins and glycans such as hyaluronan fragments and glycan conjugates. DAMPs may also be nucleic acids, such as DNA, when released from tumor cells after apoptosis or necrosis. Examples of additional DAMP nucleic acids may include RNA and purine metabolites such as ATP, adenosine and uric acid present outside the nucleus or mitochondria.

The therapeutic use of DAMPs may focus on indications with an immune component such as arthritis, cancer, ischemia-reperfusion injury, myocardial infarction and stroke. In these indications, the therapeutic effects of DAMP may include the prevention of DAMP release using therapeutic strategies such as pro-apoptotic interventions, platinum and ethyl pyruvate, extracellular neutralization or blockade of DAMP release or signaling using therapeutic strategies such as anti-inflammatory drugs. HMGB1, Rasburiaspect and sRAGE, and direct or indirect blockade of DAMP receptors and downstream signaling events using therapeutic strategies such as RAGE molecule antagonists; TLR4 antagonists and antibodies to DAMP-R.

In addition, the immune response elicited by TLR agonists can be further enhanced when co-administered with a CD40 agonist antibody. For example, can co-administration of a TLR agonist such as Poly-IC: LC with a CD40 agonist antibody is synergistic to stimulate a stronger CD8 ⁺ T cell response than any of the two agonists alone.

However, the therapeutic use of PAMPs and DAMPs or other interventional mechanisms may be limited as systemic activation of PAMP and DAMP signaling pathways by cytokine syndrome-induced or cytotoxic-induced toxic shock syndrome can have life-threatening consequences. Accordingly, there is a critical need for therapeutic, clinically relevant targeted delivery of PAMP and DAMP agonists for safe and effective strategies to improve the immune response. The conjugate described herein can be used as a safe and effective strategy for improving the immune response. A conjugate may comprise an antibody construct and an immunostimulatory compound.

Antibody construct

An antibody construct may contain an antigen-binding domain. An antigen-binding domain may be a domain capable of specifically binding to an antigen. An antigen-binding domain may be an antigen-binding portion of an antibody or an antibody fragment. An antigen-binding domain may be one or more fragments of an antibody that may retain the ability to specifically bind to an antigen. An antigen binding domain may be any antigen binding fragment. An antigen-binding domain can recognize a single antigen. For example, one antigen-binding domain can recognize two, three, four, five, six, seven, eight, nine, ten or more antigens. For example, an antibody construct may contain two, three, four, five, six, seven, eight, nine, ten, or more antigen-binding domains. An antibody construct may contain two antigen-binding domains in which each antigen-binding domain can recognize the same antigen. An antibody construct can contain two antigen-binding domains in which each antigen-binding domain can recognize different antigens. An antigen-binding domain may be present in a scaffold, where a scaffold is a supportive framework for the antigen-binding domain. An antigen-binding domain may be present in a non-antibody framework. An antigen-binding domain may be present in an antibody framework. An antibody construct may comprise an antigen-binding domain in a scaffold. The antibody construct may comprise an Fc fusion protein product. In some embodiments, the antibody construct is an Fc fusion protein product.

The antigen-binding domain of an antibody construct can be selected from any domain that binds the antigen, including, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, or a functional fragment thereof, for example, a heavy chain variable domain ( V _H ) and a variable domain of a light chain (V _L ), a DARPin, an affimer, an avimers, a knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a T cell receptor or a recombinant T cell receptor. antigen binding domain of an antibody construct may be at least 80% homologous to an antigen binding domain selected from, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody or a functional fragment thereof, for example a heavy chain variable domain (V _H ) and a variable domain of a light chain (V _L ), a DARPin, an affimer, an Avimer, a Knottin, a monobody, an affinity bracket, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine , a T cell receptor or a recombinant T cell receptor.

An antigen binding domain of an antibody construct, for example an antigen binding domain from a monoclonal antibody, can comprise a light chain and a heavy chain. In one aspect, the monoclonal antibody binds to CD40 and comprises the anti-CD40 antibody light chain and the anti-CD40 antibody heavy chain that bind a CD40 antigen. In another aspect, the monoclonal antibody binds to a tumor antigen and comprises the tumor antigen antibody light chain and the tumor antigen antibody heavy chain that bind the tumor antigen.

An antibody construct can be an antibody. An antibody can consist of two identical light protein chains and two identical heavy protein chains, all covalently held together by precise localized disulfide bonds. The N-terminal regions of the light and heavy chains may together form the antigen recognition site of an antibody. Structurally, various functions of an antibody may be limited to discrete protein domains (ie, regions). The sites that can recognize and bind antigen can be made up of three complementarity-determining regions (CDRs) which may be within the heavy chain variable region and the light chain variable region region at the N-terminal end of the heavy chain and the light chain. The constant domains may provide the general framework of the antibody and may not be directly involved in the binding of the antibody to an antigen, but may be involved in various effector functions, such as the antibody's involvement in antibody-dependent cellular cytotoxicity, and may be Fc Bind receptors.

The domains of natural light and heavy chains may have the same general structures, and each domain may comprise four framework regions, the sequences of which may be conserved slightly, linked by three hypervariable regions or CDRs. The four framework regions can largely adopt a β-sheet conformation and the CDRs can form loops that interconnect, and in some aspects form part of, the β-sheet structure. The CDRs in each chain can be held in close proximity by the framework regions and can contribute to CDRs from the other chain for the formation of the antigen binding site.

An antibody of an antibody construct may comprise any antibody of any kind that is useful for various classes of immunoglobins, e.g. As IgA, IgD, IgE, IgG and IgM can be assigned. Several different classes may continue in isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. An antibody may further comprise a light chain and a heavy chain, often more than one chain. The heavy chain constant regions (Fc) corresponding to the different classes of immunoglobulins may be α, δ, ε, γ, and μ, respectively. The light chains may be either kappa or k and lambda or λ based on the amino acid sequences of the constant domains. The Fc region may contain a Fc domain. An Fc receptor can bind to an Fc domain. Antibody constructs may also include any fragment or recombinant forms thereof, including, but not limited to, single-chain variable fragments (scFvs), "T-bodies", anti-calines, centyrins, affibodies, domain antibodies or peptibodies.

An antibody may comprise an antigen-binding domain, which may refer to a portion of an antibody comprising the antigen-recognition region, i. H. an antigenic determining variable region of an antibody sufficient to cause recognition of the antigen and binding of the antigen binding region to a target, such as an antigen, i. H. to lend the epitope. Examples of antibody binding domains may include but are not limited to Fab, scFv, variable Fv fragment and other antibody fragments, combinations of fragments or types of fragments known or well-known to those skilled in the art.

An antibody construct may comprise an antigen-binding domain of an antibody. An antigen-binding domain of an antibody may comprise one or more light chain CDRs (LC) and one or more heavy chain CDRs (HC). For example, an antibody binding domain of an antibody may comprise one or more of the following: a complementarity determining region 1 of the light chain (LC CDR1), a complementarity determining region 2 of the light chain (LC CDR2) or a complementarity determining region 3 of the light chain (LC CDR3). As another example, an antibody binding domain may include one or more of: a heavy chain complementarity determining region (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), or a heavy chain complementarity determining region (HC CDR3) , As an additional example, an antibody binding domain of an antibody may include one or more of the following: LC CDR1, LC CDR2, LC CDR3, HC CDR1, HC CDR2 and HC CDR3.

An antibody construct may comprise an antibody fragment. An antibody fragment may be (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , C _L and C _H1 domains; (ii) an F (ab ') ₂ fragment, a bivalent fragment comprising two Fab fragments joined by a disulfide bond at the hinge region; and (iii) an Fv fragment consisting of the V _L and V _H domains of a single arm of an antibody. Although the two domains of the Fv fragment, V _L and V _H , can be encoded by separate genes, they can be linked by a synthetic linker to be produced as a single protein chain in which the V _L and V _H regions pair to form monovalent molecules.

F (ab ') ₂ and Fab' residues can be prepared by treating immunoglobulin (eg, monoclonal antibody) with a protease such as pepsin and papain, and can comprise an antibody fragment that is close to that present by digestion of immunoglobulin Disulfide bonds is generated, which are present between the hinge areas in each of the two H chains. The Fab fragment can also be the comprising the light chain constant domain and the first constant domain (C _H1 ) of the heavy chain. Fab 'fragments may differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain C _H1 domain, including one or more cysteine (s) from the antibody hinge region.

An Fv may be the minimal antibody fragment containing a complete antigen recognition and antigen binding site. This region may consist of a dimer of a heavy chain variable domain and one of the light chain in close, non-covalent association. In this configuration, the three hypervariable regions of each variable domain can interact to define an antigen binding site on the surface of the V _H -V _L dimer. A single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) can recognize and bind antigen, although the binding may have a lower affinity than the affinity of the entire binding site.

An antibody used herein may be "humanized". Humanized forms of non-human (e.g., murine) antibodies may be chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab ', F (ab') _2, or other targeting subdomains of antibodies) which may contain minimal sequences. that are derived from non-human immunoglobulin. In general, the humanized antibody may comprise substantially all of at least one and typically two variable domains, wherein all or substantially all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all FR regions are those of a human immunoglobulin sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.

An antibody described herein may be a human antibody. As used herein, "human antibodies" may include antibodies having, for example, the amino acid sequence of a human immunoglobulin, and may include antibodies isolated from human immunoglobulin libraries or from animals that are transgenic for one or more human immunoglobulins and that are not endogenous Express immunoglobulins. Human antibodies can be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes. Fully human antibodies recognizing a selected epitope can be generated by guided selection. In this approach, a selected non-human monoclonal antibody, e.g. A mouse antibody, can be used to direct the selection of a fully human antibody that recognizes the same epitope.

An antibody described herein may be a bispecific antibody or a two variable domain (DVD) antibody. Bispecific and DVD antibodies may be monoclonal, often human or humanized, antibodies that may have binding specificities for at least two different antigens.

An antibody described herein may be a derivatized antibody. For example, derivatized antibodies can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to a cellular ligand, or other protein.

An antibody described herein may have a sequence that has been modified to alter at least one constant region-mediated biological effector function compared to the corresponding wild-type sequence. For example, in some embodiments, the antibody may be modified to reduce at least one constant region-mediated biological effector function compared to an unmodified antibody, e.g. B. a reduced binding to the Fc receptor (FcR). For example, FcR binding can be reduced by, for example, mutating the antibody constant-immunoglobulin region segment in certain regions necessary for FcR interactions.

An antibody described herein may be modified to acquire or enhance at least one constant region-mediated biological effector function relative to an unmodified antibody, e.g. B. FcγR-Wechselschützen to reinforce. For example, an antibody having a constant region that binds FcγRIIA, FcγRIIB, and / or FcγRIIIA with greater affinity than the corresponding wild type constant-region can be prepared according to the methods described herein.

An antibody described herein can bind to tumor cells, such as an antibody to a cell surface receptor or a tumor antigen. An antibody described herein can bind to CD40, such as an antibody that can be a CD40 agonist and bind to CD40.

As used herein, the abbreviations for the natural 1-enantiomeric amino acids are conventional and may be as follows: alanine (A, Ala); Arginine (R, Arg); Asparagine (N, Asn); Aspartic acid (D, Asp); Cysteine (C, Cys); Glutamic acid (E, Glu); Glutamine (Q, Gin); Glycine (G, Gly); Histidine (H, His); Isoleucine (I, Ile); Leucine (L, Leu); Lysine (K, Lys); Methionine (M, Met); Phenylalanine (F, Phe); Proline (P, Pro); Serine (S, Ser); Threonine (T, Thr); Tryptophan (W, Trp); Tyrosine (Y, Tyr); Valine (V, Val). Unless otherwise stated, X may indicate any amino acid. In some aspects, X may be asparagine (N), glutamine (Q), histidine (H), lysine (K) or arginine (R).

exprimiert werden. Eine leichte Kette eines Anti-CD40-Antikörpers kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 1 umfasst. Eine variable Region einer leichten Kette eines Anti-CD40-Antikörpers kann aus einer DNA-Sequenz umfassend

exprimiert werden. Eine variable Region einer leichten Kette eines Anti-CD40-Antikörpers kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 3 umfasst. Zusätzlich können Anti-CD40-Antikörper, die aus SEQ ID NO: 1 exprimiert werden oder aus einer DNA-Sequenz exprimiert werden, die mehr als 70% Homologie zu SEQ ID NO: 1 umfasst, eine Dissoziationskonstante aufweisen (K_d) für CD40, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die aus SEQ ID NO: 1 exprimiert werden oder aus einer DNA-Sequenz exprimiert werden, die mehr als 70% Homologie zu SEQ ID NO: 1 umfasst, eine Dissoziationskonstante (K_d) für CD40 aufweisen, die kleiner als 1 nM ist, kleiner als 100 PM, kleiner als 10 pM, kleiner als 1 pM oder kleiner als 0,1 pM ist. Die leichte anti-CD40-Kette kann mit jeglicher schweren anti-CD40-Kette oder Fragment davon exprimiert werden. Die leichte anti-CD40-Kette kann auch mit jeglicher schweren anti-CD40 Kette oder Fragment davon exprimiert werden, um einen anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein.An antibody construct may comprise an anti-CD40 antibody. An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of anti-CD40 antibody can be comprised of a DNA sequence

are expressed. A light chain of anti-CD40 antibody can be expressed from a DNA sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater as 99% homology to SEQ ID NO: 1. A light chain variable region of an anti-CD40 antibody can be comprised of a DNA sequence

are expressed. A light chain variable region of an anti-CD40 antibody can be expressed from a DNA sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% % or more than 99% homology to SEQ ID NO: 3. In addition, anti-CD40 antibodies expressed from SEQ ID NO: 1 or expressed from a DNA sequence greater than 70% homology to SEQ ID NO: 1 may have a dissociation constant (K _d ) for CD40. the smaller than 10 nM. Anti-CD40 antibodies expressed from SEQ ID NO: 1 or expressed from a DNA sequence comprising more than 70% homology to SEQ ID NO: 1, having a dissociation constant (K _d ) for CD40 smaller than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. The light anti-CD40 chain can be expressed with any heavy anti-CD40 chain or fragment thereof. The light anti-CD40 chain can also be expressed with any heavy anti-CD40 chain or fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct.

umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 4 umfassen. Eine variable Region einer leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz

umfassen. Eine variable Region einer leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 6 umfassen. Zusätzlich können Anti-CD40-Antikörper, die SEQ ID NO: 4 umfassen oder eine Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 4 umfassen, eine Dissoziationskonstante aufweisen (K_d) für CD40, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die SEQ ID NO: 4 umfassen oder eine Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 4 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die kleiner als 1 nM, kleiner als 100 PM, kleiner als 10 pM, kleiner als 1 pM oder kleiner als 0,1 pM ist. Die leichte anti-CD40-Kette kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Die leichte anti-CD40-Kette kann mit jeglicher schweren anti-CD40-Kette oder Fragment davon exprimiert werden. Die leichte anti-CD40-Kette kann auch mit jeglicher schweren anti-CD40 Kette oder Fragment davon exprimiert werden, um einen anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Darüber hinaus würde ein Fachmann erkennen, dass diese Konzepte für Anti-CD40-Antikörper gelten könnten, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden.A light chain of an anti-CD40 antibody may have an amino acid sequence

include. A light chain of anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 4 include. A variable region of a light chain of an anti-CD40 antibody may have an amino acid sequence

include. A light chain variable region of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99%. Homology to SEQ ID NO: 6. In addition, anti-CD40 antibodies comprising SEQ ID NO: 4 or having an amino acid sequence greater than 70% homology to SEQ ID NO: 4 may have a dissociation constant (K _d ) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SEQ ID NO: 4 or comprising an amino acid sequence having greater than 70% homology to SEQ ID NO: 4 may have a dissociation constant (K _d ) for CD40 that is less than 1 nM, less than 100 PM, less than 10 pM, less than 1 pM or less than 0.1 pM. The light anti-CD40 chain can be purified and combined with a pharmaceutically acceptable carrier. The light anti-CD40 chain can be expressed with any heavy anti-CD40 chain or fragment thereof. The light anti-CD40 chain can also be expressed with any heavy anti-CD40 chain or fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, one skilled in the art would recognize that these concepts could apply to anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

umfassen. SBT-040 VL-Ck kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 26 umfassen.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of anti-CD40 antibody may be SBT-040 VL-Ck. SBT-040 VL-Ck can be a DNA amino acid derivative

include. SBT-040 VL-Ck may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 26 include.

A light chain of an anti-CD40 antibody may comprise a CDR. A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence QGIYSW (SEQ ID NO: 27). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence TAS (SEQ ID NO: 28). A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence QQANIFPLT (SEQ ID NO: 29). A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 27. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 28. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 29.

umfassen. Dacetuzumab kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 38 umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GYSFTGYY (SEQ ID NO: 39) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz VIPNAGGT (SEQ ID NO: 40) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz AREGIYW (SEQ ID NO: 41) umfassen. Ein CDR einer schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 39 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 40 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80% umfassen, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 41 umfassen. Die zweidimensionale Struktur der schweren Kette von Dacetuzumab ist in 16 gezeigt.An antibody construct can comprise a heavy antibody chain. A heavy chain may be a heavy chain of an anti-CD40 antibody capable of binding a CD40 antigen. A heavy chain of anti-CD40 antibody may be an IgG1 isotype. A heavy chain of an anti-CD40 antibody may be Dacetuzumab. Dacetuzumab may have an amino acid sequence

include. Dacetuzumab may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 38. A heavy chain of an anti-CD40 antibody may comprise a CDR. A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence GYSFTGYY (SEQ ID NO: 39). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence VIPNAGGT (SEQ ID NO: 40). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence AREGIYW (SEQ ID NO: 41). A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 39. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 40. A heavy chain CDR of an anti-CD40 antibody may comprise an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99%. Homology to SEQ ID NO: 41. The two-dimensional structure of the heavy chain of Dacetuzumab is in 16 shown.

Dacetuzumab kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 42 umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz QSLVHSNGNTF (SEQ ID NO: 43) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz TVS (SEQ ID NO: 44) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz SQTTHVPWT (SEQ ID NO: 45) umfassen. Eine CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 43 umfassen. Eine CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 44 umfassen. Eine CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 45 umfassen. Die zweidimensionale Struktur der leichten Kette von Dacetuzumab ist in 17 gezeigt.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of an anti-CD40 antibody may be dacetuzumab. Dacetuzumab may have an amino acid sequence

Dacetuzumab may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 42. A light chain of an anti-CD40 antibody may comprise a CDR. A light chain of an anti-CD40 antibody can a CDR having an amino acid sequence QSLVHSNGNTF (SEQ ID NO: 43). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence TVS (SEQ ID NO: 44). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence SQTTHVPWT (SEQ ID NO: 45). A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 43. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 44. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 45. The two-dimensional structure of the light chain of Dacetuzumab is in 17 shown.

umfassen. Bleselumab kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 46 umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GGSISSPGYY (SEQ ID NO: 47) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz IYKSGST (SEQ ID NO: 48) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz TRPVVRYFGWFDP (SEQ ID NO: 49) umfassen. Eine CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 47 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 48 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 49 umfassen. Die zweidimensionale Struktur der schweren Kette von Bleselumab ist in 18 gezeigt.An antibody construct can comprise a heavy antibody chain. A heavy chain can be a heavy one Chain of an anti-CD40 antibody that can bind a CD40 antigen. A heavy chain of anti-CD40 antibody may be an IgG4 isotype. A heavy chain of an anti-CD40 antibody may be bleselumab. Bleselumab can be an amino acid sequence

include. Bleselumab may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 46. A heavy chain of an anti-CD40 antibody may comprise a CDR. A heavy chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence GGSISSPGYY (SEQ ID NO: 47). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence IYKSGST (SEQ ID NO: 48). A heavy chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence TRPVVRYFGWFDP (SEQ ID NO: 49). A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 47. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 48. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 49. The two-dimensional structure of bleselumab heavy chain is in 18 shown.

umfassen. Bleselumab kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 50 umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz QGISSA (SEQ ID NO: 51) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz DAS (SEQ ID NO: 52) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz QQFNSYPT (SEQ ID NO: 53) umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 51. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 52 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 53 umfassen. Die zweidimensionale Struktur der leichten Kette von Bleselumab ist in 19 gezeigt.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of an anti-CD40 antibody may be bleselumab. Bleselumab can be an amino acid sequence

include. Bleselumab may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 50. A light chain of an anti-CD40 antibody may comprise a CDR. A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence QGISSA (SEQ ID NO: 51). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence DAS (SEQ ID NO: 52). A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence QQFNSYPT (SEQ ID NO: 53). A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 51. An anti-CD40 antibody light chain CDR may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% % or greater than 99% homology to SEQ ID NO: 52. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 53. The two-dimensional structure of the bleselumab light chain is in 19 shown.

umfassen. Lucatumumab kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 54 umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GFTFSSYG (SEQ ID NO: 55) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz ISYEESNR (SEQ ID NO: 56) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz ARDGGIAAPGPDY (SEQ ID NO: 57) umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 55 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 56 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 57 umfassen. Die zweidimensionale Struktur der schweren Kette von Lucatumumab ist in 20 gezeigt.An antibody construct can comprise a heavy antibody chain. A heavy chain may be a heavy chain of an anti-CD40 antibody capable of binding a CD40 antigen. A heavy chain of anti-CD40 antibody may be an IgG1 isotype. Lucatumumab may have an amino acid sequence

include. Lucatumumab may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 54. A heavy chain of an anti-CD40 antibody may comprise a CDR. A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence GFTFSSYG (SEQ ID NO: 55). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence ISYEESNR (SEQ ID NO: 56). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence ARDGGIAAPGPDY (SEQ ID NO: 57). A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 55. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 56. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 57. The two-dimensional structure of the lucatumumab heavy chain is in 20 shown.

umfassen. Lucatumumab kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 59 umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz LGS (SEQ ID NO: 60) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz MQARQTPFT (SEQ ID NO: 61) umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 59 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 60 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 61 umfassen. Die zweidimensionale Struktur der leichten Kette von Lucatumumab ist in 21 gezeigt.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of an anti-CD40 antibody may be lucatumumab. Lucatumumab may have an amino acid sequence

include. Lucatumumab may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 59. A light chain of an anti-CD40 antibody may be a CDR with an amino acid sequence LGS (SEQ ID NO: 1). 60). A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence MQARQTPFT (SEQ ID NO: 61). A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 59. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 60. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 61. The two-dimensional structure of the lucatumumab light chain is in 21 shown.

umfassen. ADC-1013 kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 62 umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GFTFSTYG (SEQ ID NO: 63) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz ISGGSSYI (SEQ ID NO: 64) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz ARILRGGSGMDL (SEQ ID NO: 65) umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 63 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 64 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 65 umfassen. Die zweidimensionale Struktur der schweren Kette von ADC-1013 ist in 22 gezeigt.An antibody construct can comprise a heavy antibody chain. A heavy chain may be a heavy chain of an anti-CD40 antibody capable of binding a CD40 antigen. A heavy chain one Anti-CD40 antibody may be an IgG1 isotype. A heavy chain of an anti-CD40 antibody may be ADC-1013. ADC-1013 may have an amino acid sequence

include. ADC-1013 may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 62. A heavy chain of an anti-CD40 antibody may comprise a CDR. A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence GFTFSTYG (SEQ ID NO: 63). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence ISGGSSYI (SEQ ID NO: 64). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence ARILRGGSGMDL (SEQ ID NO: 65). A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 63. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 64. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence of greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 65 include. The two-dimensional structure of the heavy chain of ADC-1013 is in 22 shown.

umfassen. ADC-1013 kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 66 umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz SSNIGAGYN (SEQ ID NO: 67) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GNI (SEQ ID NO: 68) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz AAWDKSISGLV (SEQ ID NO: 69) umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 67 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 68 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 69 umfassen. Die zweidimensionale Struktur der leichten Kette von ADC-1013 ist in 23 gezeigt.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of an anti-CD40 antibody may be ADC-1013. ADC-1013 may have an amino acid sequence

include. ADC-1013 may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 66. A light chain of an anti-CD40 antibody may comprise a CDR. A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence SSNIGAGYN (SEQ ID NO: 67). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence GNI (SEQ ID NO: 68). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence AAWDKSISGLV (SEQ ID NO: 69). A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 67. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 68. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 69. The two-dimensional structure of the light chain of ADC-1013 is in 23 shown.

umfassen. APX005 kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 70 umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GFSFSSTY (SEQ ID NO: 71) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz IYTGDGTN (SEQ ID NO: 72) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz ARPDITYGFAINFW (SEQ ID NO: 73) umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 71 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 72 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 73 umfassen. Die zweidimensionale Struktur der schweren Kette von APX005 ist in 24 gezeigt.An antibody construct may comprise a heavy chain of the antibody. A heavy chain may be a heavy chain of an anti-CD40 antibody capable of binding a CD40 antigen. A heavy chain of an anti-CD40 antibody may be the humanized rabbit antibody APX005. APX005 can have an amino acid sequence

include. APX005 may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 70. A heavy chain of an anti-CD40 antibody may comprise a CDR. A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence GFSFSSTY (SEQ ID NO: 71). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence IYTGDGTN (SEQ ID NO: 72). A heavy chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence ARPDITYGFAINFW (SEQ ID NO: 73). A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 71. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 72. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 73. The two-dimensional structure of the APX005 heavy chain is in 24 shown.

umfassen. APX005 kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 74 umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz QSISSR (SEQ ID NO: 75) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz RAS (SEQ ID NO: 76) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz QCTGYGISWP (SEQ ID NO: 77) umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 75 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 76 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 77 umfassen. Die zweidimensionale Struktur der leichten Kette APX005 ist in 25 gezeigt.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of an anti-CD40 antibody may be the humanized rabbit antibody APX005. APX005 can have an amino acid sequence

include. APX005 may comprise an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 74. A light chain of an anti-CD40 antibody may comprise a CDR. A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence QSISSR (SEQ ID NO: 75). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence RAS (SEQ ID NO: 76). A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence QCTGYGISWP (SEQ ID NO: 77). A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 75. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 76. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 77. The two-dimensional structure of light chain APX005 is in 25 shown.

umfassen. Chi Lob 7/4 kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 78 umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GYTFTEYI (SEQ ID NO: 79) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz IIPNNGGT (SEQ ID NO: 80) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz TRREVYGRNYYALDY (SEQ ID NO: 81) umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 79 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 80 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 81 umfassen. Die zweidimensionale Struktur der schweren Kette von Chi Lob 7/4 ist in 26 gezeigt.An antibody construct can comprise a heavy antibody antibody. A heavy chain may be a heavy chain of an anti-CD40 antibody capable of binding a CD40 antigen. A heavy chain of anti-CD40 antibody may be Chi Lob 7/4. Chi LOB 7/4 can be an amino acid sequence

include. Chi Lob 7/4 may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 78 include. A heavy chain of an anti-virus CD40 antibody may include a CDR. A heavy chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence GYTFTEYI (SEQ ID NO: 79). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence IIPNNGGT (SEQ ID NO: 80). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence TRREVYGRNYYALDY (SEQ ID NO: 81). A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 79. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 80. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 81. The two-dimensional structure of the heavy chain of Chi praise 7/4 is in 26 shown.

umfassen. Chi LoB 7/4 kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 82. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz QGINNY (SEQ ID NO: 83) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz YTS (SEQ ID NO: 84) umfassen. Eine leichte Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz QQYSNLPYT (SEQ ID NO: 85) umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 83 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 84 umfassen. Ein CDR der leichten Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 85 umfassen. Die zweidimensionale Struktur der leichte Kette von Chi Lob 7/4 ist in 27 gezeigt.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an anti-CD40 antibody capable of binding a CD40 antigen. A light chain of anti-CD40 antibody may be Chi Lob 7/4. Chi LOB 7/4 can be an amino acid sequence

include. Chi LoB 7/4 may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 82 A light chain of anti-CD40 antibody may comprise a CDR. A light chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence QGINNY (SEQ ID NO: 83). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence YTS (SEQ ID NO: 84). A light chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence QQYSNLPYT (SEQ ID NO: 85). A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 83. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 84. A light chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 85. The two-dimensional structure of the light chain of Chi praise 7/4 is in 27 shown.

expemiert werden. SBT-040-G1WT kann von einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 8 umfasst. Eine variable Region von SBT-040-G1WT kann von einer DNA-Sequenz umfassend

exprimiert werden. Eine variable Region von SBT-040-G1WT kann von einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 13 umfasst. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1WT umfassen, die aus SEQ ID NO: 8 exprimiert werden oder aus einer DNA-Sequenz exprimiert werden, die mehr als 70% Homologie zu SEQ ID NO: 8 umfasst, eine Dissoziationskonstante (K_d) für CD40 haben, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die SBT-040-G1WT umfassen, die aus einer DNA-Sequenz exprimiert werden, die SEQ ID NO: 8 oder mehr als 70% Homologie zu SEQ ID NO: 8 umfasst, können eine Dissoziationskonstante (K_d) für CD40 haben, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G1WT kann mit jeglicher anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden. SBT-040-G1WT kann auch mit jeglicher anti-CD40-leichten Kette oder Fragment davon exprimiert werden, um einen Anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.An antibody construct may comprise a heavy chain of the antibody. A heavy chain may be a heavy chain of an anti-CD40 antibody capable of binding a CD40 antigen. A heavy chain of anti-CD40 antibody may be an IgG1 isotype. A heavy chain of anti-CD40 antibody may be SBT-040-G1WT. SBT-040-G1WT can be comprised of a DNA sequence

be expelled. SBT-040-G1WT can be expressed from a DNA sequence that is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 8. A variable region of SBT-040-G1WT can be comprised of a DNA sequence

are expressed. A variable region of SBT-040-G1WT can be expressed from a DNA sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater as 99% homology to SEQ ID NO: 13. In addition, anti-CD40 antibodies comprising SBT-040-G1WT expressed from SEQ ID NO: 8 or expressed from a DNA sequence greater than 70% homology to SEQ ID NO: 8 may have a dissociation constant (K _d ) for CD40 which is smaller than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1WT expressed from a DNA sequence comprising SEQ ID NO: 8 or greater than 70% homology to SEQ ID NO: 8 may have a dissociation constant (K _d ). for CD40 which is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1WT can be expressed with any anti-CD40 light chain or a fragment thereof. SBT-040-G1WT may also be expressed with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

umfassen. SBT-040-G1WT kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 15 umfassen. SBT-040-G1WT kann eine Aminosäuresequenz

umfassen. Eine variable Region von SBT-040-G1WT kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 20 umfassen. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1WT mit SEQ ID NO: 15 oder mit einer Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 15 umfassen, eine Dissoziationskonstante (K_d) für CD40 haben, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die SBT-040-G1WT mit SEQ ID NO: 15 oder mit einer Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 15 umfassen, können eine Dissoziationskonstante (K_d) für CD40 haben, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM ist. SBT-040-G1WT kann gereinigt werden. SBT-040-G1WT kann mit jeglicher anti-CD40-leichten Kette oder einem Fragment davon kombiniert werden, um einen Anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.SBT-040-G1WT may have an amino acid sequence

include. SBT-040-G1WT may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 15 include. SBT-040-G1WT may have an amino acid sequence

include. A variable region of SBT-040-G1WT may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20 include. In addition, anti-CD40 antibodies comprising SBT-040-G1WT of SEQ ID NO: 15 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 15 may have a dissociation constant (K _d ) for CD40 smaller than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1WT of SEQ ID NO: 15 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 15 may have a dissociation constant (K _d ) for CD40 less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1WT can be cleaned. SBT-040-G1WT can be combined with any anti-CD40 light chain or a fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz GYTFTYY (SEQ ID NO: 23) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz INPDSGGT (SEQ ID NO: 24) umfassen. Eine schwere Kette eines Anti-CD40-Antikörpers kann ein CDR mit einer Aminosäuresequenz ARDQPLGYCTNGVCSYFDY (SEQ ID NO: 25) umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 23 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 24 umfassen. Ein CDR der schweren Kette eines Anti-CD40-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 25 umfassen.An antibody construct can comprise a heavy antibody chain. A heavy chain may be a heavy chain of an anti-CD40 antibody capable of binding a CD40 antigen. A heavy chain of anti-CD40 antibody may be an IgG1 isotype. A heavy chain of anti-CD40 antibody may be SBT-040 VH-hIgG1 wt. SBT-040 VH-hIgG1 wt may have an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to an amino acid sequence

include. A heavy chain of an anti-CD40 antibody may comprise a CDR. A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence GYTFTYY (SEQ ID NO: 23). A heavy chain of an anti-CD40 antibody may comprise a CDR having an amino acid sequence INPDSGGT (SEQ ID NO: 24). A heavy chain of anti-CD40 antibody may comprise a CDR having an amino acid sequence ARDQPLGYCTNGVCSYFDY (SEQ ID NO: 25). A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 23. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 24. A heavy chain CDR of an anti-CD40 antibody may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 25.

exprimiert werden. SBT-040-G2 kann von einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ist ID NO: 7 umfasst. Eine variable Region von SBT-040-G2 kann aus einer DNA-Sequenz, die SEQ ID NO: 13 umfasst, exprimiert werden. Eine variable Region von SBT-040-G2 kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 13 umfasst. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G2 umfassen, das aus SEQ ID NO: 7 exprimiert wurde oder aus einer DNA-Sequenz, die mehr als 70% Homologie zu SEQ ID NO: 7 umfasst, exprimiert wurde, eine Dissoziationskonstante (K_d) für CD40, die weniger als 10 nM beträgt, aufweisen. Anti-CD40-Antikörper, die SBT-040-G2 umfassen, das aus SEQ ID NO: 7 exprimiert wurde oder aus einer DNA-Sequenz, die mehr als 70% Homologie zu SEQ ID NO: 7 umfasst, exprimiert wurde, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G2 kann mit jeglicher anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden. SBT-040-G2 kann auch mit jeglicher anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden, um einen anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.A heavy chain of an anti-CD40 antibody may be an IgG2 isotype. A heavy chain of anti-CD40 antibody may be SBT-040-G2. Comprising SBT-040-G2 from a DNA sequence

are expressed. SBT-040-G2 can be expressed from a DNA sequence that is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 7 is included. A variable region of SBT-040-G2 can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G2 can be expressed from a DNA sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater as 99% homology to SEQ ID NO: 13. In addition, anti-CD40 antibodies comprising SBT-040-G2 expressed from SEQ ID NO: 7 or expressed from a DNA sequence comprising more than 70% homology to SEQ ID NO: 7 may be used Dissociation constant (K _d ) for CD40 which is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G2 expressed from SEQ ID NO: 7 or expressed from a DNA sequence comprising more than 70% homology to SEQ ID NO: 7 may have a dissociation constant (K _d ) for CD40 which is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G2 can be expressed with any anti-CD40 light chain or a fragment thereof. SBT-040-G2 may also be expressed with any anti-CD40 light chain or a fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 14 umfassen. SBT-040-G1WT kann eine Aminosäuresequenz SEQ ID NO: 20 umfassen. Eine variable Region von SBT-040-G2 kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 20 umfassen. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G2 mit SEQ ID NO: 14 oder mit einer Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 14 umfassen, eine Dissoziationskonstante (K) für CD40 aufweisen, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die SBT-040-G2 mit SEQ ID NO: 14 oder mit einer Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 14 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM ist. SBT-040-G2 kann gereinigt werden. SBT-040-G2 kann mit jeglicher anti-CD40-leichten Kette oder einem Fragment davon kombiniert werden, um einen Anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.SBT-040-G2 may have an amino acid sequence

70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95% or more than 99% homology to SEQ ID NO: 14. SBT-040-G1WT may comprise an amino acid sequence of SEQ ID NO: 20. A variable region of SBT-040-G2 may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 20 include. In addition, anti-CD40 antibodies comprising SBT-040-G2 of SEQ ID NO: 14 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 14 may have a CD40 dissociation constant (K) smaller than 10 nM is. Anti-CD40 antibodies comprising SBT-040-G2 of SEQ ID NO: 14 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 14 may have a dissociation constant (K _d ) for CD40 less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G2 can be cleaned. SBT-040-G2 can be combined with any anti-CD40 light chain or a fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

An antibody construct may comprise an antibody with modifications that occur on at least one amino acid residue. Modifications may be substitutions, supplements, mutations, deletions or the like. An antibody modification can be an insertion of an unnatural amino acid.

An antibody construct may include a light chain of an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine, or ten modifications, but not more than 40, 35, 30, 25, 20, 15, or 10 modifications Amino acid sequence relative to the natural or original amino acid sequence include. An antibody construct may comprise a heavy chain of an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine, or ten modifications, but not more than 40, 35, 30, 25, 20, 15, or 10 modifications Amino acid sequence relative to the natural or original amino acid sequence include. A heavy chain may be the heavy chain of an anti-CD40 antibody capable of binding to the CD40 antigen.

An antibody construct may be an IgG1 isotype. An antibody construct may be an IgG2 isotype. An antibody construct may be an IgG3 isotype. An antibody construct may be an IgG4 isotype. An antibody construct may consist of a hybrid isotype comprising constant regions of two or more isotypes. An antibody construct may be an anti-CD40 antibody in which the anti-CD40 antibody may be a monoclonal human antibody comprising a wild-type sequence of an IgG1 isoform, particularly at an Fc region of the antibody.

Additional anti-CD40 antibody sequences that may be used in the antibody construct include any sequence shown below in TABLE 1 or a combination thereof:

Additional anti-CD40 antibody sequences that may be used in the antibody construct may have a sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%. or more than 99% homology to any sequence in TABLE 1.

Antibody constructs disclosed herein may not be natural, designed and / or constructed. Antibody constructs disclosed herein may not be natural, designed and / or engineered scaffolds that comprise an antigen-binding domain. Antibody constructs disclosed herein may not be natural, designed and / or constructed antibodies. Antibody constructs can be monoclonal antibodies. Antibody constructs can be human antibodies. Antibody constructs can be humanized antibodies. Antibody constructs can be monoclonal humanized antibodies. Antibody constructs can be recombinant antibodies.

An antigen-binding domain of an antibody construct can be selected to recognize an antigen. For example, an antigen may be a cell surface marker on a target cell associated with a disease or condition. An antigen can be expressed on an immune cell. An antigen may be a peptide or fragment thereof. An antigen can be expressed on an antigen-presenting cell. An antigen can be expressed on a dendritic cell, a macrophage or a B cell. An antigen may be a peptide that is presented in a large histocompatibility complex by cell. As another example, a cell surface marker recognized by the antigen-binding domain may comprise macromolecules associated with viral and bacterial diseases or infections, autoimmune diseases, and cancers. An antigen may be CD40 and an antigen-binding domain may recognize a CD40 antigen. An antigen can be a tumor antigen or a fragment thereof. A tumor antigen can be any antigen listed in tumor antigen databases, such as TANTIGEN, or peptide databases for T-cell-defined tumor antigens, such as the Cancer Immunity Peptide Database. A tumor antigen can also be any antigen that is described in the review by Chen ( Chen, Cancer Immune 2004 [updated March 10, 2004, cited April 1, 2004] ) is listed. Note that the "antibody" can recognize the "tumor antigen" or a peptide derived therefrom bound to an MHC molecule. An antigen can be CDS, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, BCMA, CS-1, PD-L1 , B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate binding protein, A33, G250, prostate-specific membrane antigen, ferritin GD2, GD3, GM2, Le ^y , CA-125, CA19- 9, epidermal growth factor, p185HER2, IL-2 receptor, de2-7 EGFR, fibroblast activating protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6E7, EGFRvIII, Her-2 / neu, Idiotype, MAGE A3, p53 non-mutant, NY-ESO-1, PMSA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, Bcr-abl, tyrosinase, survivin, PSA, HTERT, sarcoma Translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE A1, SLe ( animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carboanhydr ase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, leguminium, Tie3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2 , ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE or Fos-related antigen 1, or have at least 80% homology to it. An antigen-binding domain may be capable of recognizing a single antigen. An antigen-binding domain may be capable of recognizing two or more different antigens.

umfassen. SBT-050 VH-hIgG1 wt kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 30 umfassen. Eine schwere Kette eines Anti-HER2-Antikörpers kann ein CDR umfassen. Eine schwere Kette eines Anti-HER2-Antikörpers kann ein CDR mit einer Aminosäuresequenz GFTFTDYT (SEQ ID NO: 31) umfassen. Eine schwere Kette eines Anti-HER2-Antikörpers kann ein CDR mit einer Aminosäuresequenz VNPNSGGS (SEQ ID NO: 32) umfassen. Eine schwere Kette eines Anti-HER2-Antikörpers kann ein CDR mit einer Aminosäuresequenz ARNLGPSFYFDY (SEQ ID NO: 33) umfassen. Ein CDR der schweren Kette eines Anti-HER2-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 31 umfassen. Ein CDR der schweren Kette eines Anti-HER2-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 32 umfassen. Ein CDR der schweren Kette eines Anti-HER2-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 33 umfassen.An antibody construct can comprise a heavy antibody chain. A heavy chain may be a heavy chain of an anti-HER2 monoclonal antibody capable of binding an HER2 antigen. A heavy chain of anti-HER2 antibody may be an IgG1 isotype. A heavy chain of anti-HER2 antibody may be SBT-050 VH-hIgG1 wt (pertuzumab). SBT-050 VH-hIgG1 wt may have an amino acid sequence

include. SBT-050 VH-hIgG1 wt may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO : 30 include. A heavy chain of anti-HER2 antibody may comprise a CDR. A heavy chain of anti-HER2 antibody may comprise a CDR having an amino acid sequence GFTFTDYT (SEQ ID NO: 31). A heavy chain of anti-HER2 antibody may comprise a CDR having an amino acid sequence VNPNSGGS (SEQ ID NO: 32). A heavy chain of anti-HER2 antibody may comprise a CDR having an amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 33). An anti-HER2 antibody heavy chain CDR may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 31. An anti-HER2 antibody heavy chain CDR may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 32. An anti-HER2 antibody heavy chain CDR may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 33.

umfassen. SBT-050 VL-Ck kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 34 umfassen. Eine leichte Kette eines Anti-HER2-Antikörpers kann ein CDR umfassen. Eine leichte Kette eines Anti-HER2-Antikörpers kann ein CDR mit einer Aminosäuresequenz QDVSIG (SEQ ID NO: 35) umfassen. Eine leichte Kette eines Anti-HER2-Antikörpers kann ein CDR mit einer Aminosäuresequenz SAS (SEQ ID NO: 36) umfassen. Eine leichte Kette eines Anti-HER2-Antikörpers kann ein CDR mit einer Aminosäuresequenz QQYYIYPYT (SEQ ID NO: 37) umfassen. Ein CDR der leichten Kette eines Anti-HER2-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 35 umfassen. Ein CDR der leichten Kette eines Anti-HER2-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 36. Ein CDR der leichten Kette eines Anti-HER2-Antikörpers kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 37 umfassen.An antibody construct may comprise a light antibody chain. A light chain may be a light chain of an HER2 monoclonal antibody capable of binding an HER2 antigen. A light chain of anti-HER2 antibody may be SBT-050 VL-Ck (pertuzumab). SBT-050 VL-Ck can be an amino acid sequence

include. SBT-050 VL-Ck may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 34 include. A light chain of anti-HER2 antibody may comprise a CDR. A light chain of anti-HER2 antibody may comprise a CDR having an amino acid sequence QDVSIG (SEQ ID NO: 35). A light chain of anti-HER2 antibody may comprise a CDR having an amino acid sequence SAS (SEQ ID NO: 36). A light chain of anti-HER2 antibody may comprise a CDR having an amino acid sequence QQYYIYPYT (SEQ ID NO: 37). An anti-HER2 antibody light chain CDR may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 35. An anti-HER2 antibody light chain CDR may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 36. An anti-HER2 antibody light chain CDR may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% % or greater than 99% homology to SEQ ID NO: 37.

An antibody construct may include an Fc region having an Fc domain. An Fc domain is a structure that can bind to Fc receptors (FcRs). An antibody construct may comprise an Fc domain. Fc domains can be bound by FcRs. An Fc domain may be of an antibody. An Fc domain may be at least 80% homologous to an Fc domain of an antibody. An Fc region can be in a scaffold. An Fc region with an Fc domain may be in an antibody framework. An Fc region with an Fc domain may be in a non-antibody framework. An antibody construct may include an Fc region having an Fc domain in an antibody framework. An antibody construct may comprise an Fc region having a Fc domain in a non-antibody framework. An Fc domain may be in a scaffold. An Fc domain may be in an antibody framework. An Fc domain may be in a non-antibody framework. An antibody construct may comprise an Fc domain in an antibody framework. An antibody construct may comprise an Fc domain in a non-antibody framework. Fc domains of antibodies, including those of the present disclosure, can be linked by FcRs. Fc domains may be part of the Fc region of an antibody. FcRs can bind to an Fc domain of an antibody. FcRs can bind to an Fc domain of an antibody bound to an antigen. FcRs can be organized into classes (eg, gamma (γ), alpha (α) and epsilon (ε)) based on the class of antibody that the FcR recognizes. The FcαR class can bind to IgA and contains several isoforms, FcαRI (CD89) and FcαμR. The FcγR class can bind to IgG and contains several isoforms, FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a) and FcγRIIIB (CD16b). An FcγRIIIA (CD16a) may be a FcγRIIIA (CD16a) F158 variant. An FcγRIIIA (CD16a) may have a FcγRIIIA (CD16a) V158 Be variant. Each FcγR isoform may differ depending on the Fc region of the IgG antibody. For example, FcγRI can bind to IgG with greater affinity than FcγRII or FcγRIII. The affinity of a particular FcγR isoform to IgG can be partially controlled by a glycan (eg, oligosaccharide) at position CH ₂ 84.4 of the IgG antibody. For example, fucose containing CH ₂ 84.4 glycans can reduce IgG affinity for FcγRIIIA. In addition, G0-glucans may have an increased affinity for FcγRIIIA due to the lack of galactose and terminal G1cNAc moiety.

The binding of an Fc domain to an FcR may enhance an immune response. FcR-mediated signaling, which can result from an Fc region that binds to an FcR, can lead to the maturation of immune cells. FcR-mediated signaling, which may result from an Fc domain that binds to an FcR, can lead to the maturation of dendritic cells. FcR-mediated signaling that can result from an Fc domain that binds to an FcR can lead to more efficient antigen uptake and processing by immune cells. FcR-mediated signaling that can result from an Fc region that binds to an FcR can lead to more efficient antigen uptake and processing by dendritic cells. FcR-mediated signaling that can result from an Fc region that binds to an FcR can increase antigen presentation. FcR-mediated signaling that can result from an Fc region that binds to an FcR can increase antigen presentation by immune cells. FcR-mediated signaling that can result from an Fc domain that binds to an FcR can increase antigen presentation by antigen presenting cells. FcR-mediated signaling that can result from an Fc region that binds to an FcR can increase antigen presentation by dendritic cells. FcR-mediated signaling, which may result from an Fc domain that binds to an FcR, may promote the expansion and activation of T cells. FcR-mediated signaling, which may result from an Fc domain that binds to an FcR, may promote the expansion and activation of CDB ⁺ T cells. FcR-mediated signaling, which may result from an Fc domain that binds to an FcR, may affect immune cell regulation of T cell responses. FcR-mediated signaling, which may result from an Fc domain that binds to an FcR, may affect immune cell regulation of T cell responses. FcR-mediated signaling, which may result from an Fc domain that binds to an FcR, may affect the regulation of T cell responses by dendritic cells. FcR-mediated signaling that can result from an Fc domain that binds to an FcR can affect the functional polarization of T cells (eg, polarization can lead to a TH1 cell response).

modifizierte Fc-Domäne kann eine Substitution einer Aminosäure im Vergleich zu SEQ ID NO: 162 umfassen. Eine Modifikation kann Bindung eines FcR an einen Ort auf der Fc-Region eines Antikörperkonstrukts ermöglichen, an dem die FcR sonst nicht binden kann. Eine Modifikation kann die Bindungsaffinität eines FcR an die Fc-Domäne eines Antikörperkonstrukts erhöhen, für das die FcR eine reduzierte Bindungsaffinität aufweisen kann. Eine Modifikation kann die Bindungsaffinität eines FcR zu einer Stelle auf der Fc-Domäne eines Antikörperkonstrukts verringern, für die die FcR eine erhöhte Bindungsaffinität aufweisen kann. Eine Modifikation kann die nachfolgende FcR-vermittelte Signalgebung nach Fc-Bindung an eine FcR erhöhen.The profile of FcRs on a DC can affect the ability of the DC to respond to stimulation. For example, most DCs can express both CD32a and CD32b, which may have opposite effects on IgG-mediated maturation and function of DCs: IgG binding to CD32a, unlike CD32b DCs, can mature and activate DCs Phosphorylation of immunoreceptor tyrosine-based inhibition motif (ITIM) after CD32b binding of IgG. Therefore, the activity of these two receptors can set a threshold of DC activation. In addition, the difference in functional avidity of these IgG receptors may shift their functional balance. Therefore, altering Fc domain binding to FcRs may also shift their functional balance, allowing for interference (either increased activity or enhanced inhibition) of the DC immune response. Modification in the amino acid sequence of the antibody construct can alter the recognition and binding of an FcR for the Fc domain. For example, modification of the Fc domain amino acid sequence in an antibody construct may increase the binding affinity and / or avidity of the Fc domain for FcRs. This increase in binding affinity and / or avidity may be specific for one type of FcR. However, such modifications may still allow FcR-mediated signaling. A modification may be a substitution of one amino acid on one residue (eg, wild-type) with another amino acid on that residue. For example, a wild-type Fc domain

Modified Fc domain may include a substitution of an amino acid as compared to SEQ ID NO: 162. One modification may be attachment of an FcR to a location on the Fc region of an antibody construct otherwise the FcR can not bind. A modification may increase the binding affinity of an FcR to the Fc domain of an antibody construct for which the FcR may have a reduced binding affinity. A modification may reduce the binding affinity of an FcR to a site on the Fc domain of an antibody construct for which the FcR may have increased binding affinity. One modification may increase subsequent FcR-mediated signaling after Fc binding to an FcR.

An antibody construct may comprise an Fc region having at least one amino acid change compared to the sequence of the wild-type Fc region. A wild-type Fc region may include SEQ ID NO: 162. An antibody construct may comprise an Fc domain having at least one amino acid change compared to the sequence of the wild-type Fc domain. A wild-type Fc domain may include SEQ ID NO: 162. For example, an antibody construct may comprise an Fc domain having at least one amino acid change as compared to the wild-type Fc domain sequence comprising SEQ ID NO: 162. An amino acid change in an Fc region of an antibody construct may allow the antibody construct to bind to at least one Fc receptor with a greater affinity compared to a wild-type Fc region. An amino acid change in an Fc domain of an antibody construct may allow the antibody to bind to at least one Fc receptor with a greater affinity compared to a wild-type Fc domain. An Fc region may have an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine, or ten modifications, but not more than 40, 35, 30, 25, 20, 15, or 10 amino acid sequence modifications relative to the natural or original amino acid sequence. An Fc domain may have an amino acid sequence having at least one, two, three, four, five, six, seven, eight, nine, or ten modifications, but not more than 40, 35, 30, 25, 20, 15, or 10 amino acid sequence modifications relative to the natural or original amino acid sequence. An Fc region may be an Fc region of an anti-CD40 antibody. An Fc domain may be an Fc domain of an anti-CD40 antibody. An Fc region can contain a Fc domain. An Fc region can be an Fc domain.

An antibody construct may be an antibody comprising a sequence of the IgG1 isoform modified from the wild-type IgG1 sequence. A wild-type IgG1 sequence may comprise SEQ ID NO: 162. One modification may involve substitution with more than one amino acid residue, such as 5 different amino acid residues, including L235V / F243L / R292P / Y300L / P396L (G1VLPLL). The numbering of amino acid residues described here can be done according to the EU index as in Kabat. The 5 amino acid residues may be located in a portion of an antibody sequence which may encode an Fc region of the antibody and, in particular, may be located in portions of the Fc region capable of binding to Fc receptors (i.e., the Fc domain). One modification may involve substitution with more than one amino acid residue, such as 2 different amino acid residues including S239D / I332E (G1DE). The 2 amino acid residues may be located in a portion of an antibody sequence encoding an Fc region of the antibody, particularly those located in portions of the Fc region capable of binding to Fc receptors (i.e., the Fc domain). One modification may involve substitution with more than one amino acid residue, such as 3 different amino acid residues, including S298A / E333A / K334A (G1AAA). The 3 amino acid residues may be located in a portion of an antibody sequence which may encode a Fc region of the antibody and, in particular, may be located in portions of the Fc region capable of binding Fc receptors (i.e., the Fc domain).

An antibody construct may be a monoclonal human anti-CD40 antibody comprising a sequence of the IgG1 isoform modified from the wild-type IgG1 sequence. A wild-type IgG1 sequence may comprise SEQ ID NO: 15. One modification may involve substitution with more than one amino acid residue, such as 5 different amino acid residues, including L235V / F243L / R292P / Y300L / P396L (SBT-040-G1VLPLL). The numbering of amino acid residues described here can be done according to the EU index as in Kabat. The 5 amino acid residues may be located in a portion of an antibody sequence which may encode an Fc region of the antibody and, in particular, may be located in portions of the Fc region capable of binding to Fc receptors (i.e., the Fc domain). One modification may involve substitution with more than one amino acid residue, such as 2 different amino acid residues including S239D / I332E (SBT-040-G1DE). The 2 amino acid residues may be located in a portion of an antibody sequence encoding an Fc region of the antibody, particularly those located in portions of the Fc region capable of binding to Fc receptors (i.e., the Fc domain). A modification may involve substitution with more than one amino acid residue, such as 3 different amino acid residues, including S298A / E333A / K334A (SBT-040-G1AAA). The 3 amino acid residues may be located in a portion of an antibody sequence which may encode a Fc region of the antibody and, in particular, may be located in portions of the Fc region capable of binding Fc receptors (i.e., the Fc domain).

The binding of Fc receptors to an Fc region can be affected by amino acid substitutions. For example, shows 4C SBT-040-G1VLPLL, which is an antibody having an amino acid sequence (SEQ ID NO: 16) of a human monoclonal anti-CD40 monoclonal antibody heavy chain with modifications to a wild-type IgG1 Fc domain (L235V / F243L / R292P / Y300L / P396L). The binding of some Fc receptors to the Fc region of SBT-040-G1VLPLL may be increased as compared to wild-type as a result of the amino acid modifications L235V / F243L / R292P / Y300L / P396L. However, the binding of other Fc receptors to the Fc region of SBT-040-G1VLPLL may be reduced by the amino acid modifications L235V / F243L / R292P / Y300L / P396L as compared to wild-type. For example, the binding affinities of SBT-040-G1VLPLL to FcγRIIIA and FcγRIIA may be increased compared to wild-type, while the binding affinity of SBT-040-G1VLPLL to FcγRIIB may be reduced compared to wild-type. 4D *** "" represents an SBT-040 G1DE antibody which is an antibody having an amino acid sequence (SEQ ID NO: 17) of a human anti-CD40 monoclonal antibody heavy chain with modifications to a wild-type IgG1 Fc domain (S239D / I332E). The binding of Fc receptors to the Fc region of SBT-040-DE may be increased as compared to wild type as a result of amino acid modifications S239D / I332E. However, binding of some Fc receptors to the Fc region of SBT-040-G1DE may be reduced by amino acid modifications S239D / I332E as compared to wild-type. For example, the binding affinities of SBT-040-G1DE to FcγRIIIA and FcγRIIB may be increased compared to wild-type. The binding of Fc receptors to an Fc region is affected by amino acid substitutions. 4E *** "" represents an SBT-040 G1AAA antibody which is an antibody having an amino acid sequence (SEQ ID NO: 18) of a human monoclonal anti-CD40 monoclonal antibody heavy chain, with modifications to a wild-type IgG1 Fc domain (S298A / E333A / K334A). The binding of Fc receptors to an Fc region of SBT-040-G1AAA may be increased as compared to wild type as a result of amino acid modifications S298A / E333A / K334A. However, binding of some Fc receptors to the Fc region of SBT-040-G1AAA may be reduced by amino acid modifications S298A / E333A / K334A compared to wild-type. The binding affinities of SBT-040-G1AAA to FcγRIIIA may be increased compared to wild-type, while the binding affinity of SBT-040-G1AAA to FcγRIIB may be reduced compared to wild-type.

In some embodiments, the heavy chain of a human IgG2 antibody may be mutated to cysteines as positions 127, 232 or 233. In some embodiments, the light chain of a human IgG2 antibody may be mutated on a cysteine at position 214. The mutations in the heavy and light chains of the human IgG2 antibody may be from a cysteine residue to a serine residue.

wobei die DNA-Sequenz DNA-Nukleotidmodifikationen aufweist, die Aminosäurerestmodifikationen L235V, F243L, R292P, Y300L und P396L im Vergleich zu einer Wildtyp-DNA-Sequenz entsprechen. SBT-040-G1VLPLL kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 9 umfasst. Eine variable Region von SBT-040-G1VLPLL kann aus einer DNA-Sequenz, die SEQ ID NO: 13 umfasst, exprimiert werden. Eine variable Region von SBT-040-G1VLPLL kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 13 umfasst. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1VLPLL exprimiert aus DNA-Sequenz umfassend SEQ ID NO: 9 oder mit mehr als 70% Homologie zu SEQ ID NO: 9 umfassen, eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 10 nM beträgt. Anti-CD40-Antikörper, die SBT-040-G1VLPLL exprimiert aus DNA-Sequenz umfassend SEQ ID NO: 9 oder mit mehr als 70% Homologie zu SEQ ID NO: 9 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G1VLPLL kann mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden. SBT-040-G1VLPLL kann auch mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden, um einen anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.An antibody construct may be a heavy chain of an anti-CD40 antibody. A heavy chain of anti-CD40 antibody may be SBT-040-G1VLPLL. SBT-040-G1VLPLL are expressed from a DNA sequence comprising

wherein the DNA sequence has DNA nucleotide modifications corresponding to L235V, F243L, R292P, Y300L and P396L amino acid residue modifications as compared to a wild-type DNA sequence. SBT-040-G1VLPLL can be expressed from a DNA sequence that is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 9. A variable region of SBT-040-G1VLPLL can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G1VLPLL can be expressed from a DNA sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater as 99% homology to SEQ ID NO: 13. In addition, anti-CD40 antibodies expressing SBT-040 G1VLPLL comprising DNA sequence comprising SEQ ID NO: 9 or having greater than 70% homology to SEQ ID NO: 9 may have a dissociation constant (K _d ) for CD40, which is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1VLPLL expressed from DNA sequence comprising SEQ ID NO: 9 or having more than 70% homology to SEQ ID NO: 9 may have a dissociation constant (K _d ) for CD40 which is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1VLPLL can be expressed with any anti-CD40 light chain or a fragment thereof. SBT-040-G1VLPLL can also be used with any anti-CD40 light chain or a fragment thereof be expressed to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

umfassen, wobei die Aminosäuresequenz Aminosäuremodifikationen L235V, F243L, R292P, Y300L, und P396L im Vergleich zu einer Wildtyp-Aminosäure-Sequenz umfasst. SBT-040-G1VLPLL kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 16 umfassen. SBT-040-G1VLPLL kann eine Aminosäuresequenz SEQ ID NO: 20 umfassen. Eine variable Region von SBT-040-G1VLPLL kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 20 umfassen. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1VLPLL mit SEQ ID NO: 16 oder mit einem Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 16 umfassen, eine Dissoziationskonstante (K_d) für CD40 aufweisen, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die SBT-040-G1VLPLL mit SEQ ID NO: 16 oder mit einer Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 16 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G1VLPLL kann gereinigt werden. SBT-040-G1VLPLL kann mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon kombiniert werden, um einen Anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.SBT-040-G1VLPLL may have an amino acid sequence

wherein the amino acid sequence comprises amino acid modifications L235V, F243L, R292P, Y300L, and P396L as compared to a wild-type amino acid sequence. SBT-040-G1VLPLL may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 16 include. SBT-040-G1VLPLL may comprise an amino acid sequence of SEQ ID NO: 20. A variable region of SBT-040-G1VLPLL may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 20 include. Additionally, anti-CD40 antibodies comprising SBT-040 G1VLPLL of SEQ ID NO: 16 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 16 may have a dissociation constant (K _d ) for CD40 smaller than 10 nM. Anti-CD40 antibodies comprising SBT-040 G1VLPLL of SEQ ID NO: 16 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 16 may have a dissociation constant (K _d ) for CD40 less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1VLPLL can be cleaned. SBT-040-G1VLPLL can be combined with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

wobei die DNA-Sequenz DNA-Nukleotidmodifikationen aufweist, die Aminosäurerestmodifikationen S239D und I332E im Vergleich zu einer Wildtyp-DNA-Sequenz entsprechen. SBT-040-G1DE kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 10 umfasst. Eine variable Region von SBT-040-G1DE kann aus einer DNA-Sequenz, die SEQ ID NO: 13 umfasst, exprimiert werden. Eine variable Region von SBT-040-G1DE kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 13 umfasst. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1DE exprimiert aus DNA-Sequenz umfassend SEQ ID NO: 10 oder mit mehr als 70% Homologie zu SEQ ID NO: 10 umfassen, eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 10 nM beträgt. Anti-CD40-Antikörper, die SBT-040-G1DE exprimiert aus DNA-Sequenz umfassend SEQ ID NO: 10 oder mit mehr als 70% Homologie zu SEQ ID NO: 10 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G1DE kann mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden. SBT-040-G1DE kann auch mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden, um einen anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.A heavy chain of anti-CD40 antibody may be SBT-040-G1DE. SBT-040-G1DE are expressed from a DNA sequence comprising

wherein the DNA sequence has DNA nucleotide modifications corresponding to amino acid residue modifications S239D and I332E as compared to a wild-type DNA sequence. SBT-040-G1DE can be expressed from a DNA sequence that is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 10. A variable region of SBT-040-G1DE can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G1DE can be expressed from a DNA sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater as 99% homology to SEQ ID NO: 13. In addition, anti-CD40 antibodies expressing SBT-040 G1DE comprising DNA sequence comprising SEQ ID NO: 10 or having greater than 70% homology to SEQ ID NO: 10 may have a dissociation constant (K _d ) for CD40. which is less than 10 nM. Anti-CD40 antibodies expressing SBT-040-G1DE comprising DNA sequence comprising SEQ ID NO: 10 or having greater than 70% homology to SEQ ID NO: 10 may have a dissociation constant (K _d ) for CD40 which is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1DE can be expressed with any anti-CD40 light chain or a fragment thereof. SBT-040-G1DE can also be expressed with any anti-CD40 light chain or a fragment thereof to produce an anti-CD40 antibody or to form a fragment of it. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

Aminosäuremodifikationen S239D und I332E im Vergleich zu einer Wildtyp-Aminosäure-Sequenz umfasst. SBT-040-G1DE kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 17 umfassen. SBT-040-G1DE kann eine Aminosäuresequenz SEQ ID NO: 20 umfassen. Eine variable Region von SBT-040-G1DE kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 20 umfassen. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1DE mit SEQ ID NO: 17 oder mit einem Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 17 umfassen, eine Dissoziationskonstante (K_d) für CD40 aufweisen, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die SBT-040-G1DE mit SEQ ID NO: 17 oder mit einer Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 17 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G1DE kann gereinigt werden. SBT-040-G1DE kann mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon kombiniert werden, um einen Anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.SBT-040G1DE can be an amino acid sequence

Amino acid modifications S239D and I332E compared to a wild-type amino acid sequence comprises. SBT-040-G1DE may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 17 include. SBT-040-G1DE may comprise an amino acid sequence of SEQ ID NO: 20. A variable region of SBT-040-G1DE may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20 include. In addition, anti-CD40 antibodies comprising SBT-040 G1DE of SEQ ID NO: 17 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 17 may have a dissociation constant (K _d ) for CD40 smaller than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1DE of SEQ ID NO: 17 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 17 may have a dissociation constant (K _d ) for CD40 less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1DE can be cleaned. SBT-040-G1DE can be combined with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

wobei die DNA-Sequenz DNA-Nukleotidmodifikationen aufweist, die Aminosäurerestmodifikationen S298A, E333A, und K334A im Vergleich zu einer Wildtyp-DNA-Sequenz entsprechen. SBT-040-G1AAA kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 11 umfasst. Eine variable Region von SBT-040-G1AAA kann aus einer DNA-Sequenz, die SEQ ID NO: 13 umfasst, exprimiert werden. Eine variable Region von SBT-040-G1AAA kann aus einer DNA-Sequenz exprimiert werden, die mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 13 umfasst. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1AAA exprimiert aus DNA-Sequenz umfassend SEQ ID NO: 11 oder mit mehr als 70% Homologie zu SEQ ID NO: 11 umfassen, eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 10 nM beträgt. Anti-CD40-Antikörper, die SBT-040-G1AAA exprimiert aus DNA-Sequenz umfassend SEQ ID NO: 11 oder mit mehr als 70% Homologie zu SEQ ID NO: 11 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G1AAA kann mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden. SBT-040-G1AAA kann auch mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon exprimiert werden, um einen anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten. SBT-040G1AAA kann eine Aminosäuresequenz

Aminosäuremodifikationen S298A, E333A, und K334A im Vergleich zu einer Wildtyp-Aminosäure-Sequenz umfasst. SBT-040-G1AAA kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 18 umfassen. SBT-040-G1AAA kann eine Aminosäuresequenz SEQ ID NO: 20 umfassen. Eine variable Region von SBT-040-G1AAA kann eine Aminosäuresequenz mit mehr als 70%, mehr als 75%, mehr als 80%, mehr als 85%, mehr als 90%, mehr als 95% oder mehr als 99% Homologie zu SEQ ID NO: 20 umfassen. Zusätzlich können Anti-CD40-Antikörper, die SBT-040-G1AAA mit SEQ ID NO: 18 oder mit einem Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 18 umfassen, eine Dissoziationskonstante (K_d) für CD40 aufweisen, die kleiner als 10 nM ist. Anti-CD40-Antikörper, die SBT-040-G1AAA mit SEQ ID NO: 18 oder mit einer Aminosäuresequenz mit mehr als 70% Homologie zu SEQ ID NO: 18 umfassen, können eine Dissoziationskonstante (K_d) für CD40 aufweisen, die weniger als 1 nM, weniger als 100 pM, weniger als 10 pM, weniger als 1 pM oder weniger als 0,1 pM beträgt. SBT-040-G1AAA kann gereinigt werden. SBT-040-G1AAA kann mit jeglicher Anti-CD40-leichten Kette oder einem Fragment davon kombiniert werden, um einen Anti-CD40-Antikörper oder ein Fragment davon zu bilden. Der Anti-CD40-Antikörper oder das Fragment davon kann gereinigt werden und kann mit einem pharmazeutisch annehmbaren Träger kombiniert werden. Der Anti-CD40-Antikörper kann ein Antikörperkonstrukt sein. Zusätzlich würde der Fachmann erkennen, dass diese Konzepte für Antikörperkonstrukte, die Anti-CD40-Antikörper umfassen, die für die Verwendung in den Veterinärwissenschaften und/oder in Labortieren geschaffen wurden, gelten könnten.A heavy chain of anti-CD40 antibody may be SBT-040-G1AAA. SBT-040-G1AAA are expressed from a DNA sequence comprising

wherein the DNA sequence has DNA nucleotide modifications corresponding to amino acid residue modifications S298A, E333A, and K334A as compared to a wild-type DNA sequence. SBT-040-G1AAA can be expressed from a DNA sequence that is greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 11. A variable region of SBT-040-G1AAA can be expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of SBT-040-G1AAA can be expressed from a DNA sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater as 99% homology to SEQ ID NO: 13. In addition, anti-CD40 antibodies expressing SBT-040 G1AAA comprising DNA sequence comprising SEQ ID NO: 11 or having greater than 70% homology to SEQ ID NO: 11 may have a dissociation constant (K _d ) for CD40, which is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1AAA comprised of DNA sequence comprising SEQ ID NO: 11 or having greater than 70% homology to SEQ ID NO: 11 may have a dissociation constant (K _d ) for CD40 which is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1AAA can be expressed with any anti-CD40 light chain or a fragment thereof. SBT-040-G1AAA can also be expressed with any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment it can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals. SBT-040G1AAA may have an amino acid sequence

Amino acid modifications S298A, E333A, and K334A compared to a wild-type amino acid sequence. SBT-040-G1AAA may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 18 include. SBT-040-G1AAA may comprise an amino acid sequence of SEQ ID NO: 20. A variable region of SBT-040-G1AAA may have an amino acid sequence greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20 include. Additionally, anti-CD40 antibodies comprising SBT-040-G1AAA of SEQ ID NO: 18 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 18 may have a CD40 dissociation constant (K _d ) smaller than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1AAA of SEQ ID NO: 18 or having an amino acid sequence of greater than 70% homology to SEQ ID NO: 18 may have a dissociation constant (K _d ) for CD40 less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM or less than 0.1 pM. SBT-040-G1AAA can be cleaned. SBT-040-G1AAA can be combined with any anti-CD40 light chain or a fragment thereof to form an anti-CD40 antibody or a fragment thereof. The anti-CD40 antibody or fragment thereof can be purified and combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody may be an antibody construct. In addition, those skilled in the art would recognize that these concepts could apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

While an antibody construct of the present disclosure may comprise an anti-CD40 antibody having wild-type or modified amino acid sequences encoding the Fc region or the Fc domain, the modifications of the Fc region or Fc domain from the wild-type sequence may be binding and / or affinity of the anti-CD40 antibody for CD40 also did not change significantly. For example, the binding and / or affinity of SBT-040-G1WT, SBT-040-G1VLPLL, SBT-040-G1DE and SBT-040-G1AAA can not be significantly altered even by modification of an Fc region or Fc domain amino acid sequence with a wild-type sequence. Modifications of an Fc region or a Fc domain from a wild-type sequence also can not alter the binding and / or affinity of antibodies that bind to CD40 in an antibody construct. In addition, the binding and / or affinity of the antibodies described herein which bind to CD40 and are antibody constructs, e.g., SBT-040-G1WT, SBT-040-G1VLPLL, SBT-040-G1DE, and SBT-040-G1AAA may also be comparable to that Binding and / or affinity of wild-type antibodies capable of binding to CD40.

Sequences that can be used to make antibodies to antibody constructs may contain leader sequences. Leader sequences can be signal sequences. Leader sequences useful in the compositions and methods described herein include, but are not limited to, a DNA sequence comprising ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTTCCAGATGC (SEQ ID NO: 2) or ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC (SEQ ID NO: 12), or an amino acid sequence comprising MRLPAQLLGLLLLWFPGSRC (SEQ ID NO: 5) and MDWTWRILFLVAAATGAHS (SEQ ID NO: 19). The leader sequence may contain a DNA sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 2 or Include SEQ ID NO: 12. The leader sequence may have an amino acid sequence of greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% homology to SEQ ID NO: 5 or SEQ ID NO: 19 include. Any of the sequences described herein can be used with or without a leader sequence. In addition, those skilled in the art would recognize that these concepts may apply to antibody constructs comprising anti-CD40 antibodies designed for use in veterinary sciences and / or laboratory animals.

Targeting binding domain

An antibody construct may further comprise a targeting binding domain. A targeting domain may comprise a domain that binds to a target. A target can be an antigen. A targeting domain may include an antigen binding domain. A targeting domain may be a domain that can specifically bind to an antigen. A targeting domain may be an antigen-binding portion of an antibody or antibody fragment. A targeting domain can be one or more fragments of an antibody that can retain the ability to specifically bind to an antigen. A targeting domain can be any antigen binding fragment. A targeting domain may be in a framework in which a framework is a supportive framework for the antigen binding domain. A targeting domain may comprise an antigen binding domain in a scaffold.

A targeting domain may comprise an antigen binding domain, which may refer to a portion of an antibody comprising the antigen recognition portion, i. H. an antigen-determining variable region of an antibody sufficient to confer recognition and binding of the antigen-recognition portion to a target such as an antigen, d. H. the epitope. Examples of a targeting domain may include, but are not limited to, Fab, a single chain variable fragment (scFv), variable Fv fragment and other fragments, combinations of fragments or types of fragments known or well-known to those skilled in the art. A targeting domain may comprise an antigen binding domain, which may refer to a portion of an antibody comprising the antigen recognition portion, i. H. an antigen-determining variable region of an antibody sufficient to confer recognition and binding of the antigen-recognition portion to a target such as an antigen, d. H. the epitope. Examples of a targeting domain may include, but are not limited to, Fab, a single chain variable fragment (scFv), variable Fv fragment and other fragments, combinations of fragments or types of fragments known or well-known to those skilled in the art.

A targeting domain may comprise an antigen-binding domain of an antibody. An antigen-binding domain of an antibody may comprise one or more light chain CDRs (LC) and one or more heavy chain CDRs (HC). For example, an antibody binding domain of an antibody may include one or more of the following: a complementarity determining region 1 (LC CDR1) of the light chain, a complementarity determining region 2 (LC CDR2) of the light chain, or a complementarity determining region 3 of the light chain (LC CDR3). As another example, an antibody binding domain may include one or more of the following: a complementarity determining region 1 (HC CDR1) of the heavy chain, a complementarity determining region 2 (HC CDR2) of the heavy chain or a complementarity determining region 3 of the heavy chain (HC CDR3).

An antibody construct may comprise an antibody fragment. An antibody fragment may be (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , C _L and C _H1 domains; (ii) an F (ab ') ₂ fragment, a bivalent fragment comprising two Fab fragments joined by a disulfide bond at the hinge region; and (iii) an Fv fragment consisting of the V _L and V _H domains of a single arm of an antibody. Although the two domains of the Fv fragment, V _L and V _H , can be encoded by separate genes, they can be linked by a synthetic linker to be produced as a single protein chain in which the V _L and V _H Pair regions to form monovalent molecules.

F (ab ') ₂ and Fab' groups can be prepared by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain and can contain an antibody fragment obtained by digestion of immunoglobulin near the disulfide bonds that occur between the Hinge regions are present in each of the two H chains is generated.

An Fv may be the minimal antibody fragment containing a complete antigen recognition and antigen binding site. This region may consist of a dimer of a heavy chain variable domain and one of the light chain in close noncovalent association. In this configuration, the three hypervariable regions of each variable domain can interact to define an antigen binding site on the surface of the V _H -V _L dimer. A single variable domain (or half of a Fv comprising only three hypervariable regions specific for an antigen) can recognize and bind antigen, albeit at a lower affinity than the entire binding site.

A targeting domain may be at least 80% homologous to an antigen binding domain selected from, but not limited to, a monoclonal antibody, a polyclonal antibody, a recombinant antibody or a functional fragment thereof, for example a heavy chain variable domain (V _H ) and light chain variable domain (V _L ), a DARPin, an affimer, an avimer, a knottin, a monobody, an affinity bracket, an ectodomain, a receptor ectodomain, a receptor, a cytokine, a ligand, an immunocytokine, a T cell receptor or a recombinant T cell receptor.

A targeting domain may comprise an antigen binding domain comprising a monoclonal antibody light chain and heavy chain. In one aspect, a targeting domain binds to CD40 and comprises the anti-CD40 antibody light chain and the anti-CD40 antibody heavy chain that bind a CD40 antigen. In another aspect, the targeting domain binds to a tumor antigen comprising the tumor antigen antibody light chain and the tumor antigen antibody heavy chain that binds the tumor antigen.

A targeting domain may be attached to an antibody construct. For example, an antibody construct can be fused to a targeting binding domain to generate an antibody construct targeting binding domain fusion. The antibody construct targeting binding domain fusion may be the result of expression of the nucleic acid sequence of the targeting binding domain in frame with the nucleic acid sequence of the antibody construct. The antibody construct targeting binding domain fusion may be the result of an in-frame genetic nucleotide sequence or a contiguous peptide sequence encoding the antibody construct with the targeting binding domain. As another example, a targeting-binding domain can be linked to an antibody construct. A targeting-binding domain can be linked to an antibody construct by chemical conjugation. For example, the targeting binding domain can target the antibody construct to a particular cell or cell type. A targeting binding domain of an antibody construct can be selected to recognize an antigen. For example, an antigen can be expressed on an immune cell. An antigen may be a peptide or fragment thereof. An antigen can be expressed on an antigen-presenting cell. An antigen can be expressed on a dendritic cell, a macrophage or a B cell. An antigen can be CD40 and a targeting binding domain can recognize a CD40 antigen. A targeting binding domain may be a CD40 agonist. For example, a targeting domain can recognize CD40 on an antigen presenting cell. As another example, an antigen can be a tumor antigen. The tumor antigen may be any tumor antigen described herein.

Immunostimulatory compounds

Pattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). A PRR can be membrane bound. A PRR can be cytosolic. A PRR can be a toll-like receptor (TLR). A PRR may be a RIG-I-like receptor. A PRR can be a receptor kinase. A PRR can be a C-type lectin receptor. A PRR can be an NOD-like receptor. A PRR can be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

A PRR agonist may be a pathogen-associated molecular pattern (PAMP) molecule. A PAMP molecule can be a toll-like receptor agonist. A PRR agonist may be a toll-like receptor agonist. A toll-like receptor agonist may be any molecule that acts as an agonist to at least one toll-like receptor. A toll-like receptor agonist may be bacterial lipoprotein. Toll-like receptor agonist may be bacterial peptidoglycans. A toll-like receptor agonist may be double-stranded RNA. A toll-like receptor agonist may be lipopolysaccharides. A toll-like receptor agonist may be bacterial flagella. A toll-like receptor agonist may be single-stranded RNA. A toll-like receptor may be CpG DNA. A toll-like receptor agonist may be imiquimod. A toll-like receptor agonist may be CL307. A Toll-like receptor agonist may be S-27609. A toll-like receptor agonist may be resiquimod. A great-like Receptor agonist may be UC-IV 150. A toll-like receptor agonist may be gardiquimod. A toll-like receptor agonist may be motolimod. A toll-like receptor agonist may be VTX-1463. A toll-like receptor agonist may be GS-9620. A toll-like receptor agonist may be GSK2245035. A toll-like receptor agonist may be TMX-101. A toll-like receptor agonist may be TMX-201. A toll-like receptor agonist may be TMX-202. A toll-like receptor agonist may be isatoribin. A toll-like receptor agonist may be AZD8848. A toll-like receptor agonist may be MEDI9197. A toll-like receptor agonist may be 3M-051. A toll-like receptor agonist may be 3M-852. A toll-like receptor agonist may be 3M-052. A toll-like receptor agonist may be 3M-854A. A toll-like receptor agonist may be S-34240. A toll-like receptor agonist may be CL663. An RIG-I agonist may be KIN1148. An RIG-I agonist may be SB-9200. An RIG-I agonist may be KIN700, KIN600, KIN500, KIN100, KIN101, KIN400 or KIN2000. A Toll-like receptor agonist may be KU34B.

A PRR agonist may be a damage associated molecular pattern (DAMP) molecule. A DAMP molecule can be an intracellular protein. A DAMP molecule can be a heat shock protein. A DAMP molecule can be an HMGB1 protein. A DAMP molecule may be a protein derived from the extracellular matrix produced after tissue injury. A DAMP molecule can be a hyaluronan fragment. A DAMP molecule can be DNA. A DAMP molecule can be RNA. A DAMP molecule can be an S100 molecule. A DAMP molecule can be nucleotides. A DAMP molecule can be an ATP. A DAMP molecule can be nucleosides. A DAMP molecule can be an adenosine. A DAMP molecule can be uric acid.

wobei in manchen Ausführungsformen X₁=X₂=O; X₃=G; X₄=G; X₅=CO(CH₂)₁₂CH₃, X₆=2 TEAH; in manchen Ausführungsformen X₁=X₂=S [R_p,R_p]; X₃=G; X₄=A; X₅=H; X₆=2 TEAH; in manchen Ausführungsformen X₁=X₂=S [R_p,R_p]; X₃=A; X₄=A; X₅=H; X₆=2 Na; in manchen Ausführungsformen X₁=X₂=S [R_p,R_p]; X₃=A; X₄=A; X₅=H; X₆=2 NH₄; und in manchen Ausführungsformen X₁=X₂=O; X₃=G; X₄=A; X₅=H; X₆=2 TEAH,

wobei R₁=R₂=H; R₁=Propargyl, R₂=H; R₁=H, R₂=Propargyl; R₁=Allyl, R₂=H; R₁=H, R₂=Allyl; R₁=Methyl, R₂=H; R₁=H, R₂=Methyl; R₁=Ethyl, R₂=H; R₁=H, R₂=Ethyl; R₁=Propyl, R₂=H; R₁=H, R₂=Propyl; R₁=Benzyl, R₂=H; R₁=H, R₂=Benzyl; R₁=Myristoyl, R₂=H; R₁=H, R₂=Myristoyl; R₁=R₂=Heptanoyl; R₁=R₂=Hexanoyl; oder R₁=R₂=Pentanoyl,

wobei R₁=R₂=H; R₁=Propargyl, R₂=H; R₁=H, R₂=Propargyl; R₁=Allyl, R₂=H; R₁=H, R₂=Allyl; R₁=Methyl, R₂=H; R₁=H, R₂=Methyl; R₁=Ethyl, R₂=H; R₁=H, R₂=Ethyl; R₁=Propyl, R₂=H; R₁=H, R₂=Propyl; R₁=Benzyl, R₂=H; R₁=H, R₂=Benzyl; R₁=Myristoyl, R₂=H; R₁=H, R₂=Myristoyl; R₁=R₂=Heptanoyl; R₁=R₂=Hexanoyl; oder R₁=R₂=Pentanoyl,

wobei R₁=R₂=H; R₁=Propargyl, R₂=H; R₁=H, R₂=Propargyl; R₁=Allyl, R₂=H; R₁=H, R₂=Allyl; R₁=Methyl, R₂=H; R₁=H, R₂=Methyl; R₁=Ethyl, R₂=H; R₁=H, R₂=Ethyl; R₁=Propyl, R₂=H; R₁=H, R₂=Propyl; R₁=Benzyl, R₂=H; R₁=H, R₂=Benzyl; R₁=Myristoyl, R₂=H; R₁=H, R₂=Myristoyl; R₁=R₂=Heptanoyl; R₁=R₂=Hexanoyl; oder R₁=R₂=Pentanoyl,

wobei jedes X unabhängig O oder S ist, und R3 und R4 jedes unabhängig H oder ein optional substituiertes geradketiges Alkyl aus von 1 bis 18 Kohlenstoffen und von 0 bis 3 Heteroatomen, ein optional substituiertes Alkenyl aus von 1-9 Kohlenstoffen, ein optional substituiertes Alkinyl aus von 1-9 Kohlenstoffen or ein optional substituiertes Aryl sind, wobei Substitution(en), falls vorhanden, unabhängig ausgewählt werden können aus der Gruppe bestehend aus verzweigtem oder geradkettigem C_1-6-Alkyl, Benzyl, Halogen, Trihalomethyl, C_1-6-Alkoxy, -NO₂, -NH₂, -OH, =O, -COOR' wobei R' H oder niederes Alkyl ist, -CH₂OH, und -CONH₂, wobei R3 und R4 nicht beide H sind,

wobei X₁=X₂=O; X₁=X₂=S; oder X₁=O und X₂=S,

Additionally, the stimulator of interferon genes (STING) may act as a cytosolic DNA sensor, recognizing cytosolic DNA and unique bacterial nucleic acids called cyclic dinucleotides that are STING agonists. Interferon regulatory factor (IRF) agonist may be KIN-100. Non-limiting examples of STING agonists include:

wherein in some embodiments X ₁ = X ₂ = O; X ₃ = G; X ₄ = G; X ₅ = CO (CH ₂ ) ₁₂ CH ₃ , X ₆ = 2 TEAH; in some embodiments X ₁ = X ₂ = S [R _p , R _p ]; X ₃ = G; X ₄ = A; X ₅ = H; X ₆ = 2 TEAH; in some embodiments X ₁ = X ₂ = S [R _p , R _p ]; X ₃ = A; X ₄ = A; X ₅ = H; X ₆ = 2 Na; in some embodiments X ₁ = X ₂ = S [R _p , R _p ]; X ₃ = A; X ₄ = A; X ₅ = H; X ₆ = 2 NH ₄ ; and in some embodiments X ₁ = X ₂ = O; X ₃ = G; X ₄ = A; X ₅ = H; X ₆ = 2 TEAH,

wherein R ₁ = R ₂ = H; R ₁ = propargyl, R ₂ = H; R ₁ = H, R ₂ = propargyl; R ₁ = allyl, R ₂ = H; R ₁ = H, R ₂ = allyl; R ₁ = methyl, R ₂ = H; R ₁ = H, R ₂ = methyl; R ₁ = ethyl, R ₂ = H; R ₁ = H, R ₂ = ethyl; R ₁ = propyl, R ₂ = H; R ₁ = H, R ₂ = propyl; R ₁ = benzyl, R ₂ = H; R ₁ = H, R ₂ = benzyl; R ₁ = myristoyl, R ₂ = H; R ₁ = H, R ₂ = myristoyl; R ₁ = R ₂ = heptanoyl; R ₁ = R ₂ = hexanoyl; or R ₁ = R ₂ = pentanoyl,

wherein R ₁ = R ₂ = H; R ₁ = propargyl, R ₂ = H; R ₁ = H, R ₂ = propargyl; R ₁ = allyl, R ₂ = H; R ₁ = H, R ₂ = allyl; R ₁ = methyl, R ₂ = H; R ₁ = H, R ₂ = methyl; R ₁ = ethyl, R ₂ = H; R ₁ = H, R ₂ = ethyl; R ₁ = propyl, R ₂ = H; R ₁ = H, R ₂ = propyl; R ₁ = benzyl, R ₂ = H; R ₁ = H, R ₂ = benzyl; R ₁ = myristoyl, R ₂ = H; R ₁ = H, R ₂ = myristoyl; R ₁ = R ₂ = heptanoyl; R ₁ = R ₂ = hexanoyl; or R ₁ = R ₂ = pentanoyl,

wherein R ₁ = R ₂ = H; R ₁ = propargyl, R ₂ = H; R ₁ = H, R ₂ = propargyl; R ₁ = allyl, R ₂ = H; R ₁ = H, R ₂ = allyl; R ₁ = methyl, R ₂ = H; R ₁ = H, R ₂ = methyl; R ₁ = ethyl, R ₂ = H; R ₁ = H, R ₂ = ethyl; R ₁ = propyl, R ₂ = H; R ₁ = H, R ₂ = propyl; R ₁ = benzyl, R ₂ = H; R ₁ = H, R ₂ = benzyl; R ₁ = myristoyl, R ₂ = H; R ₁ = H, R ₂ = myristoyl; R ₁ = R ₂ = heptanoyl; R ₁ = R ₂ = hexanoyl; or R ₁ = R ₂ = pentanoyl,

wherein each X is independently O or S, and R3 and R4 are each independently H or an optionally substituted straight chain alkyl of from 1 to 18 carbons and from 0 to 3 heteroatoms, an optionally substituted alkenyl of from 1-9 carbons, an optionally substituted alkynyl are from 1-9 carbons or an optionally substituted aryl, said substitution (s), if present, can be independently selected from the group consisting of branched or straight chain C _1-6 alkyl, benzyl, halogen, trihalomethyl consisting, C _{1- 6} -alkoxy, -NO ₂ , -NH ₂ , -OH, = O, -COOR 'where R' is H or lower alkyl, -CH ₂ OH, and -CONH ₂ , where R3 and R4 are not both H,

where X ₁ = X ₂ = O; X ₁ = X ₂ = S; or X ₁ = O and X ₂ = S,

An immunostimulatory compound may be a PRR agonist. An immunostimulatory compound may be a PAMP. An immunostimulatory compound may be a DAMP. An immunostimulatory compound may be a TLR agonist. An immunostimulatory compound may be a STING agonist. An immunostimulatory compound may be a cyclic dinucleotide.

An immunostimulatory compound can be a drug.

The specificity of the antigen-binding domain for an antigen of an antibody construct in a conjugate of antibody construct and immunostimulatory compound as disclosed herein may be affected by the presence of an immunostimulatory compound. The antigen-binding domain of the antibody construct in a conjugate of antibody construct and immunostimulatory compound may be linked to an antigen of at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%. , about 85%, about 90%, about 95%, or about 100% of a specificity of the antigen-binding domain for the antigen in the absence of the immunostimulatory compound.

The specificity of the Fc domain for an Fc receptor of an antibody construct in a conjugate of antibody construct and immunostimulatory compound as disclosed herein can be understood by the Presence of an immunostimulatory compound can be influenced. The Fc domain of the antibody construct in a conjugate of antibody construct and immunostimulatory compound may be linked to an Fc receptor having at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60% 70%, about 80% %, about 85%, about 90%, about 95%, or about 100% of a specificity of the Fc domain for the Fc receptor in the absence of the immunostimulatory compound.

The affinity of the antigen-binding domain to an antigen of an antibody construct in a conjugate of antibody construct and immunostimulatory compound as disclosed herein may be affected by the presence of an immunostimulatory compound. The antigen-binding domain of the antibody construct in a conjugate of antibody construct and immunostimulatory compound may be linked to an antigen of at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%. , about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of an antigen-binding domain affinity to the antigen in the absence of the immunostimulatory compound.

The affinity of the Fc domain for an Fc receptor of an antibody construct in a conjugate of antibody construct and immunostimulatory compound as disclosed herein may be affected by the presence of an immunostimulatory compound. The Fc domain of the antibody construct in a conjugate of antibody construct and immunostimulatory compound may be linked to an Fc receptor with at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, conjugate about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of an Fc domain affinity to the Fc receptor in the absence of the immunostimulatory compound.

The K _d for the binding of an antigen-binding domain of a conjugate of antibody construct and immunostimulatory compound to an antigen in the presence of an immunostimulatory compound may be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, for example 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times , about 80 times, about 90 times, about 100 times, about 110 times or about 120 times greater than the K _d for the binding of the antigen-binding domain to the antigen of an antibody construct in the absence of the immunostimulatory compound. The K _d for the binding of an antigen-binding domain of a conjugate of antibody construct and immunostimulatory compound to an antigen in the presence of the immunostimulatory compound may be less than 10 nM. The K _d for the binding of an antigen-binding domain of a conjugate of antibody construct and immunostimulatory compound to an antigen in the presence of the immunostimulatory compound may be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM or less than 0.1 nM.

The K _d for the binding of an Fc domain of a conjugate of antibody construct and immunostimulatory compound to an Fc receptor in the presence of the immunostimulatory compound may be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, for example 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times , about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the K _d for the binding of the Fc domain to the Fc receptor in the absence of the immunostimulatory compound. The K _d for the binding of an Fc domain of a conjugate of antibody construct and immunostimulatory compound to an Fc receptor in the presence of the immunostimulatory compound may be less than 10 nM. The K _d for the binding of an Fc domain of a conjugate of antibody construct and immunostimulatory compound to an Fc receptor in the presence of the immunostimulatory compound may be less than 10 μM, less than 1 μM, less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM or less than 0.1 nM.

Affinity can be the strength of the sum of the noncovalent interactions between a single binding site of a molecule, such as an antibody, and the binding partner of the molecule, such as an antigen. The affinity can also measure the strength of an interaction between an Fc portion of an antibody and the Fc receptor. Unless otherwise stated, "binding affinity" as used herein may refer to an intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen or Fc domain and Fc receptor) , The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K _d ). The affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

In some embodiments, an antibody contained therein may have a dissociation constant (K _d ) of about 1 μM, about 100 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 0.5 nM, about 0.1 nM , about 0.05 nM, about 0.01 nM, or about 0.001 nM or less (eg, 10 ^-8 M or less, eg, from 10 ^-8 M to 10 ^-13 M, eg, from 10 ^-9 M to 10 ^-13 M). An affinity matured antibody can be an antibody having one or more changes in one or more complementarity determining regions (CDRs) compared to a parent antibody that may not possess such alterations, such changes leading to an improvement in the affinity of the antibody for antigen. These antibodies can bind to their antigen with a K _d of about 5 × 10 ⁹ M, about 2 × 10 ^-9 M, about 1 × 10 ^-9 M, about 5 × 10 ^-1 M, about 2 × 10 ^-9 M, about 1 × 10 ^-10 M, about 5 × 10 ^-11 M, about 1 × 10 ^-11 M, about 5 × 10 ^-12 M, about 1 × 10 ^-12 M or less. In some In embodiments, the antibody construct may have an increased affinity of at least 1.5, 2, 2.5, 3, 4, 5, 10, 20, or more compared to one Antibody construct without changes in one or more complementarity determining regions.

K _d can be measured by any suitable test. For example, K _d can be measured by a radiolabeled antigen binding assay (RIA). For example, K _d can be measured using surface plasmon resonance assays (eg., Using a BIACORE ^® -2000 or a BIACORE ^® -3000).

Agonism can be described as the binding of a chemical to a receptor to induce a biological response. A chemical can be, for example, a small molecule, a compound or a protein. An agonist causes a response, an antagonist can block the action of an agonist, and an inverse agonist can elicit a response that is opposite to that of the agonist. A receptor can be activated by either endogenous or exogenous agonists.

The molar ratio of a conjugate of antibody construct and immunostimulatory compound can refer to the average number of immunostimulatory compounds conjugated to the antibody construct in a preparation of a conjugate of antibody construct and immunostimulatory compound. The molar ratio can be determined, for example, by liquid chromatography / mass spectrometry (LC / MS), whereby the number of immunostimulatory compounds conjugated to the antibody construct can be determined directly. In addition, as non-limiting examples, the molar ratio may be determined on the basis of the hydrophobic interaction chromatography (HIC) peak area, coupled by liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS), by UV / Vis spectroscopy, by reverse phase chromatography. HPLC (RP-HPLC) or matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

In some embodiments, the molar ratio of immunostimulatory compound to antibody may be less than 8. In other embodiments, the molar ratio of immunostimulatory compound to antibody may be 8, 7, 6, 5, 4, 3, 2, or 1.

oder ein pharmazeutisch annehmbares Salz davon, wobei:
X¹ ausgewählt ist aus -OR² und -SR²;
X² ausgewählt ist aus -OR³ und -SR³;
B¹ und B² unabhängig ausgewählt sind aus gegebenenfalls substituierten stickstoffhaltigen Basen;
Y ausgewählt ist aus -OR⁴, -NR⁴R⁴, und Halogen;
R¹, R², R³ und R⁴ bei jedem Auftreten unabhängig ausgewählt sind aus Wasserstoff, -C(=O)R¹⁰⁰, -C(=O)OR¹⁰⁰ und -C(=O)NR¹⁰⁰; C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, von denen jedes bei jedem Auftreten unabhängig gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus; und C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, wobei jeder C_3-10-Carbozyklus und 3- bis 10-gliedrige Heterozyklus in R¹, R², R³ und R⁴ unabhängig gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)2, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_1-6-Alkyl, C_2-6-Alkenyl, und C_2-6-Alkinyl; und
R¹⁰⁰ bei jedem Auftreten unabhängig ausgewählt ist aus Wasserstoff; und C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, C_3-10-Carbozyklus, und 3- bis 10-gliedrigem Heterozyklus, von denen jedes bei jedem Auftreten unabhängig gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -CN, -NO₂, =O, =S, und Haloalkyl.In some aspects, the present disclosure provides a compound represented by formula (I):

or a pharmaceutically acceptable salt thereof, wherein:
X ^{1 is} selected from -OR ² and -SR ² ;
X ^{2 is} selected from -OR ³ and -SR ³ ;
B ¹ and B ^{2 are} independently selected from optionally substituted nitrogenous bases;
Y is selected from -OR ⁴ , -NR ⁴ R ⁴ , and halogen;
R ¹ , R ² , R ³ and R ⁴ at each occurrence are independently selected from hydrogen, -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each independently independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle, and _3-10 -linked heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carboxylic cycle and 3- to 10-membered heterocycle in R ¹ , R ² , R ³ and R ^{4 is} independently optionally substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) 2, -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl; and
R ^{100 is} independently selected at each occurrence from hydrogen; and C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbo cycle, and _3-10 membered heterocycle each independently independently substituted on each occurrence with one or more substituents selected from halogen, -CN, -NO ₂ , = O, = S, and haloalkyl.

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound of formula (I) is represented by formula (IA):

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon.In an alternative embodiment, the compound of formula (I) is represented by formula (IB):

or a pharmaceutically acceptable salt thereof.

In bestimmten Ausführungsformen sind B¹ und B² unabhängig ausgewählt aus gegebenenfalls substituierten Pyrimidinen.In various embodiments, B ¹ and B ^{2 are} independently selected from optionally substituted purines. In certain embodiments, B ¹ and B ^{2 are} independently selected from:

In certain embodiments, B ¹ and B ^{2 are} independently selected from optionally substituted pyrimidines.

gegebenenfalls substituiert durch ein oder mehr zusätzliche Substituenten.In some embodiments, optionally substituted purines may include optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxaanthin, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid and optionally substituted isoguanine. In certain embodiments, B ¹ and B ^{2 are} independently selected from:

optionally substituted by one or more additional substituents.

, wobei der Anknüpfungspunkt von B¹ an den Rest der Verbindung durch dargestellt ist.In certain embodiments, B ¹ and B ^{2 are} independently selected from:

, wherein the point of attachment of B ¹ to the rest of the compound is represented by.

, gegebenenfalls weiter substituiert mit einem oder mehr Substituenten. In bestimmten Ausführungsformen sind B¹ und B² unabhängig ausgewählt aus:

und

In a preferred embodiment, B ¹ and B ^{2 are} independently selected from optionally substituted adenine and optionally substituted guanine. In certain embodiments, B ¹ and B ^{2 are} independently selected from:

optionally further substituted with one or more substituents. In certain embodiments, B ¹ and B ^{2 are} independently selected from:

and

In some embodiments, B ¹ and B ^{2 are} independently optionally substituted with one or more substituents, with the optional substituents on B ¹ and B ² independently selected on each occurrence from halogen, = O, = S, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰⁾ _2, -S (O) R ^100, -S (O) 2 R ^100, -C (O) R ^100, -C (O) OR ^100, -OC (O) R ^100, - NO ₂ , -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ and -CN; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each independently independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰⁾ _2, -S (O) R ^100, -S (O) 2 R ^100, -C (O) R ^100, -C (O) OR ^100, -OC (O) R ^100, -NO _2, = O, = S, = N (R ^100), -P (O) (OR ¹⁰⁰⁾ _2, -OP (O) (OR ¹⁰⁰⁾ _2, -CN, C _3-10 -Carbozyklus and 3- to 10- hetero-cyclic; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carbo-cycle and 3- to 10-membered heterocycle in R ¹ , R ² , R ³ and R ^{4 is} independently optionally substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl.

In some embodiments, B ¹ and B ^{2 are} independently optionally substituted with one or more substituents, wherein the optional substituents on B ¹ and B ^{2 are} independently selected on each occurrence from halogen, = S, -OR ¹⁰⁰ , -SR ¹⁰⁰ , N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN and C _1-10 alkyl.

In bestimmten Ausführungsformen ist

wobei der Anknüpfungspunkt von B¹ an den Rest der Verbindung durch

dargestellt ist. In manchen Ausführungsformen ist B¹ ein gegebenenfalls substituiertes Adenin. In bestimmten Ausführungsformen ist

In bestimmten Ausführungsformen ist

wobei der Anknüpfungspunkt von B¹ an den Rest der Verbindung durch

dargestellt ist.In some embodiments, B ^{1 is} an optionally substituted guanine. In certain embodiments

In certain embodiments

wherein the point of attachment of B ¹ to the rest of the compound

is shown. In some embodiments, B ^{1 is} an optionally substituted adenine. In certain embodiments

In certain embodiments

wherein the point of attachment of B ¹ to the remainder of the compound by

is shown.

In bestimmten Ausführungsformen ist

wobei der Anknüpfungspunkt von B² an den Rest der Verbindung durch

dargestellt ist. In manchen Ausführungsformen ist B² ein gegebenenfalls substituiertes Adenin. In bestimmten Ausführungsformen ist

In bestimmten Ausführungsformen ist B²

wobei der Anknüpfungspunkt von B² an den Rest der Verbindung durch

dargestellt ist.In some embodiments, B ² is an optionally substituted guanine. In certain embodiments

In certain embodiments

wherein the point of attachment of B ² to the rest of the compound

is shown. In some embodiments, B ^{2 is} an optionally substituted adenine. In certain embodiments

In certain embodiments, B is ²

wherein the point of attachment of B ² to the rest of the compound

is shown.

In some embodiments, B ¹ is an optionally substituted guanine and B ² is an optionally substituted guanine. In some embodiments, B ^{1 is} an optionally substituted adenine and B ^{2 is} an optionally substituted guanine.

In various embodiments, X ^{I is} selected from -OH and -SH. For example, X ¹ can be -OH. In various embodiments, X ^{2 is} selected from -OH and -SH. For example, X ² can be -OH. In some embodiments, X ^{1 is} -OH and X ^{2 is} -OH. In some embodiments, X ^{1 is} -SH and X ^{2 is} -SH.

In various embodiments, Y is selected from -OH, -OC _1-10 alkyl, -NH (C _1-10 alkyl), and -NH ₂ . For example, Y can be -OH.

In various embodiments, R ^{100 is} independently selected from hydrogen and C _1-10 alkyl at each occurrence, optionally substituted at each occurrence with one or more substituents selected from halogen, -CN, -NO ₂ ; = O and = S.

oder ein pharmazeutisch annehmbares Salz davon. In manchen Ausführungsformen wird die Verbindung der Formel (I) dargestellt durch Formel (ID):

oder ein pharmazeutisch annehmbares Salz davon.In various embodiments, the compound of formula (I) is represented by formula (IC):

or a pharmaceutically acceptable salt thereof. In some embodiments, the compound of formula (I) is represented by formula (ID):

or a pharmaceutically acceptable salt thereof.

In various embodiments, the compound is a pharmaceutically acceptable salt. In some embodiments, the compound or salt is a modulator of a stimulator of interferon genes (STING). The compound or salt may agonize a stimulator of interferon genes (STING). In certain embodiments, the compound or salt may cause STING to coordinate multiple immune responses to infection, including the induction of interferons and a STAT6-dependent response and a selective autophagy response. In certain embodiments, the compound or salt may cause STING to mediate type I interferon production.

In some aspects, the present disclosure provides an antibody-drug conjugate comprising a compound described above or a salt, an antibody and a linker group, wherein the compound or salt is linked to the antibody through the linker group, e.g. B. covalently bonded, is. The linker group may be selected from a cleavable or non-cleavable linker. In some embodiments, the linker group is cleavable. In alternative embodiments, the linker group is not cleavable. Linkers are further described in the present application in the following section, any of which may be used to link an antibody to a compound described herein.

oder ein pharmazeutisch annehmbares Salz davon, wobei
X¹ ausgewählt ist aus -OR² und -SR²;
X² ausgewählt ist aus -OR³ und -SR³;
B¹ und B² unabhängig ausgewählt sind aus gegebenenfalls substituierten stickstoffhaltigen Basen, wobei jeder optionale Substituent unabhängig ausgewählt ist aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -CN, R⁶, und -X³;
Y ausgewählt ist aus -OR⁴, -SR⁴, -NR⁴R⁴, und Halogen;
Z ausgewählt ist aus -OR⁵, -SR⁵, und -NR⁵R⁵;
R¹, R², R³, R⁴, und R⁵ unabhängig ausgewählt sind aus einem -X³; Wasserstoff, -C(=O)R¹⁰⁰, -C(=O)OR¹⁰⁰ und -C(=O)NR¹⁰⁰; C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, von denen jedes bei jedem Auftreten unabhängig gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus; und C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, wobei jeder C_3-10-Carbozyklus und 3- bis 10-gliedrige Heterozyklus in R¹, R², R³, R⁴, und R⁵ gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰ -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_1-6-Alkyl, C_2-6-Alkenyl, C_2-6-Alkinyl;
R⁶ unabhängig ausgewählt ist aus -C(=O)R¹⁰⁰, -C(=O)OR¹⁰⁰ und -C(=O)NR¹⁰⁰; C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, von denen jedes bei jedem Auftreten unabhängig gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OR¹⁰⁰, -SR¹⁰⁰, -N(R¹⁰⁰)₂, -S(O)R¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus; und C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, wobei jeder C_3-10-Carbozyklus und 3- bis 10-gliedrige Heterozyklus in R⁶ gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -OOR¹⁰⁰, -SOR¹⁰⁰, -N(OR¹⁰⁰)₂, -S(O)OR¹⁰⁰, -S(O)₂R¹⁰⁰, -C(O)R¹⁰⁰, -C(O)OR¹⁰⁰, -OC(O)R¹⁰⁰, -NO₂, =O, =S, =N(R¹⁰⁰), -P(O)(OR¹⁰⁰)₂, -OP(O)(OR¹⁰⁰)₂, -CN, C_1-6-Alkyl, C_2-6-Alkenyl, C_2-6-Alkinyl;
R¹⁰⁰ bei jedem Auftreten unabhängig ausgewählt ist aus Wasserstoff; und C_1-10-Alkyl, C_2-10-Alkenyl, C_2-10-Alkinyl, C_3-10-Carbozyklus und 3- bis 10-gliedrigem Heterozyklus, von denen jedes bei jedem Auftreten unabhängig gegebenenfalls substituiert ist mit einem oder mehr Substituenten ausgewählt aus Halogen, -CN, -NO₂, =O, =S, und Haloalkyl; und
X³ eine Linkergruppe ist, wobei mindestens eines von R¹, R², R³, R⁴, R⁵, X¹, X², einem B¹-Substituent und einem B²-Substituent -X³ ist.In some aspects, the present disclosure provides a compound represented by formula (II):

or a pharmaceutically acceptable salt thereof, wherein
X ^{1 is} selected from -OR ² and -SR ² ;
X ^{2 is} selected from -OR ³ and -SR ³ ;
B ¹ and B ^{2 are} independently selected from optionally substituted nitrogenous bases, wherein each optional substituent is independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN , R ⁶ , and -X ³ ;
Y is selected from -OR ⁴ , -SR ⁴ , -NR ⁴ R ⁴ , and halogen;
Z is selected from -OR ⁵ , -SR ⁵ , and -NR ⁵ R ⁵ ;
R ¹ , R ² , R ³ , R ⁴ , and R ^{5 are} independently selected from -X ³ ; Hydrogen, -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each independently independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle, and _3-10 -linked heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carboxylic cycle and 3- to 10-membered heterocycle are optionally substituted in R ¹ , R ² , R ³ , R ⁴ , and R ⁵ is one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) ( OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl;
R ^{6 is} independently selected from -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each independently independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle, and _3-10 -linked heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carboxylic cycle and 3- to 10-membered heterocycle in R ^{6 is} optionally substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SOR ¹⁰⁰ , -N (OR ¹⁰⁰ ) ₂ , -S (O) OR ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O ) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 Alkyl, C _2-6 alkenyl, C _2-6 alkynyl;
R ^{100 is} independently selected at each occurrence from hydrogen; and C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbo cycle, and _3-10 membered heterocycle, each of which is independently independently optionally substituted with one or more on each occurrence more substituents selected from halo, -CN, -NO ₂ , = O, = S, and haloalkyl; and
X ^{3 is} a linker group, wherein at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , X ¹ , X ² , a B ¹ substituent and a B ² substituent is -X ³ .

oder ein pharmazeutisch annehmbares Salz davon.In various embodiments, the compound of formula (II) is represented by formula (IIA):

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon.In various embodiments, the compound of formula (II) is represented by formula (IIB):

or a pharmaceutically acceptable salt thereof.

In various embodiments, B ¹ and B ^{2 are} independently selected from optionally substituted purines. Each of B ¹ and B ² , independently selected, may be adenine, guanine and derivatives thereof. B ¹ and B ² may be independently selected from optionally substituted adenine, optionally substituted guanine, optionally substituted xanthine, optionally substituted hypoxanthine, optionally substituted theobromine, optionally substituted caffeine, optionally substituted uric acid and optionally substituted isoguanine. In a preferred embodiment, B ¹ and B ^{2 are} independently selected from optionally substituted adenine and optionally substituted guanine.

dargestellt werden, wobei B¹ gegebenenfalls mit einem oder mehr Substituenten substituiert ist.In various embodiments, B ^{1 is} substituted with X 3 and optionally one or more additional substituents independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN, and R ⁶ . For example, B ¹ can be given by:

where B ^{1 is} optionally substituted with one or more substituents.

dargestellt werden, wobei B² gegebenenfalls mit einem oder mehr Substituenten substituiert ist.In various embodiments, B ^{2 is} substituted with X 3 and optionally one or more additional substituents independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ . -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , - NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN, and R ⁶ . For example, B ² can be given by:

where B ^{2 is} optionally substituted with one or more substituents.

und ist B² dargestellt durch

In manchen Ausführungsformen ist B¹ dargestellt durch

und ist B² dargestellt durch

In some embodiments, B ^{1 is} represented by

and B ^{2 is} represented by

In some embodiments, B ^{1 is} represented by

and B ^{2 is} represented by

In various embodiments, X ^{1 is} selected from -O-X ³ and -SX ³ . In some embodiments, X ^{1 is} selected from -OH and -SH. In some embodiments, X ^{1 is} -SH.

In various embodiments, X ^{2 is} selected from -O-X ³ and -SX ³ . In some embodiments, X ^{2 is} selected from -OH and -SH. In some embodiments, X ^{2 is} -SH.

In some embodiments, X ^{1 is} -SH and X ^{2 is} -SH.

In certain embodiments, Y is selected from -NR ⁴ X ³ , -SX ³ , and -O-X ³ . In some embodiments, Y is selected from -OH, -SH, -OC _1-10 -alkyl, -NH (C _1-10 -alkyl), and -NH ₂ . In a preferred embodiment, Y is selected from -OH.

In certain embodiments, Z is selected from -NR ⁴ X ³ , -SX ³ , and -O-X ³ . In some embodiments, Z is selected from -OH, -SH, -OC _1-10 alkyl, -NH (C _1-10 alkyl), and -NH ₂ .

In various embodiments, -X ^{3 is} represented by the formula:

wobei RX eine reaktive Gruppe wie Maleimid umfasst.In some embodiments, -X ^{3 is} represented by the formula:

wherein RX comprises a reactive group such as maleimide.

wobei RX* eine reaktive Gruppe ist, die mit einer Gruppe auf einem Antikörper reagiert hat, um ein Antikörper-Medikamenten-Konjugat zu bilden.In some embodiments, -X ^{3 is} represented by the formula:

wherein RX * is a reactive group that has reacted with a group on an antibody to form an antibody-drug conjugate.

wobei RX eine reaktive Gruppe wie Maleimid umfasst.In some embodiments, -X ^{3 is} represented by the formula:

wherein RX comprises a reactive group such as maleimide.

wobei RX* eine reaktive Gruppe ist, die mit einer Gruppe auf einem Antikörper reagiert hat, um ein Antikörper-Medikamenten-Konjugat zu bilden.In some embodiments, -X ^{3 is} represented by the formula:

wherein RX * is a reactive group that has reacted with a group on an antibody to form an antibody-drug conjugate.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon.The compound is represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon.The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. In manchen Ausführungsformen ist die Verbindung dargestellt durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon. Die Verbindung kann dargestellt sein durch die Formel:

oder ein pharmazeutisch annehmbares Salz davon.In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof. The compound can be represented by the formula:

or a pharmaceutically acceptable salt thereof.

left

The compositions and methods described herein may include a linker, e.g. A peptide linker. Linkers of the compositions and methods described herein may not affect binding to a target of active portions of a conjugate (e.g., active portions include antigen binding domains, Fc domains, targeting binding domains, antibodies, agonists, or the like) related binding partner may be like an antigen. A linker sequence may form a link between different parts of a composition. A composition may be a conjugate. A conjugate may include multiple linkers. These linkers may be the same linker or different linker.

Attachment via a linker may involve incorporation of a linker between parts of a composition or a conjugate. A linker may be short, flexible, rigid, cleavable, non-cleavable, hydrophilic or hydrophobic. A linker may contain segments having different features, such as segments of flexibility or segments of stiffness. The linker may be chemically stable to extracellular environments, such as chemically stable in the bloodstream, or may contain compounds that are not stable. The linker may contain linkages designed to be cleaved and / or immolated or otherwise disassembled specifically or non-specifically into cells. A cleavable linker may be sensitive to enzymes. A cleavable linker can be cleaved by enzymes such as proteases. A cleavable linker may be a valine-citrulline linker or a valine-alanine linker. A valine-citrulline or valine-alanine linker may contain a pentafluorophenyl group. A valine-citrulline or valine-alanine linker may contain a succimide group. A valine-citrulline or valine-alanine linker may contain a para-aminobenzoic acid (PABA) group. A valine-citrulline or valine-alanine linker may contain a PABA group and a pentafluorophenyl group. A valine-citrulline or valine-alanine linker may contain a PABA group and a succinimide group. A non-cleavable linker may be protease insensitive. A non-cleavable linker may be a maleimidocaproyl linker. A maleimidocaproyl linker may comprise N-maleimidomethylcyclohexane-1-carboxylate. A maleimidocaproyl linker may contain a succinimide group. A maleimidocaproyl linker may contain a pentafluorophenyl group. A linker may be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. A linker may be a maleimide-PEG4 linker. A linker may be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. A linker may be a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules. A linker may contain maleimides linked to polyethylene glycol molecules in which the polyethylene glycol may allow or be used for more linker flexibility, to extend the linker. A linker may be a (maleimidocaproyl) (valine-citrulline) (para-aminobenzyloxycarbonyl) (NH ₂ ) linker. A linker may be a THIOMAB linker. A THIOMAB linker may be a (maleimidocaproyl) (valine-citrulline) (para-aminobenzyloxycarbonyl) (NH ₂ ) linker. A linker may also be an alkylene, alkenylene, alkynylene, polyether, polyester, polyamide, polyamino acids, polypeptides, cleavable peptides or aminobenzyl carbamates. A linker may contain a maleimide at one end and an N-hydroxysuccinimidyl ester at the other end. A linker may contain a lysine with an acetylated N-terminal amine and a valine citrulline cleavage site. A linker may be a compound produced by a microbial transglutaminase, which compound can be prepared between an amine-containing moiety and a moiety designed to contain glutamine as a result of catalysing bond formation between the acyl group of a glutamine side chain and the primary amine of a lysine chain. A linker may contain a reactive primary amine. A linker can be a sortase A linker. An S-acetylated gene can be created by a sortase A enzyme that fuses an LXPTG recognition motif (SEQ ID NO: 21) to an N-terminal GGG motif to regenerate a native amide bond. The generated linker can therefore link a moiety linked to the LXPTG recognition motif (SEQ ID NO: 21) to a moiety bound to the N-terminal GGG motif. A linker may be a compound formed between an unnatural amino acid on a unit that reacts with an oxime bond formed by modifying a ketone group with an alkoxyamine on another entity. A unit may be an antibody construct. A unit can be an antibody. A unit can be an immunostimulatory compound. A unit can be a targeting binding domain. A linker may be part of a linker which may be unsubstituted or substituted, for example with a substituent. A substituent may include, for example, hydroxyl, amino, nitro, cyano, azido, carboxyl, carboxaldehyde, imine, alkyl, alkenyl, alkynyl, alkoxy, acyl, acyloxy, amide and ester groups.

In the conjugate of antibody construct and immunostimulatory compound described herein, the immunostimulatory compound is linked to the antibody construct via linkers. The linker linking an immunostimulatory compound to the antibody of an antibody construct-immunostimulatory compound conjugate may be short, long, hydrophobic, hydrophilic, flexible or rigid, or consist of segments each independently having one or more of the above properties that the linker may contain segments with different properties. The linkers may be polyvalent such that they covalently link more than one immunostimulatory compound to a single site on the antibody construct, or monovalent, so as to covalently link a single immunostimulatory compound to a single site on the antibody construct.

As will be appreciated by those skilled in the art, the linkers link the immunostimulatory compound to the antibody by forming a covalent bond to the immunostimulatory compound at one site and a covalent bond to the antibody construct at another. The covalent bonds are formed by the reaction between functional groups on the linker and functional groups on the inhibitors and antibody constructs. As used herein, the term "linker" is intended to include (i) unconjugated forms of the linker comprising a functional group capable of covalently linking the linker to an immunostimulatory compound and a functional group capable of is to covalently link the linker to an antibody construct; (ii) partially conjugated forms of the linker comprising a functional group capable of covalently linking the linker to an antibody construct and covalently linked to an immunostimulatory compound, or vice versa; and (iii) fully conjugated forms of the linker covalently linked to an immunostimulatory compound and an antibody construct. In some specific embodiments of intermediate synthons and antibody construct-immunostimulatory compound conjugates described herein, moieties comprising the functional groups on the linker and covalent bonds formed between the linker and the antibody construct are specifically designated Rx or LK shown. One embodiment relates to a conjugate of antibody construct and immunostimulatory compound that binds by contacting an antibody construct that binds a cell surface receptor or a tumor-associated antigen expressed on a tumor cell with a synthon as described herein under conditions in which the synthon is covalent linked to the antibody construct. One embodiment relates to a method for producing a conjugate of antibody construct and immunostimulatory compound formed by contacting a synthon described herein under conditions in which the synthon covalently links to the antibody construct. One embodiment relates to a method of stimulating immune activity in a cell expressing CD40, comprising contacting the cell with a conjugate of antibody construct and immunostimulatory compound described herein capable of binding the cell under conditions in which the cell is expressed Conjugate of antibody construct and immunostimulatory compound binds the cell.

Exemplary polyvalent linkers that can be used to link many immunostimulatory compounds to an antibody construct are described. For example, the Fleximer ^® linker technology has the potential to become a high-DAR conjugate of antibody construct and immunostimulatory combination with good physicochemical properties to allow. As shown below, the linker Fleximer® ^® technology is based on the incorporation of drug molecules in a solubilizing polyacetal backbone via a sequence of Esterbindungen. The methodology provides highly loaded conjugates of antibody construct and immunostimulatory compound (DAR up to 20) while maintaining good physicochemical properties. This methodology could be used with an immunostimulatory compound as shown in the scheme below.

To use the one shown in the above scheme Fleximer ^® linker technology, an aliphatic alcohol in the immunostimulatory compound may be present or introduced into it. The alcohol moiety is then conjugated to an alanine residue, which is then incorporated into the synthetic Fleximer® ^® linkers. The liposomal administration of the conjugate of antibody construct and immunostimulatory compound in vitro releases the original alcohol-containing drug.

By way of example and not limitation, some cleavable and nonscleavable linkers which may be included in the antibody construct and immunostimulatory compound conjugates described herein are described.

Cleavable linkers may be cleavable in vitro and in vivo. Cleavable linkers may contain chemically or enzymatically labile or degradable bonds. Cleavable linkers may rely on processes within the cell to release an immunostimulatory compound, such as cytoplasmic depletion, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. Cleavable linkers may comprise one or more chemical bonds that are either cleavable chemically or enzymatically while the remainder of the linker is not cleavable.

A linker may contain a chemically labile group such as hydrazone and / or disulfide groups. Linkers comprising chemically labile groups can exploit different properties between the plasma and some cytoplasmic compartments. The intracellular conditions that may facilitate the release of immunostimulatory compound for hydrazone-containing linkers may be the acidic environment of endosomes and lysosomes, while the disulfide-containing linkers may be reduced in the cytosol containing high thiol concentrations, e.g. As glutathione may contain. The plasma stability of a linker containing a chemically labile group can be increased by introducing steric hindrance using substituents in the vicinity of the chemically labile group.

Acid labile groups such as hydrazone may remain intact during systemic circulation in the blood neutral pH environment (pH 7.3-7.5) and may undergo hydrolysis and may release the immunostimulatory compound once the conjugate of antibody construct and immunostimulatory compound has dissolved in mild acid endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of the cell is recorded. This pH-dependent release mechanism may be associated with non-specific release of the drug. To increase the stability of the hydrazone group of the linker, the linker may be replaced by chemical modification, e.g. Substitution, allowing for coordination to achieve more efficient release in the lysosome with minimized circulating loss.

wobei D und Ab die immunstimulatorische Verbindung bzw. das Antikörperkonstrukt darstellen und n die Anzahl der mit dem Antikörperkonstrukt verknüpften immunstimulatorischen Verbindungen-Linker darstellt. Bei bestimmten Linker wie Linker (Ig) kann der Linker zwei spaltbare Gruppen umfassen – eine Disulfid – und eine Hydrazon-Einheit. Für solche Linker kann eine effektive Freisetzung der unmodifizierten freien immunstimulatorischen Verbindung einen sauren pH- oder Disulfidreduktion und sauren pH-Wert erfordern. Linker wie (Ih) und (Ii) können mit einer einzigen Hydrazon-Spaltstelle wirksam sein.Hydrazonhaltige linkers may contain additional cleavage sites, such as. B. additional acid labile cleavage sites and / or enzymatically labile cleavage sites. Conjugates of antibody construct and immunostimulatory compound, including exemplary hydrazone-containing linkers, may include, for example, the following structures:

where D and Ab represent the immunostimulatory compound or antibody construct and n represents the number of immunostimulatory linker linked to the antibody construct. For certain linkers such as linker (Ig), the linker may comprise two cleavable groups - a disulfide moiety and a hydrazone moiety. For such linkers, effective release of the unmodified free immunostimulatory compound may require acid pH or disulfide reduction and acidic pH. Linkers such as (Ih) and (Ii) can be effective with a single hydrazone cleavage site.

Other acid labile groups that may be included in linkers include cis-aconityl-containing linkers. cis-Aconityl chemistry may use a carboxylic acid adjacent to an amide bond to accelerate amide hydrolysis under acidic conditions.

Cleavable linkers may also include a disulfide group. Disulfides can be thermodynamically stable at physiological pH and can be designed to be immunostimulatory Compound can release after uptake into cells, wherein the cytosol can provide a significantly reducing environment compared to the extracellular environment. Cleavage of disulfide bonds may require the presence of a cytoplasmic thiol cofactor, such as (reduced) glutathione (GSH), so that disulfide-containing linkers in the circulation may be sufficiently stable, with the immunostimulatory compound being selectively released in the cytosol. The intracellular enzyme protein disulfide isomerase or similar enzymes capable of cleaving disulfide bonds may also contribute to the preferential cleavage of disulfide bonds within the cells. GSH may be present in cells in the concentration range of 0.5-10 mM, compared to a significantly lower concentration of GSH or cysteine, the most abundant low molecular weight thiol, circulating at about 5 μM. Tumor cells in which an irregular blood flow can lead to a hypoxic state, can lead to an increased activity of reducing enzymes and thus to even higher glutathione concentrations. The in vivo stability of a disulfide-containing linker can be achieved by chemical modification of the linker, e.g. For example, the use of steric hindrance adjacent to the disulfide bond can be increased.

wobei D und Ab die immunstimulatorische Verbindung bzw. das Antikörperkonstrukt darstellen, n die Anzahl der mit dem Antikörperkonstrukt verknüpften immunstimulatorischen Verbindung-Linker darstellt und R bei jedem Auftreten unabhängig ausgewählt ist aus Wasserstoff oder Alkyl. Eine Erhöhung der sterischen Hinderung neben der Disulfidbindung kann die Stabilität des Linkers erhöhen. Strukturen wie (Ij) und (Il) können eine erhöhte in vivo-Stabilität zeigen, wenn eine oder mehr R-Gruppen aus einem niederen Alkyl wie Methyl ausgewählt sind.Antibody construct and immunostimulatory compound conjugates, including exemplary disulfide-containing linkers, may include the following structures:

wherein D and Ab represent the immunostimulatory compound and the antibody construct respectively, n represents the number of immunostimulatory compound linkers linked to the antibody construct, and R is independently selected on each occurrence from hydrogen or alkyl. Increasing the steric hindrance besides the disulfide bond can increase the stability of the linker. Structures such as (Ij) and (II) may show increased in vivo stability when one or more R groups are selected from a lower alkyl such as methyl.

Another type of linker that can be used is a linker that is specifically cleaved by an enzyme. For example, the linker can be cleaved by a lysosomal enzyme. Such linkers may be peptide-based or may include peptide regions that can act as substrates for enzymes. Peptide-based linkers may be more stable in the plasma and extracellular environment than chemically labile linkers.

Peptide bonds can have good serum stability since lysosomal proteolytic enzymes in the blood can have very low activity compared to lysosomes due to endogenous inhibitors and the unfavorably high pH of the blood. The release of an immunostimulatory compound from an antibody construct may be due to the action of lysosomal proteases, e.g. As cathepsin and plasmin take place. These proteases may be present in increased amounts in certain tumor tissues. The linker may be cleavable by a lysosomal enzyme. The lysosomal enzyme may be, for example, cathepsin B, β-glucuronidase or β-galactosidase.

The cleavable peptide can be selected from tetrapeptides such as Gly-Phe-Leu-Gly, Ala-Leu-Ala-Leu or dipeptides such as Val-Cit, Val-Ala and Phe-Lys. Dipeptides may have lower hydrophobicity compared to longer peptides.

A variety of dipeptide-based cleavable linkers can be used in the antibody construct and immunostimulatory compound conjugates described herein.

Enzymatically cleavable linkers may contain a self-immunogenic spacer to spatially separate the immunostimulatory compound from the site of enzymatic cleavage. Direct binding of an immunostimulatory compound to a peptide linker may result in proteolytic release of an amino acid adduct of the immunostimulatory compound, thereby impairing its activity. The use of a self-immulatory spacer may allow the elimination of the fully active, chemically unmodified immunostimulatory compound after amide bond hydrolysis.

wobei X-D die unmodifizierte immunstimulatorische Verbindung darstellt. Heterozyklische Varianten dieser selbstimmolativen Gruppe wurden ebenfalls beschrieben.A self-immolative spacer may be a bifunctional para-aminobenzyl alcohol group which can be linked to the peptide by the amino group and form an amide bond, while amine containing the immunostimulatory compounds can be linked by carbamate functionalities to the hydroxyl group of the benzyl of the linker (to form a p-amidobenzyl carbamate, PABC, to surrender). The resulting preimmune stimulatory compound can be activated in protease-mediated cleavage, resulting in a 1,6-elimination reaction that releases the unmodified immunostimulatory compound, carbon dioxide, and linker group residues. The following scheme shows the fragmentation of p-amidobenzylcarbamate and the release of the immunostimulatory compound:

where XD is the unmodified immunostimulatory compound. Heterocyclic variants of this self-immunogenic group have also been described.

The enzymatically cleavable linker may be a β-glucuronic acid-based linker. Slight release of the immunostimulatory compound can be accomplished by cleavage of the glycosidic linkage of β-glucuronide by the lysosomal enzyme β-glucuronidase. This enzyme may be abundant in lysosomes and may be overexpressed in some tumor types, while the enzyme activity outside the cells may be low. β-glucuronic acid-based linkers can be used to circumvent the tendency of a conjugate of antibody construct and immunostimulatory compound to aggregate due to the hydrophilic nature of β-glucuronides. In certain embodiments, β-glucuronic acid-based linkers may link an antibody construct to a hydrophobic immunostimulatory compound. The following scheme shows the release of an immunostimulatory compound (D) from a conjugate of antibody construct (Ab) and immunostimulatory compound containing a β-glucuronic acid-based linker:

A variety of cleavable β-glucuronic acid-based linkers useful for linking drugs such as auristatins, camptothecin and doxorubicin analogs, CBI minor groove binders and psymberin with antibodies have been described. All of these β-glucuronic acid-based linkers can be used in the ADCs described herein. In certain embodiments, the enzymatically cleavable linker is a β-galactoside-based linker. β-galactoside is abundant within lysosomes, while enzyme activity outside cells is low.

In addition, immunostimulatory compounds containing a phenolic group can be covalently linked to a linker by the phenolic oxygen. Such a linker is based on a methodology in which a diaminoethane "Space Link" is used in conjunction with traditional "PABO" -based self-immolative groups to deliver phenols.

Cleavable linkers may comprise non-cleavable moieties or segments, and / or cleavable segments or segments may be included in an otherwise non-cleavable linker to render them cleavable. For example, polyethylene glycol (PEG) and related polymers may contain cleavable groups in the polymer backbone. For example, a polyethylene glycol or polymer linker may comprise one or more cleavable groups, such as a disulfide, a hydrazone, or a dipeptide.

Other degradable linkages that may be included in linkers may include ester bonds formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on an immunostimulatory compound, which ester groups may hydrolyze under physiological conditions to release the immunostimulatory compound , Hydrolytically degradable linkages may include, but are not limited to, carbonate linkages; Imine linkages resulting from the reaction of an amine and an aldehyde; Phosphate ester linkages formed by reacting an alcohol with a phosphate group; Acetal linkages which are the reaction product of an aldehyde and an alcohol; Ortho ester linkages which are the reaction product of a formate and an alcohol; and oligonucleotide linkages formed by a phosphoramidite group, including, but not limited to, at the end of a polymer, and a 5'-hydroxyl group of an oligonucleotide.

oder ein Salz davon, wobei: peptide ein Peptid (N → C veranschaulicht, wobei peptide die Amino- und Carboxy-„Termini” umfasst), spaltbar durch ein lysosomales Enzym, darstellt; T ein Polymer umfassend eine oder mehr Ethylenglykoleinheiten oder eine Alkylenkette oder Kombinationen davon darstellt; R^a ausgewählt ist aus Wasserstoff, Alkyl, Sulfonat und Methylsulfonat; R^y Wasserstoff oder C_1-4-Alkyl-(O)_r-(C_1-4-Alkylen)_s-G¹ oder C_1-4-Alkyl-(N)-[(C_1-4-Alkylen)-G¹]₂ ist; R^z C_1-4-Alkyl-(O)_r-(C_1-4-Alkylen)_s-G² ist; G¹ SO₃H, CO₂H, PEG 4-32 oder Zuckereinheit ist; G² SO₃H, CO₂H, oder PEG 4-32-Einheit ist; r 0 oder 1 ist; s 0 oder 1 ist; p eine ganze Zahl im Bereich von 0 bis 5 ist; q 0 oder 1 ist; x 0 oder 1 ist; y 0 oder 1 ist;

den Anknüpfungspunkt des Linkers an die immunstimulatorische Verbindung darstellt; und * den Anknüpfungspunkt des Rests des Linkers darstellt.A linker may comprise an enzymatically cleavable peptide moiety, for example a linker comprising structural formula (IVa), (IVb), (IVc) or (IVd):

or a salt thereof, wherein: peptide represents a peptide (N → C, wherein peptide comprises the amino and carboxy "termini") cleavable by a lysosomal enzyme; T represents a polymer comprising one or more ethylene glycol units or an alkylene chain or combinations thereof; R ^{a is} selected from hydrogen, alkyl, sulfonate and methylsulfonate; R ^{y is} hydrogen or C _1-4 alkyl (O) _r - (C _1-4 alkylene) _s -G ¹ or C _1-4 alkyl (N) - [(C _1-4 alkylene) - G ¹ ] ₂ ; R ^{z is} C _1-4 alkyl (O) _r - (C _1-4 alkylene) _s -G ² ; G ¹ SO ₃ H, CO ₂ H, PEG 4-32 or sugar moiety; G ^{2 is} SO ₃ H, CO ₂ H, or PEG 4-32 unit; r is 0 or 1; s is 0 or 1; p is an integer in the range of 0 to 5; q is 0 or 1; x is 0 or 1; y is 0 or 1;

represents the point of attachment of the linker to the immunostimulatory compound; and * represents the point of attachment of the remainder of the linker.

In certain embodiments, the peptide may be selected from a tripeptide or a dipeptide. In particular embodiments, the dipeptide may be selected from: Val-Cit; Cit-Val; Ala-Ala; Ala-Cit; Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-Cit; Cit-Lys; Asp-Cit; Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-Ala; Phe-Cit; Cit-Phe; Leu-cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and Trp-Cit or salts thereof.

Exemplary embodiments of linkers according to structural formula (IVa) that may be included in the antibody construct and immunostimulatory compound conjugates described herein may include the linkers shown below (as illustrated, the linkers include a group that is capable of covalently linking the linker to an antibody construct suitable is):

Exemplary embodiments of linkers according to structural formula (IVb), (IVc) or (IVd) which may be included in the antibody construct and immunostimulatory compound conjugates described herein may include the linkers shown below (as illustrated, the linkers include a group comprising suitable for covalently linking the linker with an antibody construct):

oder ein Salz davon; wobei: q 0 oder 1 ist; r 0 oder 1 ist; X¹ CH₂, O oder NH ist;

den Anknüpfungspunkt des Linkers an die immunstimulatorische Verbindung darstellt; und * den Anknüpfungspunkt des Rests des Linkers darstellt.The linker may comprise an enzymatically cleavable sugar unit, for example a linker comprising the structural formula (Va), (Vb), (Vc), (Vd) or (Ve):

or a salt thereof; where: q is 0 or 1; r is 0 or 1; X ^{1 is} CH ₂ , O or NH;

represents the point of attachment of the linker to the immunostimulatory compound; and * represents the point of attachment of the remainder of the linker.

Exemplary embodiments of linkers according to structural formula (Va) that may be included in the antibody construct and immunostimulatory compound conjugates described herein may include the linkers shown below (as illustrated, the linkers include a group that is capable of covalently linking the linker to an antibody construct suitable is):

Exemplary embodiments of linkers according to structural formula (Vb) which may be included in the antibody construct and immunostimulatory compound conjugates described herein include the linkers shown below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct is):

Exemplary embodiments of linkers according to structural formula (Vc) which may be included in the antibody construct and immunostimulatory compound conjugates described herein include the linkers shown below (as illustrated, the linkers comprise a group suitable for covalently linking the linker to an antibody construct is):

Exemplary embodiments of linkers according to structural formula (Vd) which may be included in the antibody construct and immunostimulatory compound conjugates described herein include the linkers shown below (as illustrated, the linkers comprise a group suitable for covalently linking the linker to an antibody construct is):

Exemplary embodiments of linkers according to structural formula (Ve) that may be included in the antibody construct and immunostimulatory compound conjugates described herein include the linkers shown below (as illustrated, the linkers comprise a group suitable for covalently linking the linker to an antibody construct is):

Although cleavable linkers may offer certain advantages, the linkers that comprise the conjugate described herein need not be cleavable. For non-cleavable linkers, the release of the immunostimulatory compound may not depend on the differential properties between the plasma and some cytoplasmic compartments. The release of the immunostimulatory compound may be after internalization of the antibody construct / immunostimulatory compound conjugate via antigen-mediated endocytosis and transport to the lysosomal compartment, where the antibody construct can be degraded to the amino acid level by intracellular proteolytic degradation. This method can release a derivative of the immunostimulatory compound formed by the immunostimulatory compound, the linker, and the amino acid residue to which the linker is covalently attached. The derivative of the immunostimulatory compound of antibody construct and immunostimulatory conjugate with non-cleavable linker may be more hydrophilic and less membrane permeable, resulting in fewer side effects and less non-specific toxicities compared to antibody construct and immunostimulatory conjugate with a cleavable linker. Conjugates of antibody construct and immunostimulatory compound with non-cleavable linkers may have greater stability in the circulation than conjugate of antibody construct and immunostimulatory compound with cleavable linker. Non-cleavable linkers may be alkylene chains or may be polymeric, such as based on polyalkylene glycol polymers, amide polymers, or may include segments of alkylene chains, polyalkylene glycols and / or amide polymers. The linker may contain a polyethylene glycol segment having 1 to 6 ethylene glycol units.

oder Salzen davon, wobei: R^a ausgewählt ist aus Wasserstoff, Alkyl, Sulfonat und Methylsulfonat; R^x eine Einheit ist, die eine funktionelle Gruppe umfasst, die den Linker kovalent mir einem Antikörperkonstrukt verknüpfen kann; und

den Anknüpfungspunkt des Linkers an die immunstimulatorische Verbindung darstellt.The linker may be non-cleavable in vivo, for example a linker according to the following formulations:

or salts thereof, wherein: R ^{a is} selected from hydrogen, alkyl, sulfonate and methylsulfonate; R ^{x is} a moiety that includes a functional group that can covalently link the linker to an antibody construct; and

represents the point of attachment of the linker to the immunostimulatory compound.

stellt den Anknüpfungspunkt an die immunstimulatorische Verbindung dar):

Exemplary embodiments of linkers according to structural formula (VIa) - (VId) which may be included in the conjugates described herein include the linkers shown below (as illustrated, the linkers include a group suitable for covalently linking the linker to an antibody construct , and

represents the point of attachment to the immunostimulatory compound):

Linking groups used to attach the linkers to an antibody can be electrophilic in nature and include, for example, maleimide groups, activated disulfides, active esters such as NHS esters and HOBt esters, haloformates, acid halides, alkyl and benzyl halides such as Halogenoacetamides. There are also emerging technologies associated with "self-stabilizing" maleimides and "bridge disulfides" that can be used in accordance with the disclosure.

An example of a "self-stabilizing" maleimide group which hydrolyzes spontaneously under antibody construct conjugation conditions to yield a species of a conjugate of antibody construct and immunostimulatory compound with improved stability is shown in the scheme below. Thus, the maleimide-linking group is reacted with a sulfhydryl of an antibody construct to give an intermediate succinimide ring. The hydrolyzed form of the attachment group is resistant to deconjugation in the presence of plasma proteins.

A method of bridging a pair of sulfhydryl groups derived from the reduction of a native hinge disulfide bond has been disclosed and illustrated in the scheme below. An advantage of this methodology is the ability to synthesize homogeneous DAR4 conjugates of antibody construct and immunostimulatory compound by complete reduction of IgGs (to yield 4 pairs of sulfhydryls), followed by reaction with 4 equivalents of the alkylating agent. Conjugates of antibody construct and immunostimulatory compound containing "bridged disulfides" are also claimed to have enhanced stability.

Similarly, as shown below, a maleimide derivative capable of bridging a pair of sulfhydryl groups has been developed.

oder Salze davon, wobei: R^q H oder -O-(CH₂CH₂O)₁₁-CH₃ ist; x 0 oder 1 ist; y 0 oder 1 ist; G² -CH₂CH₂CH₂SO₃H oder -CH₂CH₂O-(CH₂CH₂O)₁₁-CH₃ ist; R^w -O-CH₂CH₂SO₃H oder -NH(CO)-CH₂CH₂O-(CH₂CH₂O)₁₂-CH₃ ist; und * den Anknüpfungspunkt mit dem Rest des Linkers darstellt.The linking group may contain the following structural formulas (VIIa), (VIIb) or (VIIc):

or salts thereof, wherein: R ^{q is} H or -O- (CH ₂ CH ₂ O) ₁₁ -CH ₃ ; x is 0 or 1; y is 0 or 1; G ^{2 is} -CH ₂ CH ₂ CH ₂ SO ₃ H or -CH ₂ CH ₂ O- (CH ₂ CH ₂ O) ₁₁ -CH ₃ ; R ^{w is} -O-CH ₂ CH ₂ SO ₃ H or -NH (CO) -CH ₂ CH ₂ O- (CH ₂ CH ₂ O) ₁₂ -CH ₃ ; and * represents the point of attachment with the remainder of the linker.

Exemplary embodiments of linkers according to structural formulas (VIIa) and (VIIb) which may be included in the antibody construct immunostimulatory compound conjugates described herein may include the linkers shown below (as illustrated, the linkers include a group that is useful for covalently linking the antibody Linker with an antibody construct is suitable):

Exemplary embodiments of linkers according to structural formula (VIIc) which may be included in the antibody construct immunostimulatory compound conjugates described herein may include the linkers shown below (as illustrated, the linkers comprise a group that is capable of covalently linking the linker to an antibody construct suitable is):

conjugates

A composition described herein may be a conjugate. A conjugate may be an antibody construct, an immunostimulatory compound and a linker include. A conjugate may comprise an antibody construct, a pattern recognition receptor (PRR) agonist and a linker. A conjugate may comprise an antibody construct, a pattern-associated molecular pattern (PAMP) molecule and a linker. A conjugate may comprise an antibody construct, a damage-associated molecular pattern (DAMP) molecule and a linker. A conjugate may comprise an antibody construct, a STING agonist and a linker. A conjugate may comprise an antibody construct, a Toll-like receptor agonist molecule and a linker. A conjugate may comprise an antibody construct, imiquimod and a linker. A conjugate may comprise an antibody construct, S-27609 and a linker. A conjugate may comprise an antibody construct, CL307 and a linker. A conjugate may comprise an antibody construct, resiquimod and a linker. A conjugate may comprise an antibody construct, gardiquimod and a linker. A conjugate may comprise an antibody construct, UC-IV150 and a linker. A conjugate may comprise an antibody construct, KU34B and a linker. A conjugate may comprise an antibody construct, a motolimod and a linker. A conjugate may comprise an antibody construct, VTX-1463 and a linker. A conjugate may comprise an antibody construct, GS-9620 and a linker. A conjugate may comprise an antibody construct, GSK2245035 and a linker. A conjugate may comprise an antibody construct, TMX-101 and a linker. A conjugate may comprise an antibody construct, TMX-201 and a linker. A conjugate may comprise an antibody construct, TMX-202 and a linker. A conjugate may comprise an antibody construct, isatoribine and a linker. A conjugate may comprise an antibody construct, AZD8848 and a linker. A conjugate may comprise an antibody construct, MEDI9197 and a linker. A conjugate may comprise an antibody construct, 3M-051 and a linker. A conjugate may comprise an antibody construct, 3M-852 and a linker. A conjugate may comprise an antibody construct, 3M-052 and a linker. A conjugate may comprise an antibody construct, 3M-854A and a linker. A conjugate may comprise an antibody construct, S-34240 and a linker. A conjugate may comprise an antibody construct, CL663 and a linker. A conjugate may include an antibody construct, KIN1148 and a linker. A conjugate may include an antibody construct, SB-9200 and a linker. A conjugate may include an antibody construct, KIN-100 and a linker. A conjugate may comprise an antibody construct, ADU-S100 and a linker. A conjugate may comprise an antibody construct, KU34B and a linker. An antibody construct of any of the conjugates described herein may have a modified Fc domain of the antibody construct. The modified Fc domain may comprise a substitution on more than one amino acid residue, such as 5 different amino acid residues, including L235V / F243L / R292P / Y300L / P396L, such as 2 different amino acid residues including S239D / I332E or 3 different amino acid residues including S298A / E333A / K334A. The numbering of amino acid residues described here can be done according to the EU index as in Kabat.

A conjugate may comprise an antibody construct, a targeting binding domain, an immunostimulatory compound and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, a pattern recognition receptor (PRR) agonist and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, a pattern-associated molecular pattern (PAMP) molecule, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, a damage-associated molecular pattern (DAMP) molecule, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, a STING agonist and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, a Toll-like receptor agonist molecule, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, imiquimod and a linker. A conjugate may include an antibody construct, a targeting binding domain, S-27609, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, CL307 and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, resiquimod, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, gardiquimod, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, UC-IV150 and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, a motolimod, and a linker. A conjugate may include an antibody construct, a targeting binding domain, VTX-1463, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, GS-9620 and a linker. A conjugate may include an antibody construct, a targeting binding domain, GSK2245035, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, TMX-101 and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, TMX-201 and a linker. A conjugate may include an antibody construct, a targeting binding domain, TMX-202, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, isatoribine, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, AZD8848 and a linker. A conjugate may include an antibody construct, a targeting binding domain, MEDI9197, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, 3M-051, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, 3M-852, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, 3M-052, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, 3M-854A and a linker. A conjugate may include an antibody construct, a targeting binding domain, S-34240, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, CL663 and a linker. A conjugate may include an antibody construct, a targeting binding domain, KIN1148 and a linker. A conjugate may include an antibody construct, a targeting binding domain, SB-9200, and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, KIN-100 and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, ADU-S100 and a linker. A conjugate may comprise an antibody construct, a targeting binding domain, KU34B and a linker. An antibody construct of any of the conjugates described herein may have a modified Fc domain of the antibody construct. The modified Fc domain may comprise a substitution on more than one amino acid residue, such as 5 different amino acid residues, including L235V / F243L / R292P / Y300L / P396L, as on 2 different Amino acid residues including S239D / 1332E or as at 3 different amino acid residues including S298A / E333A / K334A.

The linker may be a linker as described herein. A linker may be cleavable, non-cleavable, hydrophilic or hydrophobic. A cleavable linker may be sensitive to enzymes. A cleavable linker can be cleaved by enzymes such as proteases. A cleavable linker may be a valine citrulline or a valine-alanine linker. A valine-citrulline or valine-alanine linker may contain a pentafluorophenyl group. A valine-citrulline or valine-alanine linker may contain a succimide group. A valine-citrulline or valine-alanine linker may contain a PABA group. A valine-citrulline or valine-alanine linker may contain a PABA group and a pentafluorophenyl group. A valine-citrulline or valine-alanine linker may contain a PABA group and a succinimide group. A non-cleavable linker may be protease insensitive. A non-cleavable linker may be a maleimidocaproyl linker. A maleimidocaproyl linker may comprise N-maleimidomethylcyclohexane-1-carboxylate. A maleimidocaproyl linker may contain a succinimide group. A maleimidocaproyl linker may contain a pentafluorophenyl group. A linker may be a combination of a maleimidocaproyl group and one or more polyethylene glycol molecules. A linker may be a maleimide-PEG4 linker. A linker may be a combination of a maleimidocaproyl linker containing a succinimide group and one or more polyethylene glycol molecules. A linker may be a combination of a maleimidocaproyl linker containing a pentafluorophenyl group and one or more polyethylene glycol molecules. A linker may contain maleimides linked to polyethylene glycol molecules in which the polyethylene glycol allows for more linker flexibility or can be used to extend the linker. A linker may be a (maleimidocaproyl) (valine-citrulline) (para-aminobenzyloxycarbon) (NH ₂ ) linker. A linker may be a THIOMAB linker. A THIOMAB linker may be a (maleimidocaproyl) (valine-citrulline) (para-aminobenzyloxycarbon) (NH ₂ ) linker. A linker may also be an alkylene, alkenylene, alkynylene, polyether, polyester, polyamide, polyamino acids, polypeptides, cleavable peptides or aminobenzyl carbamates. A linker may contain a maleimide at one end and an N-hydroxysuccinimidyl ester at the other end. A linker may contain a lysine with an acetylated N-terminal amine and a valine citrulline cleavage site. A linker may be a compound produced by a microbial transglutaminase, which compound can be prepared between an amine-containing moiety and a moiety designed to contain glutamine, as a result of catalysis of bond formation between the acyl group of a glutamine side chain and the primary amine of a lysine chain. A linker may contain a reactive primary amine. A linker can be a sortase A linker. An S-acetylated gene can be created by a sortase A enzyme that fuses an LXPTG recognition motif (SEQ ID NO: 21) to an N-terminal GGG motif to regenerate a native amide bond. The generated linker can therefore link a moiety linked to the LXPTG recognition motif (SEQ ID NO: 21) to a moiety bound to the N-terminal GGG motif. A linker may be a compound formed between an unnatural amino acid on a unit that reacts with an oxime bond formed by modifying a ketone group with an alkoxyamine on another entity. A unit may be an antibody construct. A unit can be a targeting binding domain. A unit can be an antibody. A unit can be an immunostimulatory compound.

The antibody construct may be as described herein. The antibody construct may be an anti-tumor antigen antibody construct. The antibody construct may be an anti-tumor antigen antibody. An antigen recognized by the antibody construct may be CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic Antigen, TAG-72, EpCAM, MUC1, folate binding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Le ^y , CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, de2 -7 EGFR, fibroblast activating protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2 / neu, idiotype, MAGE A3, unmutated p53, NY-ESO-1 , PMSA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, Bcr-Abl, tyrosinase, survivin, PSA, HTERT, sarcoma translocation breakpoints, EphA2, PAP, ML-IHN, AFP , ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH , ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, Legumain, Tie3, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT -2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE or Fos-related antigen 1. The antibody construct can recognize an antigen that can be expressed on a cell. The antibody construct can recognize an antigen that can be expressed by a cell. The antibody construct can recognize an antigen that can be expressed in the context of a major histocompatibility complex. The antibody construct can recognize an antigen that can stimulate the activity of a cell. The antibody construct can recognize an antigen that can stimulate an immune response. The antibody construct can recognize an antigen that has a Can reduce immune response. The antibody construct recognizes an antigen that can reduce the activity of a cell. The antibody construct can recognize an antigen that can be expressed on an immune cell. The antibody construct can recognize an antigen that can be expressed by an immune cell. The antibody construct can recognize an antigen that may be in the context of a major histocompatibility complex. The antibody construct can recognize an antigen on a cell, which antigen can be involved in a stimulating activity of a cell. The antibody construct can recognize an antigen on an immune cell that may be involved in the costimulation of an immune cell. The antibody construct can recognize an antigen on an immune cell that may be involved in a costimulation of an immune cell during an immune response. The antibody construct can recognize a receptor. The antibody construct can recognize a receptor on a cell. The antibody construct can recognize a receptor ligand. The antibody construct may recognize a receptor on a cell in which the receptor may be involved in the stimulatory activity of a cell. The antibody construct can recognize a receptor on an immune cell. The antibody construct can recognize a receptor on an immune cell that may be involved in the stimulating activity of an immune cell. The antibody construct can recognize a receptor on an immune cell that may be involved in the costimulation of an immune cell. The antibody construct can recognize a receptor on an immune cell that may be involved in the costimulation of an immune cell during an immune response. The antibody construct can recognize an antigen that can be expressed on an immune cell and that can stimulate the activity of an immune cell. The antibody construct can recognize an antigen that can be expressed on an immune that can reduce the activity of an immune cell. The antibody construct may be an anti-CD40 antibody. The antibody construct may comprise a light chain of an SBT-040 antibody. The antibody construct may comprise a heavy chain of SBT-040-G1WT. The antibody construct may comprise a heavy chain of SBT-040 G1VLPLL. The antibody construct may comprise a heavy chain of SBT-040 G1DE. The antibody construct may comprise a heavy chain of SBT-040 G1AAA. The antibody construct may include an SBT-040 CDR sequence. The antibody construct can recognize a single antigen. The antibody construct may be capable of recognizing two or more antigens. The K _d for the binding of an antigen-binding domain of a conjugate of antibody construct and immunostimulatory compound to an antigen in the presence of an immunostimulatory compound may be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, for example 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times , about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the K _d for the binding of the antigen-binding domain to the antigen of an antibody construct in the absence of the immuno-intense compound. The K _d for the binding of an antigen-binding domain of a conjugate of antibody construct and immunostimulatory compound to an antigen in the presence of the immunostimulatory compound may be less than 10 nM. The K _d for the binding of an antigen-binding domain of a conjugate of antibody construct and immunostimulatory compound to an antigen in the presence of the immunostimulatory compound may be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM or less than 0.1 nM.

An antibody construct may further comprise a targeting binding domain. A targeting binding domain of an antibody construct can recognize an antigen. For example, an antigen can be expressed on an immune cell. As another example, an antigen can be expressed by a tumor or cancer cell. An antigen may be a peptide or fragment thereof. An antigen can be expressed on an antigen-presenting cell. An antigen can be expressed on a dendritic cell, a macrophage or a B cell. An antigen can be CD40 and a targeting binding domain can recognize a CD40 antigen. An antigen may be a tumor antigen, and a targeting binding domain may recognize a tumor antigen. A targeting binding domain of an antibody construct may be a CD40 agonist. A targeting binding domain of an antibody construct can bind to a tumor antigen.

The antibody construct may have an Fc domain that can bind to an FcR when linked to an immunostimulatory compound. The antibody construct may have an Fc domain that can bind to an FcR to initiate FcR-mediated signaling when linked to an immunostimulatory compound. The antibody construct can bind to its antigen when linked to an immunostimulatory compound. The antibody construct can bind to its antigen if it is linked to an immunostimulatory compound, and the Fc domain of the antibody construct can bind to an FcR when linked to an immunostimulatory compound. The antibody construct can bind to its antigen when linked to an immunostimulatory compound, and the Fc domain of the antibody can bind to an FcR to initiate FcR-mediated signaling when linked to an immunomodulatory compound. The Fc domain linked to an immunostimulatory compound can be a modified Fc domain. The modified Fc domain may comprise a substitution on more than one amino acid residue, such as 5 different amino acid residues including L235V / F243L / R292P / Y300L / P396L, such as 2 different amino acid residues including S239D / I332E or 3 different amino acid residues including S298A / E333A / K334A. The K _d for binding an Fc domain to an Fc receptor when the Fc domain is linked to an immunostimulatory compound may be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, for example 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 40 times, about 45 times , about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the K _d for the binding of the Fc domain to the Fc receptor in Absence of the immunostimulatory compound. The K _d for binding an Fc domain to an Fc receptor when linked to an immunostimulatory compound may be less than 10 nM. The K _d for binding an Fc domain to an Fc receptor when linked to an immunostimulatory compound may be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM or less than 0.1 nM.

PRR agonist may be a toll-like receptor agonist. The toll-like receptor agonist may be a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist TLR9 agonist, a TLR10 agonist, a TLR11 agonist, a TLR12 agonist or a TLR13 agonist. The toll-like receptor agonist can activate two or more TLRs. The PAMP molecule may be an RIG-I agonist.

A conjugate can be formed by a linker that can link an antibody construct to a PRR. A conjugate can be formed by a linker that can link an antibody construct to a PAMP molecule. A conjugate may be formed by a linker capable of linking an antibody construct and a DAMP molecule. A conjugate can be formed by a linker that can link an antibody construct to a PRR, and a linker that can link to an antibody construct and a targeting binding domain. A conjugate may be formed by a linker capable of linking an antibody construct to a PAMP molecule and a linker capable of linking an antibody construct and a targeting binding domain. A conjugate may be formed by a linker capable of linking an antibody construct and a DAMP molecule, and a linker capable of linking an antibody construct and a targeting binding domain.

A linker may be linked to an antibody construct by a direct link between the antibody construct and the linker. A linker may be linked to an anti-CD40 antibody construct by a direct link between the anti-CD40 antibody construct and the linker. A linker may be linked to an anti-CD40 antibody by a direct linkage between the anti-CD40 antibody and the linker. A linker may be linked to an anti-tumor antigen antibody construct by a direct linkage between the anti-tumor antigen antibody construct and the linker. A linker may be linked to an anti-tumor antigen antibody by a direct linkage between the anti-tumor antigen antibody and the linker. A direct linkage can be a covalent bond. For example, a linker may be attached to a terminus of an amino acid sequence of an antibody construct or attached to a side chain modification on the antibody construct, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, non-natural amino acid residue or glutamic acid residue. Attachment may be via any number of bonds, including, but not limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond. A linker may have at least one functional group that can be linked to the antibody. Non-limiting examples of the functional groups may include those forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond or a thioether bond; such functional groups may include, for example, amino groups; carboxyl groups; aldehyde; azide groups; Alkyne and alkene groups; ketones; carbonates; Carbonyl functionalities that are attached to leaving groups such as cyano and succinimidyl and hydroxyl groups to be. A linker may be linked to an antibody construct on a hinge cysteine. A linker may be linked to an antibody construct on a light chain constant domain lysine. A linker may be linked to an antibody construct on a light chain engineered cysteine. A linker may be linked to an antibody construct on a light chain engineered glutamine. A linker may be linked to an antibody construct on an unnatural amino acid constructed in the light chain. A linker may be linked to an antibody construct on an unnatural amino acid constructed in the heavy chain. Amino acids can be constructed into an amino acid sequence of a composition as described herein be, for example, a linker of a conjugate. Constructed amino acids can be added to a sequence of existing amino acids. Constructed amino acids may be substituted for one or more existing amino acids of a sequence of amino acids. A linker may be conjugated to an antibody construct via a sulfhydryl group. A linker may be conjugated to an antibody construct via a primary amine. A linker may be a compound made between an unnatural amino acid on an antibody construct that reacts with an oxime bond formed by modifying a ketone group with an alkoxyamine on an immunostimulatory compound. When a linker is linked to an antibody construct at the sites described herein, an Fc domain of the antibody construct can bind to Fc receptors. When a linker is linked to an antibody construct at the sites described herein, the antigen binding domain of the antibody construct can bind its antigen. When a linker is linked to an antibody construct at the sites described herein, a targeting-binding domain of the antibody construct can bind its antigen.

An antibody with engineered reactive cysteine residues (THIOMAB) can be used to link a targeting binding domain to the antibody. A linker can link an antibody construct to a targeting binding domain via sortase A linker. A sortase A linker can be created by a sortase A enzyme that fuses a recognition motif LXPTG (SEQ ID NO: 21) to an N-terminal GGG motif to regenerate a native amide bond. The generated linker can therefore associate an antibody construct linked to the recognition motif LXPTG (SEQ ID NO: 21) with a targeting binding domain bound to the N-terminal GGG motif. A targeting binding domain may be linked by a direct link to a linker. A direct linkage can be a covalent bond. For example, a linker may be attached to one terminus of an amino acid sequence of a targeting binding domain or to a side chain modification of the targeting binding domain, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural amino acid residue or glutamic acid residue. Attachment may be via any number of bonds, including, but not limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond. A linker may have at least one functional group that can be linked to the antibody. Non-limiting examples of the functional groups may include those forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond or a thioether bond; such functional groups may include, for example, amino groups; carboxyl groups; aldehyde; azide groups; Alkyne and alkene groups; ketones; carbonates; Carbonyl functionalities that are attached to leaving groups such as cyano and succinimidyl and hydroxyl groups to be. Amino acids can be constructed into an amino acid sequence of the targeting binding domain. Constructed amino acids can be added to a sequence of existing amino acids. Constructed amino acids may be substituted for one or more existing amino acids of a sequence of amino acids. A linker may be conjugated via a sulfhydryl group to a targeting binding domain. A linker may be conjugated to a targeting binding domain via a primary amine. A targeting binding domain may be conjugated to the C-terminus of an Fc domain of an antibody construct.

oderAn antibody with engineered reactive cysteine residues (THIOMAB) can be used to link an immunostimulatory compound to the antibody. A linker can link an antibody construct to an immunostimulatory link via THIOMAB linker. A linker may link an antibody construct to an immunostimulatory compound via a sortase A linker. A sortase A linker can be created by a sortase A enzyme that fuses a recognition motif LXPTG (SEQ ID NO: 21) to an N-terminal GGG motif to regenerate a native amide bond. The generated linker can therefore associate an antibody construct linked to the recognition motif LXPTG (SEQ ID NO: 21) with an immunostimulatory compound bound to the N-terminal GGG motif. A linker may be a compound made between an unnatural amino acid on an antibody construct that reacts with an oxime bond formed by modifying a ketone group with an alkoxyamine on an immunostimulatory compound. The immunostimulatory compound may comprise one or more rings selected from carbocyclic and heterocyclic rings. The immunostimulatory compound may be covalently linked to a linker by binding to an exocyclic carbon or nitrogen atom on the immunostimulatory compound. A linker may be conjugated to an immunostimulatory compound via an exocyclic nitrogen or carbon atom of an immunostimulatory compound. A linker may be linked to a STING agonist, for example:

or

A linker agonist complex may dissociate under physiological conditions to provide an active agonist.

A linker may be linked to a PRR agonist through a direct linkage between the PRR agonist and the linker. A linker can be linked to a PAMP molecule by a direct link between the PAMP molecule and the linker. A linker may be linked to a toll-like receptor agonist through a direct linkage between the toll-like receptor agonist and the linker.

Examples of toll-like receptor agonists linked in a manner with a linker to release an active toll-like receptor agonist under physiological conditions may include:

Examples of RIG-I agonists linked to a linker in a manner capable of releasing an active RIG-I agonist under physiological conditions may include:

A linker may be linked to a DAMP molecule by a direct linkage between the DAMP molecule and the linker. A direct linkage can be a covalent bond. For example, a linker may be attached to a terminus of an amino acid sequence of an antibody or may be attached to a side chain modification of the antibody, such as the side chain of a lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a nonnatural amino acid residue or glutamic acid residue. An attachment may be via any number of bindings, for example but not limited to one Amide bond, an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single double or triple bond, a disulfide bond, or a thioether bond. A linker may have at least one functional group that can be linked to the antibody. Non-limiting examples of the functional groups may include those forming an amide bond, an ester bond, an ether bond, a carbonate bond, a carbamate bond or a thioether bond; such functional groups may include, for example, amino groups; carboxyl groups; aldehyde; azide groups; Alkyne and alkene groups; ketones; carbonates; Carbonyl functionalities that are attached to leaving groups such as cyano and succinimidyl and hydroxyl groups to be.

An ATAC can be formed by conjugating a non-cleavable maleimido-PEG4 linker containing a succinimide group to an immunostimulatory compound. For example, an ATAC may be N- ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) -1- (3- (3-amino) 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -N-ethyl-3,6,9,12-tetraoxapentadecane-15-amide (ATAC11); N- (5- (2-amino-3-pentylchinolin-5-yl) pentyl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) - 3,6,9,12-tetraoxapentadecane-15-amide (ATAC12); 1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -N- (3-pentylchinolin-2-yl) -3,6,9,12-tetraoxapentadecan -15-amide (ATAC13); 1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -N- (1-isobutyl-1H-imidazo [4,5-c] quinolin-4- yl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC 14); 1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -N-methyl-N- (2- (3- (7-methyl-benzo [1,2- d: 3,4-d '] bis (thiazole) -2-yl) ureido) ethyl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC15); (S) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -N- (1 - ((7-methylbenzo [1,2-d: 3,4-d '] bis (thiazole) -2-yl) amino) -1-oxo-3-phenylpropan-2-yl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC16); N- (benzo [d] thiazol-2-yl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -N - ((8-hydroxyquinoline -7-yl) (4- (trifluoromethoxy) phenyl) methyl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC17); N - ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -2,9-bis (2-amino-6-oxo-1H-purin-9 (6H) -yl) -5,10 , 12-trihydroxy-5,12-dioxidodecahydrodifuro [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetra-oxadiphosphacyclododecin-3-yl) -1- ( 3- (2,5-dioxo-2,5-dihydro-1 H -pyrrol-1-yl) -propanamido) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC18); N - ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -2,9-bis (2-amino-6-oxo-1H-purin-9 (6H) -yl) -10-hydroxy -5,12-dimercapto-5,12-dioxidodecahydrodifuro [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetraoxadiphosphacyclododecin-3-yl) -1- ( 3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC19); N- (9 - ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -9- (2-Amino-6-oxo-1H-purin-9 (6H) -yl) -3,5 , 10,12-tetrahydroxy-5,12-dioxidodecahydrodifuro [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetra-oxadiphosphacyclododecin-2-yl) -9 -purin-6-yl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -3,6,9,12-tetraoxapentadecan-15-amide (ATAC20); or N- (9 - ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -9- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -3, 5,10,12-tetrahydroxy-5,12-dioxidodecahydrodifuro [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetraoxadiphosphacyclododecin-2-yl) -9 purin-6-yl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -3,6,9,12-tetraoxapentadecane-15-amide ( ATAC21).

An ATAC can be formed by conjugating a cleavable valine-alanine or valine-citrulline linker containing a PABA group and a succinimide group with an immunostimulatory compound. For example, an ATAC 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3- methylbutanamido) propanamido) benzyl - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H -imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamate (ATAC22 ); 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methyl-butanamido) propanamido ) benzyl- (5- (2-amino-3-pentyl-quinolin-5-yl) -pentyl) -carbamate (ATAC23); 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methyl-butan-amido) - 5-ureidopentanamido) benzyl- (5- (2-amino-3-pentylquinolin-5-yl) pentyl) -carbamate (ATAC24); 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5 ureidopentanamido) benzyl ((4-amino-1- (2-hydroxy-2-methylpropyl) -1 H -imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamate TFA salt (ATAC25) ; 2- (3- {2- [N-methyl ({p - [(S) -2 - {(S) -2- [6- (2,5-dioxo-1H-pyrrol-1-yl) hexanoylamino] 3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonyl) amino] ethyl} ureido) -7-methyl-1,6-dithia-3,8-diaza-as-indacene (ATAC26); 2 - {[(8-hydroxy-7-quinolyl) (p-trifluoromethoxyphenyl) methyl] ({p - [(S) -2 - {(S) -2- [6- (2,5-dioxo-1H- pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonyl) amino} -1,3-benzothiazole (ATAC27); (1R, 6R, 8R, 9S, 10S, 15R, 17R, 18S) -18 - ({p - [(S) -2 - {(S) -2- [6- (2,5-dioxo-1H- pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonylamino) -8.17-bis (2-amino-6-oxo-1,9-dihydro-9-yl) -3, 12-dihydroxy-9-hydroxy-2.4.7.11.13.16 hexaoxa-3λ5.12λ5 diphosphatricyclo [13.3.0.06,10] octadecane-3,12-dione (ATAC28); (1R, 6R, 8R, 9S, 10S, 15R, 17R, 18S) -18 - ({p - {(S) -2 - {(S) -2- [6- (2,5-dioxo-1H- pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} propionylamino] -phenyl} methoxycarbonylamino) -8.17-bis (2-amino-6-oxo-1,9-dihydro-9-yl) -3.12-dihydroxy -9-hydroxy-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadecane-3,12-dione (ATAC29); (1R, 6R, 8R, 9S, 10S, 15R, 17R, 18S) -18 - ({p - [(S) -2 - {(S) -2- [6- (2,5-dioxo-1H- pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonylamino) -8.17-bis (2-amino-6-oxo-1,9-dihydro-9-yl) -9- hydroxy-3,12-dimercapto-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadecane-3,12-dione (ATAC30); {P - [(S) -2 - {(S) -2- [6- (2,5-dioxo-1H-pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methyl -9 - {(1S, 6R, 8R, 9S, 10S, 15R, 17R, 18S) -8- (2-amino-6-oxo-1,9-dihydro-9-yl) -3.12-dihydroxy- 9,18-dihydroxy-3,12-dioxo-2.4.7.11.13.16-hexaoxa-3λ5.12λ5- diphosphatricyclo [13.2.1.06,10] octadec-17-yl} -9a-adenincarboxylate (ATAC31); 1- {6 - [({7-Amino-3- (2-hydroxy-2-methylpropyl) -3.5.8-triazatricyclo [7.4.0.02,6] trideca-1 (9), 2 (6 4), 7,10,12-hexa-4-yl} methyl) -N-ethylamino] -6-oxohexyl} -1H-pyrrole-2,5-dione (ATAC32); 1 - {[4 - ({6 - [({7-amino-3- (2-hydroxy-2-methylpropyl) -3,5,8-triazatricyclo [7.4.0.02,6] trideca-1 (9), 2 ( 6), 4,7,10,12-hexa-4-yl} methyl) -N-ethylamino] -6-oxohexylamino} carbonyl) cyclohexyl] methyl} -1H-pyrrole-2,5-dione (ATAC33); or 1 - [(4 - {[({7-amino-3- (2-hydroxy-2-methylpropyl) -3,5,8-triazatricyclo [7.4.0.02,6] trideca-1 (9), 2 (6) , 4,7,10,12-hexaen-4-yl} methyl) -N-ethylamino] -carbonyl} cyclohexyl) methyl] -1H-pyrrole-2,5-dione (ATAC34).

An ATAC can be formed by conjugating a non-cleavable maleimido-PEG4 linker containing an activated ester such as a pentafluorophenyl group or an N-hydroxysuccinimide group to an immunostimulatory compound. For example, an ATAC may be pentafluorophenyl 25- (2-amino-3-pentylquinolin-5-yl) -19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC1); Perfluorophenyl-3 - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) -4-oxo-7,10,13 , 16,19-pentaoxa-3-azadocosan-22-oat (ATAC2); Pentafluorophenyl 25- (2-amino-3-pentylquinolin-5-yl) -19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC3); or 2,5-dioxopyrrolidin-1-yl-3 - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) 4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oat (ATAC4).

An ATAC can be formed by conjugating a cleavable valine-alanine or valine-citrulline linker containing a PABA group and an activated ester such as a pentafluorophenyl group or an N-hydroxysuccinimide group to an immunostimulatory compound. For example, an ATAC can be 2,5-dioxopyrrolidin-1-yl-6 - (((S) -1 - (((S) -1 - ((4 - ((((5- (2-amino-3-) pentylquinolin-5-yl) pentyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoate (ATAC5 ); 2,5-dioxopyrrolidin-1-yl-7 - (((S) -1 - (((S) -1 - ((4 - (((((4-amino-1- (2-hydroxy-2- methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl- 1-oxobutan-2-yl) amino) -7-oxohepanoate (ATAC6); 2,5-dioxopyrrolidin-1-yl-7 - (((S) -1 - (((S) -1 - ((4 - (((((4-amino-1- (2-hydroxy-2- methylpropyl) -1H-imidazo [4.5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) - 3-methyl-1-oxobutan-2-yl) amino) -7-oxohepanoate (ATAC7); Perfluorophenyl-6 - (((S) -1 - (((S) -1 - ((4 - ((((5- (2-amino-3-pentylchinolin-5-yl) pentyl) carbamoyl) oxy) methyl ) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoate (ATAC8); Perfluorophenyl-7 - (((S) -1 - (((S) -1 - ((4 - (((((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4 , 5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -7-oxoheptanoate (ATAC9); or perfluorophenyl-7 - (((S) -1 - (((S) -1 - ((4 - (((((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [ 4,5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan -2-yl) amino) -7-oxoheptanoate (ATAC10).

An antibody construct may comprise an anti-CD40 antibody. An anti-CD40 antibody may comprise two heavy chains of SBT-040-G1WT and two SBT-040 antibody light chain, which may be termed SBT-040-WT or SBT-040-G1. An anti-CD40 antibody may comprise two heavy chains of SBT-040 G1VLPLL and two light chains of an SBT-040 antibody, which may be referred to as SBT-040 VLPLL. An anti-CD40 antibody can comprise two heavy chains of SBT-040 G1DE and two light chains of an SBT-040 antibody, which can be termed SBT-040-DE. An anti-CD40 antibody can comprise two heavy chains of SBT-040 G1AAA and two light chains of an SBT-040 antibody, which can be referred to as SBT-040-AAA. An anti-CD40 antibody may comprise two heavy IgG2 chains and two SBT-040 antibody light chain, which may be termed SBT-040-G2.

One Conjugate may include SBT-040-WT-ATAC1. A conjugate may include SBT-040-WT-ATAC2. A conjugate may include SBT-040-WT-ATAC3. A conjugate may include SBT-040-WT-ATAC4. A conjugate may include SBT-040-WT-ATAC5. A conjugate may include SBT-040-WT-ATAC6. A conjugate may include SBT-040-WT-ATAC7. A conjugate may include SBT-040-WT-ATAC8. A conjugate may include SBT-040-WT-ATAC9. A conjugate may include SBT-040-WT-ATAC10. A conjugate may include SBT-040-WT-ATAC11. A conjugate may include SBT-040-WT-ATAC12. A conjugate may include SBT-040-WT-ATAC13. A conjugate may include SBT-040-WT-ATAC 14. A conjugate may include SBT-040-WT-ATAC 15. A conjugate may include SBT-040-WT-ATAC 16. A conjugate may include SBT-040-WT-ATAC 17. A conjugate may include SBT-040-WT-ATAC18. A conjugate may include SBT-040-WT-ATAC19. A conjugate may include SBT-040-WT-ATAC20. A conjugate may include SBT-040-WT-ATAC21. A conjugate may include SBT-040-WT-ATAC22. A conjugate may include SBT-040-WT-ATAC23. A conjugate may include SBT-040-WT-ATAC24. A conjugate may include SBT-040-WT-ATAC25. A conjugate may include SBT-040-WT-ATAC26. A conjugate may include SBT-040-WT-ATAC27. A conjugate may include SBT-040-WT-ATAC28. A conjugate may include SBT-040-WT-ATAC29. A conjugate may include SBT-040-WT-ATAC30. A conjugate may include SBT-040-WT-ATAC31. A conjugate may include SBT-040-WT-ATAC32. A conjugate may include SBT-040-WT-ATAC33. A conjugate may consist of SBT-040-WT-ATAC34. A conjugate may include SBT-040-WT-ATAC43. A conjugate may include SBT-040 VLPLL ATAC1. A conjugate may include SBT-040 VLPLL ATAC2. A conjugate may include SBT-040 VLPLL ATAC3. A conjugate may include SBT-040 VLPLL ATAC4. A conjugate may include SBT-040 VLPLL ATAC5. A conjugate may include SBT-040 VLPLL ATAC6. A conjugate may include SBT-040 VLPLL ATAC7. A conjugate may include SBT-040 VLPLL ATAC8. A conjugate may include SBT-040 VLPLL ATAC9. A conjugate may include SBT-040 VLPLL ATAC10. A conjugate may include SBT-040 VLPLL ATAC11. A conjugate may include SBT-040 VLPLL ATAC12. A conjugate may include SBT-040 VLPLL ATAC13. A conjugate may include SBT-040 VLPLL ATAC14. A conjugate may include SBT-040 VLPLL ATAC15. A conjugate may include SBT-040 VLPLL ATAC16. A conjugate may include SBT-040 VLPLL ATAC17. A conjugate may include SBT-040 VLPLL ATAC18. A conjugate may include SBT-040 VLPLL ATAC19. A conjugate may include SBT-040 VLPLL ATAC20. A conjugate may include SBT-040 VLPLL ATAC21. A conjugate may include SBT-040 VLPLL ATAC22. A conjugate may include SBT-040 VLPLL ATAC23. A conjugate may include SBT-040 VLPLL ATAC24. A conjugate may include SBT-040 VLPLL ATAC25. A conjugate may include SBT-040 VLPLL ATAC26. A conjugate may include SBT-040 VLPLL ATAC27. A conjugate may include SBT-040 VLPLL ATAC28. A conjugate may include SBT-040 VLPLL ATAC29. A conjugate may include SBT-040 VLPLL ATAC30. A conjugate may include SBT-040 VLPLL ATAC31. A conjugate may include SBT-040 VLPLL ATAC32. A conjugate may include SBT-040 VLPLL ATAC33. A conjugate may include SBT-040 VLPLL ATAC34. A conjugate may include SBT-040 VLPLL ATAC43. A conjugate may include SBT-040-DE-ATAC1. A conjugate may include SBT-040-DE-ATAC2. A conjugate may include SBT-040-DE-ATAC3. A conjugate may include SBT-040-DE-ATAC4. A conjugate may include SBT-040-DE-ATAC5. A conjugate may include SBT-040-DE-ATAC6. A conjugate may include SBT-040-DE-ATAC7. A conjugate may include SBT-040-DE-ATAC8. A conjugate may include SBT-040-DE-ATAC9. A conjugate may include SBT-040-DE-ATAC10. A conjugate may include SBT-040-DE-ATAC11. A conjugate may include SBT-040-DE-ATAC12. A conjugate may include SBT-040-DE-ATAC13. A conjugate may include SBT-040-DE-ATAC14. A conjugate may include SBT-040-DE-ATAC15. A conjugate may include SBT-040-DE-ATAC16. A conjugate may include SBT-040-DE-ATAC17. A conjugate may include SBT-040-DE-ATAC18. A conjugate may include SBT-040-DE-ATAC19. A conjugate may include SBT-040-DE-ATAC20. A conjugate may include SBT-040-DE-ATAC21. A conjugate may include SBT-040-DE-ATAC22. A conjugate may include SBT-040-DE-ATAC23. A conjugate may include SBT-040-DE-ATAC24. A conjugate may include SBT-040-DE-ATAC25. A conjugate may include SBT-040-DE-ATAC26. A conjugate may include SBT-040-DE-ATAC27. A conjugate may include SBT-040-DE-ATAC28. A conjugate may include SBT-040-DE-ATAC29. A conjugate may include SBT-040-DE-ATAC30. A conjugate may include SBT-040-DE-ATAC31. A conjugate may include SBT-040-DE-ATAC32. A conjugate may include SBT-040-DE-ATAC33. A conjugate may include SBT-040-DE-ATAC34. A conjugate may include SBT-040-DE-ATAC43. A conjugate may include SBT-040-AAA-ATAC1. A conjugate may include SBT-040-AAA-ATAC2. A conjugate may include SBT-040-AAA-ATAC3. A conjugate may contain SBT-040-AAA-ATAC4. A conjugate may include SBT-040-AAA-ATAC5. A conjugate may include SBT-040-AAA-ATAC6. A conjugate may include SBT-040-AAA-ATAC7. A conjugate may include SBT-040-AAA-ATAC8. A conjugate may include SBT-040-AAA-ATAC9. A conjugate may include SBT-040-AAA-ATAC10. A conjugate may include SBT-040-AAA-ATAC 11. A conjugate may include SBT-040-AAA-ATAC 12. A conjugate may include SBT-040-AAA-ATAC 13. A conjugate may include SBT-040-AAA-ATAC 14. A conjugate may include SBT-040-AAA-ATAC15. A conjugate may include SBT-040-AAA-ATAC16. A conjugate may include SBT-040-AAA-ATAC17. A conjugate may include SBT-040-AAA-ATAC18. A conjugate may include SBT-040-AAA-ATAC19. A conjugate may include SBT-040-AAA-ATAC20. A conjugate may include SBT-040-AAA-ATAC21. A conjugate may include SBT-040-AAA-ATAC22. A conjugate may include SBT-040-AAA-ATAC23. A conjugate may include SBT-040-AAA-ATAC24. A conjugate may include SBT-040-AAA-ATAC25. A conjugate may include SBT-040-AAA-ATAC26. A conjugate may include SBT-040-AAA-ATAC27. A conjugate may include SBT-040-AAA-ATAC28. A conjugate may include SBT-040-AAA-ATAC29. A conjugate may include SBT-040-AAA-ATAC30. A conjugate may include SBT-040-AAA-ATAC31. A conjugate may include SBT-040-AAA-ATAC32. A conjugate may include SBT-040-AAA-ATAC33. A conjugate may include SBT-040-AAA-ATAC34. A conjugate may include SBT-040-AAA-ATAC33. A conjugate may include SBT-040-AAA-ATAC43. The K _d for binding of the CD40-binding domain of any of these conjugates to CD40 may be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 about 15 times, about 20 times, about 25 times, about 30 times about 35 times, about 40 times, about 45 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times or about 120 times greater than the K _d for binding the CD40-binding domain to CD40 in the absence of the immunostimulatory compound or ATAC. The K _d for the binding of the CD40-binding domain any of these conjugates to CD40 may be less than 10 nM. The K _d for binding the CD40 binding domain any of the conjugates to CD40 may be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM or less than 0.1 nM. The K _d for binding of the Fc domain any of the conjugates to an Fc receptor can be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times , about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 110 times, or about 120 times greater than the K _d for the binding of the Fc domain to the Fc receptor in the absence of the immunostimulatory compound or ATAC. The K _d for binding the Fc domain any of the conjugates to an Fc receptor of one may be less than 10 nM. The K _d for binding the Fc domain any of the conjugates to an Fc receptor may be less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM or less than 0.1 nM be.

In a conjugate, an antibody can be linked to an immunostimulatory compound such that the antibody can still bind to an antigen and the Fc domain of the antibody can still bind to an FcR. In a conjugate, an antibody construct is linked to an immunostimulatory compound such that the linkage does not enhance the ability of the antigen-binding domain of the antibody construct to bind antigen, the ability of the antibody's Fc domain to bind to an FcR-, or FcR-mediated Signaling resulting from binding of the Fc domain of the antibody construct to an FcR is compromised. In a conjugate, an immunostimulatory compound may be linked to an antibody construct in such a way that the linkage does not affect the ability of the immunostimulatory compound to bind to its receptor. A conjugate can produce a stronger immune stimulation and a larger therapeutic window than components of the conjugate alone. In an anti-CD40 antibody linked to a TLR agonist conjugate, the combination of CD40 agonism, TLR agonism, and an accessible Fc domain of the anti-CD40 antibody to confer FcR-mediated signaling enable greater immune stimulation and a larger therapeutic window than CD40 agonism, TLR agonism, or FcR-mediated signaling alone.

Process for the synthesis of components of antibody construct and immunostimulatory compound conjugates

Synthesis of immunostimulatory compounds

An immunostimulatory compound can be synthesized as shown in Scheme A1. Scheme A1:

Synthesis of the C-2'-amino cyclic dinucleotide (viii) can be accomplished using a multi-step synthesis as outlined in Scheme A1 above and described below in EXAMPLE 3.

Synthesis of ATAC compounds

An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC1-ATAC4, can be synthesized as shown in Scheme B1. Scheme B1:

A PEGylated carboxylic acid (i), which has been activated to form amide bonds, can be reacted with an appropriately substituted amine-containing immunostimulatory compound to give an intermediate amide. The formation of an activated ester (ii) can be accomplished by reacting the intermediate amide-containing carboxylic acid using a reagent such as N-hydroxysuccinimide or pentafluorophenol in the presence of a coupling agent such as diisopropylcarbodiimide (DIC) to provide compounds (ii).

An ATAC compound can be synthesized by various methods. For example, ATAC compounds such as ATAC5-ATAC10 can be synthesized as shown in Scheme B2. Scheme B2:

An activated carbonate such as (i) may be reacted with an appropriately substituted amine-containing immunostimulatory compound to give carbamates (ii) which can be deprotected using standard techniques based on the nature of the R ₃ ester group. The resulting carboxylic acid (iii) may then be coupled with an activating agent such as N-hydroxysuccinimide or pentafluorophenol to provide compounds (iv).

An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATACII-ATAC21, can be synthesized as shown in Scheme B3. Scheme B3:

An activated carboxylic acid ester such as (i-a) can be reacted with an appropriately substituted amine-containing immunostimulatory compound to give amides (ii). Alternatively, type (i-b) carboxylic acids can be coupled to an appropriately substituted amine-containing immunostimulatory compound in the presence of an amide bond-forming agent, such as dicyclohexycarbodiimde (DCC), to provide the desired ATAC compounds.

An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC22-ATAC31, can be synthesized as shown in Scheme B4. Scheme B4:

An activated carbonate such as (i) may be reacted with an appropriately substituted amine-containing immunostimulatory compound to provide carbamates (ii) as the target ATAC compounds.

An ATAC compound can be synthesized by various methods. For example, ATAC compounds, such as ATAC32-ATAC34, can be synthesized as shown in Scheme B5. Scheme B5:

An activated carboxylic acid such as (i-a, i-b, i-c can be reacted with an appropriately substituted amine-containing immunostimulatory compound to provide amides (i-a, ii-b, ii-c) as the target ATAC compounds.

These conjugates of antibody construct and immunostimulatory compound can be prepared by various methods. It will be understood that those skilled in the art will be able to make these compounds by similar methods or by combining other methods known to those skilled in the art. It will also be understood that one skilled in the art would be able to make similar use as described herein by using the appropriate starting materials and modifying the synthetic routes Requirement. Starting materials and reagents may be obtained from commercial manufacturers or synthesized according to sources known to those skilled in the art or prepared as described herein.

Pharmaceutical formulations

The compositions and methods described herein may be considered useful for pharmaceutical compositions for administration to a subject in need thereof. Pharmaceutical compositions may comprise at least the compositions described herein and one or more pharmaceutically acceptable carriers, diluents, adjuvants, stabilizers, dispersants, suspending agents and / or thickening agents. The composition may comprise the conjugate with an antibody construct and an agonist. The composition may comprise the conjugate with an antibody construct, a targeting binding domain and an agonist. The composition may comprise any conjugate described herein. Often, the antibody construct is an anti-CD40 antibody. A conjugate may comprise an anti-CD40 antibody and a PAMP molecule. A conjugate may comprise an anti-CD40 antibody and a DAMP molecule. A pharmaceutical composition may further comprise buffers, antibiotics, steroids, carbohydrates, medicaments (e.g., chemotherapeutics), radiation, polypeptides, chelators, adjuvants, and / or preservatives.

Pharmaceutical compositions can be formulated with one or more physiologically acceptable carriers containing excipients and auxiliaries. The formulation may be modified depending on the chosen route of administration. Pharmaceutical compositions comprising a composition described herein can be prepared, for example, by lyophilizing the conjugate, mixing, dissolving, emulsifying, encapsulating or entrapping the conjugate. The pharmaceutical compositions may also contain the compositions described herein in a form of a free base or a pharmaceutically acceptable salt form.

Methods of formulating the conjugates described herein can include the formulation of any of the conjugates described herein with one or more inert pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid composition. Solid compositions may, for. Powder, tablets, dispersible granules, and capsules, and in some aspects, the solid compositions further contain non-toxic adjuvants, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives. Alternatively, the compositions described herein may be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g. Sterile pyrogen-free water, before use

Pharmaceutical compositions of the conjugates described herein may contain at least one active ingredient. The active ingredients may be prepared in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules or poly (methyl methacrylate) microcapsules) in colloidal drug delivery systems (e.g., liposomes, albumin Microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

Pharmaceutical compositions as described herein may often comprise more than one active ingredient, as needed for the particular indication to be treated. The active ingredients may have complementary activities that do not interfere with each other. For example, the composition may comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, an anti-growth agent, an antihormonal agent, an antiangiogenic agent and / or a cardioprotectant. Such molecules may be present in combination in amounts effective for their intended purpose.

The compositions and formulations can be sterilized. Sterilization can be carried out by filtration by sterile filtration.

The compositions described herein may be formulated for administration as an injection. Non-limiting examples of formulations for injection may include a sterile suspension, solution or emulsion in oily or aqueous vehicles. Suitable oily vehicles may include, but are not limited to, lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid esters or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension. The suspension may also contain suitable stabilizers. Injections can be formulated for bolus injection or continuous infusion. Alternatively, those described herein Compositions should be lyophilised prior to use or in powder form for reconstitution with a suitable vehicle, e.g. Sterile pyrogen-free water, before use.

For parenteral administration, the conjugates may be formulated in an injectable unit dosage form (e.g., use broth, suspension, emulsion) in conjunction with a pharmaceutically acceptable parenteral vehicle. Such vehicles may be inherently nontoxic and non-therapeutic. A vehicle may be water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Non-aqueous vehicles such as solid oils and ethyl oleate may also be used. Liposomes can be used as carriers. The vehicle may contain minor amounts of additional bulking agents, such as substances that enhance isotonicity and chemical stability (eg, buffers and preservatives).

Also, sustained-release preparations can be prepared. Examples of sustained-release preparations may include semipermeable matrices of solid hydrophobic polymers that may contain the antibody, and these matrices may be in the form of shaped articles (eg, films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol)), polylactides, copolymers of L-glutamic acid, and γ-ethyl L-glutamate, nondegradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPO ^™ (ie, injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid.

Pharmaceutical formulations of the compositions herein may be prepared for storage by mixing a conjugate with a pharmaceutically acceptable carrier, excipient and / or stabilizer. This formulation may be a lyophilized formulation or an aqueous solution. Acceptable carriers, adjuvants and / or stabilizers may be non-toxic to the recipients at the dosages and concentrations used. Acceptable carriers, excipients and / or stabilizers may include buffers such as phosphate, citrate and other organic acids; Antioxidants including ascorbic acid and methionine; Preservatives, polypeptides; Proteins such as serum albumin or gelatin; hydrophilic polymers; Amino acids; Monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; Chelating agents such as EDTA; Sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; Metal complexes; and / or nonionic surfactants or polyethylene glycol.

Therapeutic applications

The compositions and methods of the present disclosure may be useful for a variety of different patients, including, but not limited to, a mammal, human, non-human mammal, a domesticated animal (e.g., laboratory animals, domestic pets, or livestock), non-domesticated animal (eg, wild animal), dog, cat, rodent, mouse, hamster, cow, bird, chicken, fish, pig, horse, goat, sheep, rabbit, and any combination thereof.

The compositions and methods described herein may be useful as a therapeutic, for example, a treatment that may be administered to a subject in need thereof. A therapeutic effect of the present disclosure may be obtained in a subject by reducing, suppressing, remitting, or eradicating a disease state, including, but not limited to, a symptom thereof. A therapeutic effect in a subject having or is or may be predisposed to having the disease or condition may be by reduction, suppression, prevention, remission, or eradication of the condition or disease , or the pre-state or pre-disease stage.

In practicing the methods described herein, therapeutically effective amounts of the compositions herein may be administered to a subject in need thereof, often for the treatment and / or prevention of a condition or progression thereof. A pharmaceutical composition may affect the physiology of the patient, such as the immune system, the inflammatory response or other physiological affects. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors.

Treatment and / or treatment may refer to any of the features of success in treating or ameliorating the condition or condition. The treatment may, for. For example, reducing, delaying or alleviating the severity of one or more symptoms of the condition or condition, or it may also include reducing the incidence at which the symptoms of a disease, defect, disorder or condition and the like are experienced by a patient. Treatment may be used herein to refer to a method that results in some degree of treatment or improvement of the disease or condition, and may take into account a number of results directed to this purpose, including, but not limited to, the following not limited to, the complete prevention of the condition.

Prevention, prevention and the like may be directed to the prevention of the disease or condition, e.g. As tumor formation, refer to the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and later does not develop the tumor or other form of cancer, then in that individual the disease is at least have been prevented over a period of time.

A therapeutically effective amount may be that amount of a composition or an active ingredient thereof sufficient to produce a beneficial effect or otherwise to reduce an adverse, non-beneficial event for the individual to whom the composition is being administered. A therapeutically effective dose may be a dose that produces one or more desired or desirable (eg, beneficial) effects for which it is administered, such administration being one or more times over a given period of time. A precise dose may depend on the purpose of the treatment and may be detectable by one skilled in the art by known techniques.

The conjugates described herein that may be used in therapy may be formulated and dosages determined in a manner consistent with good medical practice, taking into account the disorder to be treated, the condition of the individual patient, the administration site of the composition, the nature of the disease Administration and other factors known to practitioners is consistent. The conjugates described herein can be prepared according to the description of the preparation described herein.

Pharmaceutical compositions may be considered useful in the compositions described herein and methods may be administered to a subject in need thereof using a technique known to those skilled in the art that may be useful as a therapy for the disease or condition that is affecting the subject can. It will be understood by those skilled in the art that the amount, duration and frequency of administration of a pharmaceutical composition described herein to a subject in need thereof will depend on several factors including, but not limited to, the health of the subject. the specific disease or condition of the patient, the grade or stage of a specific disease or condition of the patient, the additional therapeutic agents administered to the subject, and the like.

The methods and compositions described herein may be for administration to a subject in need thereof. Often, administration of the compositions described herein may include routes of administration, non-limiting examples of routes of administration include intravenous, intraarterial, subcutaneous, subdural, intramuscular, intracranial, intrasternal, intratumoral or intraperitoneal. Additionally, a pharmaceutical composition may be administered to a subject by additional routes of administration, for example, by inhalation, oral, dermal, intranasal or intrathecal administration.

Compositions of the present disclosure may be administered to a subject in need thereof in a first administration and in one or more additional administrations. The one or more additional administrations may be administered to the subject in need of them, minutes, hours, days, weeks or months after the first administration. Any additional administration may be administered to the subject in need less than 21 days, or less than 14 days, less than 10 days, less than 7 days, less than 4 days, or less than 1 day after the first administration. One or more administrations may be more than once a day, more than once a week, or more than once a month.

Diseases, conditions and the like

The compositions and methods provided herein may be useful for the treatment of a variety of diseases, conditions, prevention of a disease or condition in a subject or other therapeutic applications for subjects in need thereof. Often they can Compositions and methods provided herein may be useful for the treatment of hyperplastic conditions, including, but not limited to, neoplasms, cancers, tumors, and the like. A condition such as cancer may be associated with the expression of a molecule on the cancerous cells. Often, the molecule expressed by the cancerous cells may comprise an extracellular portion capable of recognition by the antibody portion of the conjugate. A molecule expressed by the cancer cells may be a tumor antigen. An antibody part of the conjugate can recognize a tumor antigen. A tumor antigen may be CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG 72, EpCAM, MUC1, folate binding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Le ^y, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, de2-7 EGFR, Fibroblast activating protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2 / neu, idiotype, MAGE A3, unmutated p53, NY-ESO-1, PMSA, GD2 , CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, Bcr-Abl, tyronsinase, survivin, PSA, HTERT, sarcoma translocation breakpoints, EpbA2, PAP, ML-IAP, AFP, ERG, NA17 , PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML , NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, Sp ermaprotein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, Legumain, Tie3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE or Fos-related antigen 1.

As described herein, a portion of the antigen binding domain of the conjugate may be configured to recognize a molecule that is expressed by a cancer cell, such as a disease antigen, a tumor antigen, or a cancer antigen. Often, such antigens are known to those skilled in the art, or it is found new that they are associated with such a condition that they are often associated and / or specific to that condition. For example, a disease antigen, a tumor antigen or a cancer antigen is, but is not limited to, CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7- H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate binding protein, A33, G250, prostate specific membrane antigen, ferritin, GD2, GD3, GM2, Le ^y, CA-125, CA19-9, epidermal Growth factor, p185HER2, IL-2 receptor, de2-7 EGFR, fibroblast activating protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6E7, EGFRvIII, Her-2 / neu, idiotype, MAGE A3, non-mutant p53, NY-ESO-1, PMSA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, Bcr-Abl, tyrosinase, survivin, PSA, HTERT, sarcoma translocation Breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe (animal), CYP1B1, PLAV1, GM3, BO RIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LC K, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 3, Page 4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE or Fos-related antigen 1. In addition, such tumor antigens may be derived be derived from the following specific conditions and / or families of conditions, including, but not limited to, cancers such as cerebral cancers, skin cancers, lymphomas, sarcomas, lung cancer, liver cancer, leukemia, uterine cancer, breast cancer, ovarian cancer, cervix cancer, bladder cancer, kidney cancer, hemangiosarcoma, Bone cancers, blood cancers, testicular cancer, prostate cancer, stomach cancer, colon cancers, pancreatic cancer and other types of cancers, as well as precancerous conditions such as hyperplasia or the like.

Non-limiting examples of cancers include acute lymphoblastic leukemia (ALL); acute myeloid leukemia; adrenocortical carcinoma; Astrocytoma, cerebellar or cerebral of childhood; basal cell carcinoma; Bladder cancer; Bone tumor, osteosarcoma / malignant fibrous histiocytoma; Brain cancer; Brain tumors, such as cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, visual pathway and hypothalamic glioma; Brainstem glioma; breast cancer; Bronchial adenoma / carcinoid tumors; Burkitt's lymphoma; cerebellar astrocytoma; Cervical cancer; cholangiocarcinoma; chondrosarcoma; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; cutaneous T-cell lymphoma; endometrial cancer; ependymoma; Esophageal cancer; Eye cancer, such as B. intraocular melanoma and retinoblastoma; Gallbladder cancer; glioma; Hairy cell leukemia; Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin's lymphoma; Hypopharynxkrebs; Islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; Kidney cancer (renal cell cancer); Throat cancer; Leukemia, such as acute lymphoblastic, acute myeloid, chronic lymphocytic, chronic myelogenous and hair cell; Lip and oral cancer; liposarcoma; Lung cancer, such as Non-small cell and small cell; Lymphoma, such as AIDS-related, Burkitt; Lymphoma, cutaneous T-cell, Hodgkin's and non-Hodgkin's, macroglobulinemia, bone malignant fibrous histiocytoma / osteosarcoma; melanoma; Merkel cell cancer; mesothelioma; multiple myeloma / plasma cell neoplasia; Mycosis fungoides; myelodysplastic syndromes; myelodysplastic / myeloproliferative disorders; myeloproliferative disorders, chronic; Nasal cavity and sinus cancer; nasopharyngeal carcinoma; neuroblastoma; oligodendroglioma; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of the bone; Ovarian cancer; Pancreatic cancer; Parathyroid cancer; pharyngeal; pheochromocytoma; pituitary adenoma; Plasma cell neoplasia; pleuropulmonary blastoma; prostate cancer; cancer; Renal cell carcinoma (kidney cancer); Renal pelvis and ureter, transitional cell carcinoma; rhabdomyosarcoma; Salivary gland cancer; Sarcoma, Ewing family of tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterus; Sezary syndrome; Skin cancer (non-melanoma); Skin cancer; The small intestine; soft tissue sarcoma; Squamous cell carcinoma; Squamous cell carcinoma of the neck with occult primary, metastatic; Stomach cancer; Testicular cancer; Throat cancer; Thymoma and thymic carcinoma; thymoma; thyroid cancer; Thyroid cancer, childhood; Uterine cancer; vaginal cancer; Waldenstrom's macroglobulinemia; Wilms tumor and any combination thereof.

EXAMPLE 1

Fc receptor binding to anti-CD40 antibody

An anti-CD40 antibody consists of two heavy chains of SBT-040-G1WT and two light chains of an SBT-040 antibody called SBT-040-WT antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040 G1VLPLL and two light chains of an SBT-040 antibody called SBT-040 VLPLL antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1DE and two light chains of an SBT-040 antibody called SBT-040-DE antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1AAA and two light chains of an SBT-040 antibody called SBT-040-AAA antibody.

SBT-040 WT antibodies, SBT-040 VLPLL antibodies, SBT-040-DE antibodies and SBT-040-AAA antibodies are prepared according to standard procedures for the production of antibodies. These antibodies are purified and the affinity of each antibody for soluble glycosylated ectodomains from all human Fcγ receptors (FcγRs) is measured. These affinities are measured by surface plasmon resonance experiments. In these experiments, biotinylated soluble glycosylated FcγR ectodomains from all human FcγRs are immobilized on a streptavidin-coated surface. The ability of each antibody to bind to soluble glycated FcγR ectodomains from all human FcγRs is then measured by surface plasmon resonance using a Biacore instrument. The data from this experiment show that the Fc domain of an SBT-040 WT antibody, the Fc domain of an SBT-040 VLPLL antibody, the Fc domain of an SBT-040-DE antibody, and the Fc Domain of a SBT-040-AAA antibody are each bound by soluble glycosylated FcγR-ectodomains from all human FcγRs. Therefore, the surface plasmon resonance experiments show that the Fc domain of the SBT-040 G1WT antibody and variants of the Fc domain of an SBT-040 G1WT antibody (ie, the Fc domain of an SBT-040 G1VLPLL antibody, the Fc domain of an SBT-040-DE antibody and the Fc domain of an SBT-040-AAA antibody) are each bound by all human FcγRs. The affinity of each antibody for each human FcγR is also demonstrated by these experiments.

EXAMPLE 2

Synthesis of linkers with immunostimulatory compounds

A linker is linked to an immunostimulatory compound. A linker linked to an immunostimulatory compound is formed to form a conjugate of linker and immunostimulatory compound (ATAC). Subsequently, an ATAC is conjugated to an antibody, wherein the ATAC is any of ATAC1-ATAC34 or ATAC43 (each of which is described in the EXAMPLES below).

A linker is linked to an antibody wherein the linker is a pegylated linker, a valine-alanine linker, a valine-citrulline linker, or an N-maleimidomethylcyclohexane-1-carboxylate (MCC) linker. Subsequently, an immunostimulatory compound is conjugated to the linker linked to the antibody, the immunostimulatory compound being a TLR ligand, a nodular-like receptor ligand, a RIG-like receptor ligand, a CLR ligand, a CDS ligand , or is an inflammasome inducer.

A linker is linked to an antibody wherein the linker is a pegylated linker, a valine-alanine linker, a valine-citrulline linker, or an N-maleimidomethylcyclohexane-1-carboxylate (MCC) linker. Subsequently, an immunostimulatory compound is conjugated to the linker linked to the antibody, wherein the immunostimulatory compound is gardiquimod or an analog of a cyclic dinucleotide.

EXAMPLE 3

Synthesis of (1R, 6R, 8R, 9R, 10S, 15R, 17R, 18R) -9-amino-8,17-bis (2-amino-6-oxo-1,9-dihydropurin-9-yl) -3 , 12-Dihydroxy-18-hydroxy-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadecane-3,12-dione (Compound 21)

This example demonstrates the synthesis of (1R, 6R, 8R, 9R, 10S, 15R, 17R, 18R) -9-amino-8,17-bis (2-amino-6-oxo-1,9-dihydropurin-9- yl) -3,12-dihydroxy-18-hydroxy-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadecane-3,12-dione (Compound 21).

Guanosine (200 g, 706.71 mmol, 1.00 equiv.) Was suspended in dry pyridine (4000 mL) under a nitrogen atmosphere and TBSC1 (572 g, 5.30 mol, 7.50 equiv.) Was added dropwise at 0 ° C added. The reaction was stirred at ambient temperature for 3 hours and then cooled to 0 ° C before adding isobutyric anhydride (167 g, 1.06 mol, 1.5 equiv.) Dropwise over 20 min. The solution was allowed to warm to room temperature and stirred for 16 hours. The reaction solution was cooled to 0 ° C and the reaction was stopped by adding water (500 ml). After stirring for 20 min at 0 ° C., 1000 ml of concentrated aqueous NH ₄ OH were added dropwise at 0 ° C. After stirring for an additional 1 h at room temperature, the resulting mixture was concentrated and the residue was dissolved in 3000 ml of water and washed with 1500 ml of EtOAc. The aqueous phase was concentrated to -1000 ml, whereupon the product precipitated out of water. The product was filtered to give 174 g of N- [9 - [(2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-oxo-6,9- dihydro-1H-purin-2-yl] -2-methylpropanamide (Int 2.13-1) as a white solid. Step B: Preparation of Int 2.13-2

To a stirred suspension of Int 2.13-1 (200.0 g, 566.57 mmol, 1.00 equiv.) In pyridine (3 L) under a nitrogen atmosphere was added 4,4 '- (chloro (phenyl) methylene) bis ( methoxybenzene) (211 g, 623.23 mmol, 1.10 equiv.). The resulting mixture was stirred at room temperature for 16 hours. The reaction was quenched with methanol (100 ml) and the mixture was concentrated under vacuum. The residue was dissolved in 3000 ml of dichloromethane, washed with 2 × 1500 ml of saturated sodium bicarbonate solution and 1500 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was applied to a silica gel column with DCM / methanol (with 0.05% triethylamine) (50 / 1-20 / 1). This gave 278 g (75%) of N- [9 - [(2R, 3R, 4S, 5R) -5 - [[bis (4-methoxyphenyl) (phenyl) methoxy] methyl] -3,4-dihydroxyoxolan-2. yl] -6-oxo-6,9-dihydro-1H-purin-2-yl] -2-methylpropanamide (Int 2.13-2) as a pale yellow solid. Step C: Preparation of Int 2.13-3a and Int 2.13-3b

The compound Int 2.13-2 (150 g, 229 mmol, 1.00 equiv.) Was dissolved in 1500 ml of pyridine under a nitrogen atmosphere. 1H-Imidazole (46.71 g, 687.02 mmol, 3.00 equiv.) Was added, followed by the portionwise addition of TBS-C1 (51.6 g, 343.51 mmol, 1.50 equiv.) 25 ° C. The resulting solution was stirred at 25 ° C for 16 hours and then concentrated and dissolved in 2000 ml of dichloromethane. The organic extract was washed with 2 × 1000 ml of saturated sodium bicarbonate solution and 1000 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was applied to a silica gel column with ethyl acetate / dichloromethane (1 / 50-1 / 1) and then purified by flash under the following conditions: silica gel column; Ethyl acetate in dichloromethane with 0.05% triethylamine: 15% up to 70% within 10 minutes and 70% over 10 minutes; This resulted in 62 g (35%) of N- [9 - [(2R, 3R, 4R, 5R) -5 - [[bis (4-methoxyphenyl) (phenyl) methoxy] methyl] -3 - [(tert -butyldimethylsilyl ) oxy] -4-hydroxyoxolan-2-yl] -6-oxo-6,9-dihydro-1 H -purin-2-yl] -2-methylpropanamide (Int 2.13-3a) as a yellow solid and 44 g (25%). ) N- [9 - [(2R, 3R, 4S, 5R) -5 - [[bis (4-methoxyphenyl) (phenyl) methoxy] methyl] -4 - [(tert -butyldimethylsilyl) oxy] -3-hydroxyoxolane 2-yl] -6-oxo-6,9-dihydro-1 H -purin-2-yl] -2-methylpropanamide (Int 2.13-3b). Step D: Preparation of Int 2.13-4

Int 2.13-3a (28 g, 36.41 mmol, 1.00 equiv.) Was dissolved in 280 ml of dichloromethane under a nitrogen atmosphere. 1H-Imidazole-4,5-dicarbonitrile (12.9 g, 109.23 mmol, 3.00 equiv.) And 3- (bis [bis (propan-2-yl) amino] phosphanyloxy) propanitrile (43.84 g , 145.64 mmol, 4.00 equiv.) Were added sequentially. The resulting solution was stirred at 25 ° C for 1 h, and the resulting solution was diluted with 500 ml of dichloromethane and washed with 4 x 400 ml of saturated sodium bicarbonate solution and 1 x 400 ml of saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (C18 silica gel, mobile phase, acetonitrile in water gradient: 40% to 100% within 8 min and 100% over 10 min) to add 25 g of Int 2.13-4 as a white solid result. Step E: Preparation of Int 2.13-5

To a solution of Int 2.13-1 (190 g, 538.24 mmol) in 3000 mL of pyridine was added 1,1,3,3-tetraisopropyl-1,3-dichlorodisiloxane (152.6 g, 484.42 mmol, 0; 9 equiv.) Was added dropwise at 0 ° C. The resulting solution was stirred at 25 ° C for 16 h. The reaction was stopped by adding 30 ml of methanol and the resulting solution was concentrated under vacuum. The residue was applied to a silica gel column with dichloromethane / methanol (50 / 1-30 / 1) to give 189 g of N- [9- [(6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4, 4-tetrakis (propan-2-yl) hexahydro-2H-furo [3,2-f] [1,3,5,2,4] trioxadisilocin-8-yl] -6-oxo-6,9-dihydro 1H-purin-2-yl] -2-methylpropanamide (Int 2.13-5) as a white solid. Step F: Preparation of Int 2.13-6

A solution of 195 g of N- [9 - [(6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4, in a 250 ml three-neck round bottom flask, which was rinsed and maintained in an inert nitrogen atmosphere, 4-tetrakis (propan-2-yl) hexahydro-2H-furo [3,2-f] [1,3,5,2,4] trioxadisilocin-8-yl] -6-oxo-6,9-dihydro 1 H -purin-2-yl] -2-methylpropanamide (Int 2.13-5) in 4000 mL of dichloromethane, 129.5 g (5.00 equiv.) Of pyridine and 4-dimethylaminopyridine (20 g, 163.86 mmol, 0 , 50 equiv.). The solution was cooled to 0 ° C and treated dropwise with 184.8 g (655.5 mmol, 2.0 equiv.) Triflic anhydride. The resulting solution was stirred for 2 h at 0 ° C and then stopped by adding 4000 ml of water / ice. The resulting solution was extracted with 3 x 4000 ml of dichloromethane and the organic layers were combined. The organic extracts were washed with 2 x 4000 ml of water / ice and 1 x 4000 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to give 213 g (crude) of (6aR, 8R, 9R, 9aR) -8- [2- (2-methylpropanamido) -6-oxo-6,9-dihydro -1H-purin-9-yl] -2,2,4,4-tetrakis (propan-2-yl) hexahydro-2H-furo [3,2-f] [1,3,5,2,4] trioxadisilocin-9-yl trifluoromethanesulfonate (Int 2.13-6) as a yellow solid. Step G: Preparation of Int 2.13-7

Int 2.13-6 (213 g, crude) was dissolved in 2100 ml of DMF and treated with sodium nitrite (158.3 g, 2.29 mol, 7.00 equiv.). After stirring for 16 h, the solution was filtered and concentrated in vacuo. The resulting solution was diluted with 6000 ml of DCM and washed with 2 x 3000 ml of brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was applied to a silica gel column with dichloromethane / methanol (50/130/1) and then purified using the following conditions: C18 silica gel, 50% MeOH / water to 100% water for 10 minutes, then 100% water over 10 min to give 50g (26% for 2 steps) of Int 2.13-7 as a white solid. Step H: Preparation of Int 2.13-8

N- [9 - [(6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4,4-tetrakis (propan-2-yl) hexahydro-2H-furo [3,2-f] [ 1,3,5,2,4] trioxadisilocin-8-yl] -6-oxo-6,9-dihydro-1 H -purin-2-yl] -2-methylpropanamide (Int 2.13-7) (50 g, 84 , 03 mmol) and DMAP (30.8 g, 252.10 mmol, 3.00 equiv.) Were dissolved in 500 ml DCM and the mixture was cooled to 0 ° C. Triflic anhydride (30.8 g, 109.24 mmol, 1.30 equiv.) Was then added dropwise with stirring at 0 ° C. The resulting solution was stirred at this temperature for 1 h. The reaction was then stopped by adding 500 ml of ice / water and then extracted with 3 x 500 ml of dichloromethane and the organic layers were combined. The organic layer was washed with 2 × 100 ml of saturated brine. The solution was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product (Int 2.13-8) (55 g) was isolated and used directly in the next step. Step I: Preparation of Int 2.13-9

Int 2,13-8 (55 g, crude) was dissolved in N, N-dimethylformamide (500 ml) and then treated with sodium azide (27.8 g, 427.73 mmol, 5.1 equiv.). The resulting solution was stirred at room temperature for 16 h. The resulting mixture was filtered and concentrated in vacuo. The residue was applied to a silica gel column with dichloromethane / methanol (20/1) to give 15.0 g of Int 2, 13-9 as a yellow solid. Step J: Preparation of Int 2.13-10

Int, 2.13-9 (23 g, 1.00 equiv.) In THF (230 mL) was treated with tetrabutylammonium fluoride (37 mL, 1.0 equiv.). The resulting solution was stirred at room temperature for 10 minutes and then concentrated in vacuo. The residue was applied directly to a silica gel column with dichloromethane / methanol (100 / 1-20 / 1). This gave 12.4 g (88%) of N- [9 - [(2R, 3R, 4S, 5R) -3-azido-4-hydroxy-5- (hydroxymethyl) oxolan-2-yl] -6-oxo. 6,9-dihydro-1 H -purin-2-yl] -2-methylpropanamide (Int 2.13-10) as a white solid. Step K: Preparation of Int 2.13-11

Into a 50 ml round bottom flask was added a solution of 11 g Int 2.13-10 in pyridine (60 ml). 4,4 '- (Chloro (phenyl) methylene) bis (methoxybenzene) (14.75 g, 1.50 equiv.) Was added and the resulting solution stirred for 2 h at room temperature. The reaction was stopped by adding 20 ml of methanol and the resulting mixture was concentrated under vacuum. The residue was dissolved in 500 ml of dichloromethane, washed with 2 × 250 ml of saturated sodium bicarbonate solution and 1 × 250 ml of saturated brine. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was chromatographed with dichloromethane / methanol with 0.05% triethylamine (100 / 1-60 / 1) to give 17.8 g of Int 2, 13-11 as a white solid. Step L: Preparation of Int 2.13-12

To a 50 ml round bottom flask was added a solution of 10 g Int 2.13-11 in methanol (150 ml) and 10% anhydrous palladium on carbon (2 g, w / w). The resulting mixture was stirred under a hydrogen atmosphere for 1 hour at room temperature. The mixture was filtered through celite and concentrated to give 8.7 g of Int 2, 13-12 as a yellow solid, which was used directly without further purification. Step M: Preparation of Int 2.13-13

To a solution of 16.8 g of N- [9 - [(2R, 3R, 4S, 5R) -3-amino-5 - [[bis (4-methoxyphenyl) (phenyl) methoxy] methyl] -4-hydroxyoxolane -2-yl] -6-oxo-6,9-dihydro-1 H -purin-2-yl] -2-methylpropanamide (Int 2.13-12) in 210 mL THF / water (4/1) was added sodium bicarbonate (6, 46 g, 3.00 equiv.) Followed by the addition of Cbz-Cl (5.67 g, 1.30 equiv.). The resulting solution was stirred at room temperature for 20 minutes. The reaction was diluted with saturated sodium carbonate solution (100 ml) and extracted with 3 × 200 ml dichloromethane. The organic phase was dried over sodium sulfate, filtered and concentrated in vacuo. The residue was chromatographed with dichloromethane / methanol containing 0.05% triethylamine (100/1) and then crystallized from dichloromethane (40 ml) to add 14.8 g (86% over two steps) of Int 2.13-13 as a white solid result. Step N: Preparation of Int 2.13-14

Int 2,13-13 (14.8 g, 18.78 mmol) was dissolved in 150 ml of dichloromethane under a nitrogen atmosphere, 3- (bis [bis (propan-2-yl) amino] phosphanyloxy) propanitrile (22.6 g , 75.12 mmol, 4.00 equiv.) And 1H-imidazole-4,5-dicarbonitrile (6.64 g, 56.34 mmol 3.00 equiv.) Were added sequentially. The resulting solution was stirred for 1 h at 25 ° C and diluted with 400 ml of dichloromethane. The solution was washed with 1 x 500 ml of saturated sodium bicarbonate solution and 1 x 500 ml of saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by flash with the following conditions: column, C18 silica gel; Mobile phase, acetonitrile in water: 30% up to 80% within 8 min and 100% maintained over 10 min to give 14.8 g (75%) N- [9 - [(2R, 3R, 4R, 5R) - 5 - [[bis (4-methoxyphenyl) (phenyl) methoxy] methyl] -3 - ([[bis (propan-2-yl) amino] (2-cyanoethoxy) phosphanyl] oxy) -4 - [(tert-butyldimethylsilyl ) oxy] oxolan-2-yl] -6-oxo-6,9-dihydro-1 H -purin-2-yl] -2-methylpropanamide (Int 2.13-14) as a white solid. Step O: Preparation of Int 2.13-15

To a solution of Int 2,13-14 (7.2 g, 7.20 mmol) in acetonitrile (600 mL) and water (260 mg) was added pyridinium triflate (1.66 g, 8.38 mmol, 1.20 equiv .). The resulting solution was stirred at room temperature for 10 minutes to give a solution of Int 2.13-15, which was used directly in the next step. Step P: Preparation of Int 2.13-16

The solution containing Int 2.13-15 was treated with tert-butylamine (36 ml) for 30 minutes at room temperature. The mixture was then concentrated under vacuum to give 7.5 g of Int 2, 13-16 as a foam, which was used directly in the next step without further purification. Step Q: Preparation of Int 2.13-17

Int. 2.13-16 was dissolved in 75 ml of dichloromethane and the solution treated with 91.5 ml of 6% dichloroacetic acid in dichloromethane. Triethylsilane (150 ml) was added after 10 minutes, followed by pyridine (10 ml). The mixture was concentrated and the residue was dissolved in 50 ml of dry acetonitrile and concentrated. This process was repeated twice. The residue was finally dissolved in 20 ml of acetonitrile and used directly in the next step. Step R: Preparation of Int 2.13-18

To the above solution containing 3.0 g Int 2, 13-17 and 3.0 g 4A MS in 20 ml MeCN, a solution of Int 2, 13-4 (18 g, 18.56 mmol, 2.5 Equiv.) In 30 ml of dry acetonitrile. The reaction solution was stirred for 5 minutes at 25 ° C and used directly in the next step. Step S: Preparation of Int 2.13-19

To the above reaction solution (Int 2.13-18) was added 8 ml of tert-butyl hydroperoxide (5.5 M in decane). The resulting solution was stirred at 25 ° C for 30 minutes and then diluted with 250 ml of ethyl acetate. The resulting solution was washed with 2 × 300 ml of water and 1 × 300 ml of saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered and concentrated under vacuum to give 21 g Int 2.13-19, which was used directly in the next step. Step T: Preparation of Int 2.13-20

Int 2.13-19 (21 g, crude) was dissolved in methylene chloride (250 mL) before 210 mL of dichloroacetic acid (6% in methylene chloride) was added to the solution. After stirring for 10 minutes, 420 ml of triethylsilane were added with stirring at room temperature. Then, 50 ml of pyridine was added and the mixture was concentrated under vacuum. The resulting residue was purified by flash column chromatography (C18 gel column, mobile phase, acetonitrile / water with 0.04% ammonium bicarbonate: 20% up to 80% within 10 min, then 100% over 5 min). This gave 2.5 g (30% total for 6 steps) of Int 2.13-20 as a white solid. Step U: Preparation of Int 2.13-21

The compound 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide (1.9 g, 10.39 mmol, 4.7 eq) was added to the solution of Int 2.13-20 (2 , 5 g, 2.21 mmol) in 50 ml of pyridine and the reaction solution was stirred at 25 ° C for 15 min. The reaction was quenched by water (600 μl) followed by iodine (0.84 g, 3.31 mmol, 1.5 equiv.). After stirring for 20 minutes, 12.5 ml of saturated sodium thiosulfate solution was added. The mixture was concentrated to a foam to give 2.5 g Int 2.13-21, which was used directly in the next step. Step V: Preparation of Int 2.13-22

To a solution of 2.5 g crude Int 2.13-21 in acetonitrile (12.5 ml) was added tert-butylamine (22.5 ml). After stirring for 10 min at 25 ° C, the reaction solution was concentrated to a yellow foam and the crude product was purified by flash chromatography (C18 gel column, mobile phase, acetonitrile / water with 0.04% ammonium bicarbonate, gradient: 10% up to 50% within 15 min, 100% over 5 min) to give pure 1.2 g (50% over 2 steps) Int 2.13-22. Step W: Preparation of Int 2.13-23

A 50 ml round bottom flask was purged with argon and then loaded with a solution of Int 2, 13-22 (500 mg, 0.46 mmol) in 25 ml of methanol. 10% anhydrous palladium-carbon (250 mg, w / w) was then added and hydrogen was bubbled through the solution. The reaction mixture was at 25 ° C for 1 h stirred, then filtered through celite and concentrated to give 380 mg (87%) int 2.13-23 as a white solid. Step X: Preparation of Int 2.13-24

To a 100 ml round bottom flask was added Int 2.13-23 (380 mg, 0.40 mmol) and 25 ml of methylamine (33% in anhydrous ethanol). The resulting solution was stirred at 25 ° C for 16 h and then concentrated to give Int 2.13-24 as a white foam, which was used directly in the next step. Step Y: Preparation of compound 21

The above crude Int 2.13-24 was azeotroped three times with pyridine / triethylamine (9 ml / 3 ml) and then dissolved in 0.8 ml pyridine in a 100 ml round bottom flask. 6 ml of triethylamine and 4 ml of triethylamine trihydrofluride were added simultaneously to this solution at 55.degree. After stirring for 1 h, the bath was removed and 60 ml of anhydrous acetone were added immediately. The mixture was stirred for 20 minutes and the white solid was collected by filtration. The precipitate was washed with 5 ml of anhydrous acetone. The product was purified by preparative flash chromatography (AQ-C18 silica gel, mobile phase = acetonitrile / water with 0.04% ammonium bicarbonate, gradient 1% to 20% over 20 min, UV detector at 210 nm). The resulting solution was lyophilized to give 100 mg (36% for 2 steps) of compound 21 as a white solid. LC-MS-SVT-001-1-24: (ESI, m / z): 690 [M + H] ^{+ 1} H NMR (D ₂ O): δ 7.86 (s, 1H); 7.83 (s, 1H); 5.80-5.92 (m, 2H); 4.96-5.09 (m, 1H); 4.84-4.88 (m, 1H); 4.69-4.83 (m, 1H); 4.32-4.39 (m, 1H); 4,11-4,39 (m, 4H); 3.91-4.02 (m, 2H); [0.18 equiv TEA: 2.98-3.12 (q, J = 6.6, 1H); 1.05-1.24 (m, J = 6.6, 1.6H)]. ³¹ P NMR (D ₂ O) δ -1.28, -1.39.

EXAMPLE 4

Synthesis of ATAC1 and ATAC2

Schritt A: Herstellung von Int ATAC1-1

This example demonstrates the synthesis of pentafluorophenyl 25- (2-amino-3-pentylquinolin-5-yl) -19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC1) and perfluorophenyl-3 - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) -4-oxo-7,10,13,16, 19-pentaoxa-3-azadocosan-22-oat (ATAC2).

Step A: Preparation of Int ATAC1-1

To a solution at 0 ° C containing 271 mg (0.90 mmol) of 5- (5-aminopentyl) -3-pentylquinoline-2-amine in 4 ml of DCM was added 435 mg (1.00 mmol) of the NHS. Esters in 1 ml DCM added dropwise over 15 minutes. The reaction mixture was allowed to warm to ambient temperature for 19 hours before being concentrated and purified by reverse phase chromatography. The pure fractions were lyophilized and dissolved in 3 ml of methanol and then treated with 1 ml of 4N HCl in dioxane. The solution was stirred for 1 h and then concentrated to give the desired compound as the HCl salt, which was used directly in the next step. Step B: Preparation of ATAC1

To a stirred solution of 25- (2-amino-3-pentylquinolin-5-yl) -19-oxo-4,7,10,13,16-pentaoxa-20-azapentacanoic acid hydrochloride (130 mg, 0.198 mmol) and Pentafluorophenol (146 mg, 0.792 mmol) in DMF (2.5 mL) was added dropwise at room temperature to N, N'-diisopropylcarbodiimide (0.186 mL, 1.189 mmol) added. The reaction was stirred at room temperature for 18 hours and then concentrated. The crude product was added to a 100 g C18 gold reverse phase column and eluted with water / acetonitrile (0.1% TFA) 10-100%. The fractions were combined and concentrated, then lyophilized to give perfluorophenyl 25- (2-amino-3-pentylquinolin-5-yl) -19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate-2 To give 2,2-trifluoroacetate (110 mg, 61.7% yield) as a clear gum. ¹ H NMR (DMSO-d ⁶ ) δ 13.7 (s, 1H), 8.37-8.35 (m, 3H), 7.78 (t, J = 5.5Hz, 1H), 7.63 (t , J = 7.5 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 7.0 Hz, 1H), 3.58 (t, J = 6.0 Hz, 2H), 3.63-3.43 (m, 20H), 3, q04-2.96 (m, 6H), 2.73 (t, J = 7.5 Hz, 2H) , 2.27 (t, J = 7.5 Hz, 2H), 1.60-1.55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J = 7.5 Hz, 3H). LCMS [M + H] = 786.3.

The following compound in TABLE 2 may be prepared using a method similar to that described above for ATAC1.

EXAMPLE 5

Synthesis of ATAC3 and ATAC4

Schritt A: Herstellung von ATAC3 This example demonstrates the synthesis of pentafluorophenyl 25- (2-amino-3-pentylquinolin-5-yl) -19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC3) and 2,5 -Dioxopyrrolidin-1-yl-3 - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) -4-oxo -7,10,13,16,19-pentaoxa-3-azadocosan-22-oat (ATAC4).

Step A: Preparation of ATAC3

To a stirred solution of Int ATAC1-1 (185 mg, 0.282 mmol) and N-hydroxysuccinimide (130 mg, 1.128 mmol) in DMF (3 mL) was added N, N'-diisopropylcarbodiimide (0.221 mL, 1.411 mmol) dropwise and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered and washed with acetonitrile and the filtrate was evaporated. The resulting residue was purified by silica gel silica gel column chromatography (DCM / MeOH / HOAc) to give 65 mg of the desired product as an acetic acid salt, which was then dissolved in 2 ml DCM and treated with 2 M HCl in diethyl ether. The solution was stirred for 1 h, then concentrated and lyophilized to give the desired compound as HCl salt. ¹ H NMR _(CDCl3) δ 15.2 (s, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7 , 55 (t, J = 8.1 Hz, 1H), 6.55 (bs, 1H), 3.98 (t, J = 6.0 Hz, 2H), 3.83-3.55 (m, 18H), 3.33-3.22 (m, 2H), 2.95-2.56 (m, 11H), 2.27 (t, J = 7.5Hz, 2H), 1.60-1 , 55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J = 7.5 Hz, 3H). LCMS [M + H] = 717.3.

¹H NMR (CDCl₃) δ 14,9 (s, 1H), 8,88 (bs, 1H), 8,15 (d, 1H), 7,85 (d, 1H), 7,61 (t, 1H), 7,45 (t, 1H), 4,72 (s, 2H), 3,83 (m, 4H), 3,65-3,45 (m, 18H), 2,90-2,71 (m, 9H), 1,43 (t, J = 7,0 Hz, 3H), 1,33 (s, 6H). LCMS [M+H] = 731. The following 2,5-dioxopyrrolidin-1-yl-3 - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl ) -4-Oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oat (ATAC4) Compound can be prepared by a method similar to that described above for ATAC3.

¹ H NMR _(CDCl3) δ 14.9 (s, 1H), 8.88 (bs, 1H), 8.15 (d, 1H), 7.85 (d, 1H), 7.61 (t, 1H), 7.45 (t, 1H), 4.72 (s, 2H), 3.83 (m, 4H), 3.65-3.45 (m, 18H), 2.90-2.71 (m, 9H), 1.43 (t, J = 7.0 Hz, 3H), 1.33 (s, 6H). LCMS [M + H] = 731.

EXAMPLE 6

Synthesis of ATAC5, ATAC6 and ATAC7

Schritt A: Herstellung von Int ATAC5-1

This example shows the synthesis of 2,5-dioxopyrrolidin-1-yl-6 - (((S) -1 - (((S) -1 - ((4 - ((((5- (2-amino-3 -pentylquinolin-5-yl) pentyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoate ( ATAC5), 2,5-dioxopyrrolidin-1-yl-7 - (((S) -1 - (((S) -1 - ((4 - ((((4-amino-1- (2-hydroxy -2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3 -methyl-1-oxobutan-2-yl) amino) -7-oxoheptanoate (ATAC6) and 2,5-dioxopyrrolidin-1-yl-7 - (((S) -1 - (((S) -1- ( (4 - (((((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -7-oxoheptanoate (ATAC7).

Step A: Preparation of Int ATAC5-1

A solution of 5- (5-aminopentyl) -3-pentylquinoline-2-amine (300 mg, 1.00 mmol) in 5 mL DCM was stirred at room temperature under nitrogen for 10 min before tert-butyl adipate-valine-alanine. para-aminobenzyl-4-nitrophenyl carbonate (tBuAdip-va-PAB-OPNP, 656 mg, 1.00 mmol) and DIPEA (0.26 mL, 1.5 mmol) in 3 mL DCM were added and the mixture became overnight stirred at room temperature. The mixture was concentrated and purified by column chromatography. Clean fractions were combined and evaporated and the residue was dissolved in 2 mL DCM and treated with 2 M HCl in diethyl ether. The solution was stirred for 1 h and then concentrated and lyophilized to give the desired compound Int ATAC5-1 as HCl salt. MS m / z 761 (M) ⁺ . Step B: Preparation of ATAC5

To a stirred solution of 6 - (((S) -1 - (((S) -1 - ((4 - ((((((5- (2-amino-3-pentyl-quinolin-5-yl) -pentyl) -carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoic acid hydrochloride (221 mg, 0.282 mmol) and N- Hydroxysuccinimide (130 mg, 1.12 mmol) in DMF (3 mL) was added dropwise N, N'-diisopropylcarbodiimide (0.221 mL, 1.41 mmol) and the reaction was stirred at room temperature for 5 h. HPLC indicated that some starting material remained so that the reaction was stirred at room temperature overnight. The reaction mixture was filtered and washed with acetonitrile. The filtrate was evaporated and the residue was dissolved in DMSO and purified by reverse phase chromatography [water / acetonitrile (0.1% TFA)] of 10%, followed by a gradient of 20 to 80%. The pure fractions were converted to 2,5-dioxopyrrolidin-1-yl-6 - (((S) -1 - (((S) -1 - ((4 - ((((5- (2-amino-3 -pentylchinolin-5-yl) pentyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoat- 2,2,2-trifluoroacetate (109 mg, 40% yield) combined as a white solid. ¹ H NMR (DMSO-d ⁶⁾ δ 13.6 (s, 1H), 9.92 (s, 1H), 8.32 (d, J = 7.5Hz, 1H), 7.84 (d, J = 8.5Hz, 1H), 7.61-7.55 (m , 4H), 7.30-7.17 (m, 4H), 4.92 (s, 2H), 4.37 (t, J = 7.0 Hz, 1H), 4.18 (t, J = 7.0 Hz, 1H), 2.96 (m, 4H), 2.81-2.62 (m, 8H), 2.33-2.11 (m, 2H), 1.95 (q, J = 7.0 Hz, 1H), 1.63-1.55 (m, 8H), 1.50-1.40 (m, 2H), 1.38-1.33 (m, 4H), 1, 29 (d, J = 7.0 Hz, 3H), 0.83 (d, J = 7.0 Hz, 6H). LCMS [M + H] = 844.3.

The following ATAC6 compound and ATAC7 compound in TABLE 3 can be prepared by a method similar to that described above for ATAC5.

EXAMPLE 7

Synthesis of ATAC8, ATAC9 and ATAC10

Schritt A: Herstellung von ATAC8

This example shows the synthesis of perfluorophenyl-6 - (((S) -1 - (((S) -1 - ((4 - (((((5- (2-amino-3-pentyl-quinolin-5-yl) pentyl ) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoate (ATAC8), perfluorophenyl-7- ( ((S) -1 - (((S) -1 - ((4 - (((((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -7- oxoheptanoate (ATAC9) and perfluorophenyl-7 - (((S) -1 - (((S) -1 - ((4 - ((((4-amino-1- (2-hydroxy-2-methylpropyl) - 1H-imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamoyl) oxy) methyl) phenyl) amino) -1-oxo-5-ureidopentan-2-yl) amino) -3-methyl 1-oxobutan-2-yl) amino) -7-oxohepanoate (ATAC10).

Step A: Preparation of ATAC8

To a stirred solution of 6 - (((S) -1 - (((S) -1 - ((4 - ((((((5- (2-amino-3-pentyl-quinolin-5-yl) -pentyl) -carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoic acid hydrochloride (168 mg, 0.215 mmol) and pentafluorophenol ( 158 mg, 0.86 mmol) in DMF (3 mL) was added dropwise N, N'-diisopropylcarbodiimide (0.166 mL, 1.07 mmol) and the reaction mixture stirred at room temperature for 6 h. The reaction mixture was concentrated and the residue was dissolved in DMSO and purified by reverse phase chromatography [water / acetonitrile (0.1% TFA)] of 10%, followed by a gradient of 20 to 80%. Pure salts were combined to give perfluorophenyl-6 - (((S) -1 - (((S) -1 - ((4 - ((((5- (2-amino-3-pentyl-quinolin-5-yl) pentyl ) carbamoyl) oxy) methyl) phenyl) amino) -1-oxopropan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) amino) -6-oxohexanoate 2,2,2-trifluoroacetate ( 122 mg) as a white solid. ¹ H NMR (DMSO-d ⁶ ) δ 13.5 (s, 1H), 9.92 (s, 1H), 8.35 (bs, 3H), 8.17 (d, J = 7.0 Hz, 1H), 7.87 (d, J = 7.0Hz, 1H), 7.64-7.52 (m, 4H), 7.32-7.18 (m, 4H), 4.91 (s , 2H), 4.37 (t, J = 7.0 Hz, 1H), 4.19 (t, J = 7.0 Hz, 1H), 3.60-3.50 (m, 4H), 2 , 97 (m, 4H), 2.79 (t, J = 7.0 Hz, 2H), 2.74 (t, J = 7.0 Hz, 2H), 2.31-2.22 (m, 2H), 1.96 (q, J = 7.0 Hz, 1H), 1.71-1.51 (m, 8H), 1.45-1.38 (m, 2H), 1.40-1 , 27 (m, 9H), 0.90-0.80 (m, 9H). LCMS [M + H] = 913.4.

The following compound in TABLE 4 can be prepared by a method similar to that described above for ATAC8.

EXAMPLE 8

Synthesis of ATAC11

Schritt A: Herstellung von ATAC11

This example shows the synthesis of N - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) -1- (3 (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -N-ethyl-3,6,9,12-tetraoxapentadecane-15-amide (ATAC11).

Step A: Preparation of ATAC11

A solution of MAL-PEG4 acid (265.7 mg, 0.638 mmol) and N, N'-dicyclohexylcarbodiimide (DCC, 144.8 mg, 0.702 mmol) in dry dichloromethane / acetonitrile (1: 1.5 mL) was added Room temperature for 1 h, followed by the addition of compound 1 (100 mg, 0.319 mmol) in one portion. After stirring for 72 h, volatile organic compounds were removed under vacuum. The resulting residue was purified by flash column chromatography on silica gel, eluting with step gradients of methanol in dichloromethane in a ratio of v / v 1:20, 1:15 and 1: 9, giving the target product N - ((4-amino -1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) -1- (3- (2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl) -propane-amido) -N-ethyl-3,6,9,12-tetraoxapentadecane-15-amide (80 mg, 35% yield) as a white foamy solid oil. ¹ H NMR (300 MHz, CDCl ₃₎ δ 8.40 to 7.82 (br m, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 7 , 5 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 6.76-6.28 (br m, 2H), 4.82-4.32 (br m, 2H) , 4.08-3.64 (br m, 6H), 3.54 (br s, 14H), 3.31 (br s, 3H), 2.63 (br s, 2H), 2.38 (t , J = 6.9 Hz, 2H), 1.27 (br s, 4H), 1.20-0.68 (br m, 5H). MS (ESI +) m / z 712 (M + 1), 734 (M + Na).

EXAMPLE 9

Synthesis of ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20 and ATAC21

Schritt A: Herstellung von ATAC12

This example shows the synthesis of N- (5- (2-amino-3-pentylquinolin-5-yl) pentyl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrole-1 -yl) propanamido) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC12), 1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido ) -N- (3-pentylquinolin-2-yl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC13), 1- (3- (2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl) propanamido) -N- (1-isobutyl-1H-imidazo [4,5-c] quinolin-4-yl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC14) , 1- (3- (2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -N-methyl-N- (2- (3- (7-methylbenzo) [1,2 -d: 3,4-d '] bis (thiazole) -2-yl) ureido) ethyl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC15), (S) -1- (3-) (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -N- (1 - ((7-methylbenzo [1,2-d: 3,4-d '] bis ( thiazole) -2-yl) amino) -1-oxo-3-phenylpropan-2-yl) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC16), N- (benzo [d] thiazole-2 -yl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) propanamido) -N - ((8-hydroxyquinoline-7-yl) (4- (trifluoromethoxy ) phenyl) methyl) -3,6,9,12-tetraoxapentadec an-15-amide (ATAC17), N - ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -2,9-bis (2-amino-6-oxo-1H-purine-9 ( 6H) -yl) -5,10,12-trihydroxy-5,12-dioxidodecahydrodifuro [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetra-oxadiphosphacyclododecin 3-yl) -1- (3- (2,5-dioxo-2,5-dihydro-1 H -pyrrol-1-yl) propanamido) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC18 ), N - ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -2,9-bis (2-amino-6-oxo-1H-purine-9 (6H) -yl) -10 -hydroxy-5,12-dimercapto-5,12-dioxidodecahydrodifuro [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetraoxadiphosphacyclododecin-3-yl) -1 - (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC19), N- (9- ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -9- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -3,5,10,12- tetrahydroxy-5,12-dioxidodecahydrodifuro [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetraoxadiphosphacyclododecin-2-yl) -9H-purin-6-yl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC20) and N- ( 9 - ((2R, 3R, 3aS, 7aR, 9R, 10R, 10aS, 14aR) -9- (2-amino-6-oxo-1H-purin-9 (6H) -yl) -3,5,10, 12-tetrahydroxy-5,12-dioxidodecahydrodi-furo [3,2-d: 3 ', 2'-j] [1,3,7,9,2,8] tetraoxadiphosphacyclododecin-2-yl) -9H-purin 6-yl) -1- (3- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -propanamido) -3,6,9,12-tetraoxapentadecane-15-amide (ATAC21) ,

Step A: Preparation of ATAC12

To a stirred solution containing 100 mg (0.33 mmol) of 5- (5-aminopentyl) -3-pentylquinolin-2-amine in 13 ml of CH ₂ Cl ₂ under N ₂ was added a solution of MAL-PEG4-NHS [CAS # 756525-99-2] (171 mg, 0.33 mmol) in 3 mL of CH ₂ Cl ₂ added via syringe pump over 90 min. The reaction mixture was stirred at room temperature for 16 h and then evaporated to give a residue which was purified by silica gel chromatography (CombiFlash Gold (12 g): CH ₂ Cl ₂ / CH ₃ OH / NH ₄ OH) to give a light yellow syrup which was dissolved in 5 ml CH ₃ CN and lyophilized to give 164 mg of the desired compound. ¹ H NMR (CD ₃ OD) δ 7.95 (s, 1H), 7.38 (s, 1H), 7.37 (s, 1H), 7.07 (t, J = 8.5 Hz, 1H ), 3.78 (t, J = 6.0 Hz, 2H), 3.65 (t, J = 6.0 Hz, 2H), 3.59-3.52 (m, 12H), 3.46 (t, J = 5.5 Hz, 2H), 3.28 (t, J = 7.5 Hz, 2H), 3.18 (t, J = 7.5 Hz , 2H), 2.98 (t, J = 8.5 Hz, 2H), 2.67 (t, J = 7.5 Hz, 2H), 2.44 (t, J = 7.0 Hz, 2H ), 2.40 (t, J = 7.0 Hz, 2H), 1.76-1.68 (m, 4H), 1.58-1.52 (m, 2H), 1.46-1, 40 (m, 6H), 0.94 (t, J = 7.0 Hz, 3H). (MS (ESI +) m / z 698 (M + 1).

The following compounds in TABLE 5 can be prepared by a method similar to that described above for ATAC12.

EXAMPLE 10

Synthesis of ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30 and ATAC31

Schritt A: Herstellung von ATAC22

This example shows the synthesis of 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3 -methylbutanamido) propanamido) benzyl - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H -imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl) carbamate (ATAC22) , 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -hexanamido) -3-methyl-butanamido) propanamido) benzyl (5- (2-amino-3-pentylquinolin-5-yl) pentyl) carbamate (ATAC23), 4 - ((S) -2 - ((S) -2- (6- (2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methyl-butan-amido) -5-ureidopentanamido) benzyl- (5- (2-amino-3-pentylchinolin-5-yl) pentyl) carbamate (ATAC24), 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) (ethyl ) carbamate TFA salt (ATAC25), 2- (3- {2- [N -methyl ({p - [(S) -2 - {(S) -2- [6- (2,5-dioxo) 1H-pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonyl) amino] ethyl} ureido) -7-methyl- l-1,6-dithia-3,8-diaza-as-indacene (ATAC26), 2 - {[(8-hydroxy-7-quinolyl) (p-trifluoromethoxyphenyl) methyl] ({p - [(S) - 2 - {(S) -2- [6- (2,5-dioxo-1H-pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonyl) amino} -1,3- benzothiazole (ATAC27), (1R, 6R, 8R, 95,10S, 15R, 17R, 18S) -18 - ({p - {(S) -2 - {(5) -2- [6- (2.5 dioxo-1H-pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonylamino) -8.17-bis (2-amino-6-oxo-1,9-dihydro-9-yl) -3.12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa-3λ5. 12λ5-diphosphatricyclo [13.3.0.06,10] octadecane-3,12-dione (ATAC28), (1R, 6R, 8R, 95,10S, 15R, 17R, 18S) -18 - ({p - [(5) - 2 - {(S) -2- [6- (2,5-dioxo-1H-pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} propionylamino] -phenyl} methoxycarbonylamino) -8.17-bis (2-amino -6-oxo-1,9-dihydro-9-yl) -3.12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadecane-3, 12-dione (ATAC29), (1R, 6R, 8R, 9S, 10S, 15R, 17R, 18S) -18 - ({p - [(S) -2 - {(S) -2- [6- (2 , 5-dioxo-1H-pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methoxycarbonylamino) -8.17-bis (2-amino-6-oxo-1,9-dihydropurin- 9-yl) -9-hydroxy-3,12-dimercapto-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadecane-3,12-dione (ATAC30) and {p- [ (S) -2 - {(S) -2- [6- (2,5-dioxo-1H-pyrrol-1-yl) hexanoylamino] -3-methylbutyrylamino} -5-ureidovalerylamino] phenyl} methyl-9- { (1S, 6R, 8R, 9S, 10S, 15R, 17R, 18S) -8- (2-amino-6-oxo-1,9-dihydro-9-yl) -3.12-dihydroxy-9,18-dihydroxy-3,12-dioxo-2.4.7.11.13.16 hexaoxa-3λ5.12λ5-diphosphatricyclo [13.2.1.06,10] octadec-17-yl} -9a-adenine carboxylate (ATAC31).

Step A: Preparation of ATAC22

A solution of compound 1 (150 mg, 0.479 mmol) and N, N'-diisopropylethylamine (145.4 mg, 1.437 mmol) in dry DMF was stirred at room temperature for 5 min, followed by the addition of maleimidocaproyl-valine-alanine-p -Aminobenzyl alcohol p-nitrophenyl carbonate (MC-Val-Ala-PAB-PNP, 343.6 mg, 0.527 mmol). After stirring for 24 hours, volatile organic compounds were removed under vacuum. The resulting residue was triturated with dry acetonitrile. The precipitated solid was collected by filtration, washed with acetonitrile and dried under vacuum to give unreacted MC-Val-Ala-PAB-PNP (130 mg) as a beige solid. The filtrate and washes were combined and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel eluting with step gradients of MeOH in dichloromethane in a ratio of v / v 1:20, 1:15 and 1:10 to give the target product mc-Val-Ala-PAB -GDQ (70 mg, 18% yield) as a beige foamy solid. ¹ H NMR (DMSO-d ⁶ ) δ 10.1-9.75 (br m, 1H), 8.58-8.24 (br m, 1H), 8.15 (d, J = 6.6 Hz , 1H), 8.01 (brs, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.65- 7.48 (m, 2H), 7.46-7.34 (m, 2H), 7.29 (br s, 1H), 7.18 (br s, 1H), 6.99 (s, 2H) , 5.03 (brs, 2H), 4.96 (brs, 1H), 4.72 (brs, 1H), 4.48-4.26 (m, 1H), 4.26-4, 04 (m, 1H), 2.22-2.02 (m, 2H), 2.02-1.80 (m, 1H), 1.58-1.37 (m, 4H), 1.36- 0.92 (br m, 15H), 0.92-0.53 (br m, 7H). MS (ESI +) m / z 826 (M + 1).

¹H NMR (CD₃OD) δ 8,35 (s, 1H), 7,63 (t, J = 8.5 Hz, 1H), 7,55 (d, J = 8,0 Hz, 1H), 7,47 (d, J = 8,0 Hz, 1H), 7,33 (d, J = 8,0 Hz, 1H), 7,27 (d, J = 8,0 Hz, 1H), 6,78 (s, 2H), 5,00 (s, 2H), 4,46 (q, J = 7,0 Hz, 2H), 4,13 (d, J = 7,0 Hz, 1H), 3,47-3,4 (m, 3H), 3,17 (t, J = 7,0 Hz, 2H), 3,05 (t, J = 7,0 Hz, 2H), 2,75 (t, J = 7,5 Hz, 2H), 2,27 (t, J = 7,5 Hz, 2H), 2,07 (q, J = 7,0 Hz, 1H), 1,72-1,51 (m, 10H), 1,46-1,35 (m, 8H), 1,32-1,26 (m, 3H), 1,00-0,92 (m, 9H). LCMS [M+H] = 812,4. ATAC24: 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl-(5-(2-Amino-3-pentylchinolin-5-yl)pentyl)-carbamat

¹H NMR (DMSO-d⁶) δ 13,5 (bs, 1H), 10,0 (s, 1H), 8,40 (m, 3H), 8,07 (d, J = 7,5 Hz, 1H), 7,80 (d, J = 8,5 Hz, 1H), 7,6-7,5 (m, 4H), 7,35-7,25 (m, 2H), 6,01 (m, 1H), 5,42 (s, 1H), 4,89 (s, 2H), 4,41 (q, J = 7,0 Hz, 1H), 4,18 (t, J = 7,0 Hz, 1H), 3,10-2,90 (m, 6H), 2,75 (t, J = 7,5 Hz, 2H), 2,27 (t, J = 7,5 Hz, 2H), 2,07 (q, J = 7,0 Hz, 1H), 1,72-1,51 (m, 10H), 1,46-1,35 (m, 8H), 1,32-1,26 (m, 3H), 1,00-0,92 (m, 9H). LCMS [M+H] = 898. ATAC25: 4-((S)-2-((S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl-((4-Amino-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]chinolin-2-yl)methyl)(ethyl)-carbamat-TFA-Salz

¹H NMR (DMSO-d⁶) δ 13,4 (bs, 1H), 9,99-9,89 (br m, 1H), 9,09-8,40 (m, 3H), 8,07 (d, J = 7,5 Hz, 1H), 7,80 (d, J = 8,5 Hz, 1H), 7,68 (t, J = 8,0 Hz, 1H), 7,59 (bs, 1H), 7,51 (t, J = 8,5 Hz, 1H), 7,46-7,14 (m, 2H), 7,00 (s, 1H), 5,99 (br s, 1H), 5,05 (br s, 1H), 4,95 (br s, 1H), 4,37 (q, J = 7,0 Hz, 1H), 4,18 (t, J = 7,0 Hz, 1H), 3,37 (t, J = 7,0 Hz, 2H), 3,03-2,93 (m, 2H), 2,22-2,07 (m, 2H), 1,99-1,92 (m, 1H), 1,75-1,05 (br m, 20H), 0,85 (d, J = 8,5 Hz, 3H), 0,81 (d, J = 8,5 Hz, 3H). MS (ESI+) m/z 912,5 (M+1).

The following ATAC30, ATAC31, ATAC32, ATAC33, ATAC34, ATAC35, ATAC36, ATAC37, ATAC38, ATAC39, ATAC40, ATAC41 and ATAC42 can be prepared by a method similar to that described above for ATAC29. ATAC23: 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) propanamido ) benzyl- (5- (2-amino-3-pentylchinolin-5-yl) pentyl) -carbamate

¹ H NMR (CD ₃ OD) δ 8.35 (s, 1H), 7.63 (t, J = 8.5 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7, 47 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 6.78 ( s, 2H), 5.00 (s, 2H), 4.46 (q, J = 7.0 Hz, 2H), 4.13 (d, J = 7.0 Hz, 1H), 3.47- 3.4 (m, 3H), 3.17 (t, J = 7.0 Hz, 2H), 3.05 (t, J = 7.0 Hz, 2H), 2.75 (t, J = 7 , 5 Hz, 2H), 2.27 (t, J = 7.5 Hz, 2H), 2.07 (q, J = 7.0 Hz, 1H), 1.72-1.51 (m, 10H ), 1.46-1.35 (m, 8H), 1.32-1.26 (m, 3H), 1.00-0.92 (m, 9H). LCMS [M + H] = 812.4. ATAC24: 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamido) -3-methylbutanamido) - 5-ureidopentanamido) benzyl- (5- (2-amino-3-pentylchinolin-5-yl) pentyl) -carbamate

¹ H NMR (DMSO-d ⁶ ) δ 13.5 (bs, 1H), 10.0 (s, 1H), 8.40 (m, 3H), 8.07 (d, J = 7.5 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.6-7.5 (m, 4H), 7.35-7.25 (m, 2H), 6.01 (m , 1H), 5.42 (s, 1H), 4.89 (s, 2H), 4.41 (q, J = 7.0 Hz, 1H), 4.18 (t, J = 7.0 Hz , 1H), 3.10-2.90 (m, 6H), 2.75 (t, J = 7.5Hz, 2H), 2.27 (t, J = 7.5Hz, 2H), 2 , 07 (q, J = 7.0 Hz, 1H), 1.72-1.51 (m, 10H), 1.46-1.35 (m, 8H), 1.32-1.26 (m , 3H), 1.00-0.92 (m, 9H). LCMS [M + H] = 898. ATAC25: 4 - ((S) -2 - ((S) -2- (6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl ) hexanamido) -3-methylbutanamido) -5-ureidopentanamido) benzyl - ((4-amino-1- (2-hydroxy-2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl ) (ethyl) carbamate TFA salt

¹ H NMR (DMSO-d ⁶ ) δ 13.4 (bs, 1H), 9.99-9.89 (br m, 1H), 9.09-8.40 (m, 3H), 8.07 ( d, J = 7.5 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.59 (bs, 1H), 7.51 (t, J = 8.5 Hz, 1H), 7.46-7.14 (m, 2H), 7.00 (s, 1H), 5.99 (br s, 1H) , 5.05 (brs, 1H), 4.95 (brs, 1H), 4.37 (q, J = 7.0 Hz, 1H), 4.18 (t, J = 7.0 Hz, 1H), 3.37 (t, J = 7.0Hz, 2H), 3.03-2.93 (m, 2H), 2.22-2.07 (m, 2H), 1.99-1 , 92 (m, 1H), 1.75-1.05 (br m, 20H), 0.85 (d, J = 8.5 Hz, 3H), 0.81 (d, J = 8.5 Hz , 3H). MS (ESI +) m / z 912.5 (M + 1).

EXAMPLE 11

Synthesis of ATAC32

Schritt A: Herstellung von ATAC32

This example demonstrates the synthesis of 1- {6 - [({7-amino-3- (2-hydroxy-2-methylpropyl) -3,5,8-triazatricyclo [7.4.0.02,6] trideca-1 (9), 2 (6), 4,7,10,12-hexaen-4-yl} methyl) -N-ethylamino] -6-oxohexyl} -1H-pyrrole-2,5-dione (ATAC32).

Step A: Preparation of ATAC32

To an ice-cold solution of 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -hexanoic acid (0.034 g, 0.16 mmol) in DCM (0.800 ml) was added 1-chloro N, N, 2-trimethylprop-1-en-1-amine (0.021 mL, 0.160 mmol) was added dropwise. This was stirred at 0 ° C for 1 h and then added to an ice-cold mixture of Compound 1 (50 mg, 0.160 mmol) and triethylamine (66.7 μL, 0.479 mmol) in DCM (800 μL). Total molar 0.1M. The mixture was stirred at room temperature overnight and then chromatographed without working up (DCM to 20% MeOH / DCM). Fractions containing the product were combined and evaporated, then dissolved in 1 ml of acetonitrile and treated with 0.1 ml of trifluoroacetic acid. The resulting material was evaporated to an oil, then redissolved in CH ₃ CN, and the sample lyophilized to give ATAC 32 (65 mg) as a white solid. ¹ H NMR (400 MHz, (DMSO-d ⁶ ) δ 13.3 (s, 1H), 8.54-8.50 (m, 3H), 7.81 (d, J = 8.5 Hz, 1H ), 7.76 (d, J = 7.5Hz, 1H), 7.51 (d, J = 7.5Hz, 1H), 6.99 (s, 1H), 6.95 (s, 1H ), 3.51 (q, J = 7.0 Hz, 2H), 3.43-3.31 (m, 3H), 2.36-2.30 (m, 2H), 1.54-1, 41 (m, 4H), 1.25-1, q00 (m, 10H) ¹⁹ F NMR (DMSO-d ⁶ ) δ -74.0 LCMS [M + H] ⁺ = 507.1.

EXAMPLE 12

Synthesis of ATAC33

Schritt A: Herstellung von ATAC33

This example shows the synthesis of 1 - {[4- ({6 - [({7-amino-3- (2-hydroxy-2-methylpropyl) -3,5,8-triazatricyclo [7.4.0.02,6] trideca-1 (9), 2 (6), 4,7,10,12-hexaene-4-yl} methyl) -N-ethylamino] -6-oxohexylamino} carbonyl) cyclohexyl] methyl} -1H-pyrrole-2,5- dion (ATAC33).

Step A: Preparation of ATAC33

To a stirred solution of 1- (4-amino-2 - ((ethylamino) methyl) -1H-imidazo [4,5- c] quinolin-1-yl) -2-methylpropan-2-ol (100 mg, 0.319 mmol) in DCM (10 ml) under nitrogen, a solution of 2,5-dioxopyrrolidin-1-yl-6- (4 - ((2,5-dioxo-2,5-dihydro-1H-pyrrole) 1-yl) methyl) cyclohexane-1-carboxamido) hexanoate (143 mg, 0.319 mmol) in DCM (5 mL) over 3.5 h. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated and the residue was purified by reverse phase column chromatography. Pure fractions identified by HPLC analysis were pooled and concentrated. The residue was lyophilized from CH ₃ CN to give a white solid (52.8 mg) as the TFA salt of ATAC33 as a mixture of cis and trans isomers. ¹ H NMR (400 MHz, (CD ₃ OD) δ 8.54 and 8.48 (d, J = 8.3 Hz, 1H), 7.81-7.71 (m, 2H), 7.62- 7.55 (m, 1H), 6.80 (s, 2H), 3.66 (q, J = 7.0 Hz, 2H), 3.13 and 3.08 (t, J = 7.0 Hz , 2H), 2.45 and 2.38 (t, J = 7.5 Hz, 2H), 2.1-2.0 (m, 1H), 1.8-1.47 (m, 10H), 1.46-1.15 (m, 16H), 1.54-1.41 (m, 4H), 1.25-1.00 (m, 10H), LCMS [M + H] ⁺ = 646.3 ,

EXAMPLE 13

Synthesis of ATAC34

Schritt A: Herstellung von ATAC34

This example demonstrates the synthesis of 1 - [(4 - {[({7-amino-3- (2-hydroxy-2-methylpropyl) -3,5,8-triazacyclo [7.4.0.02,6] trideca-1 (9) , 2 (6), 4,7,10,12-hexa-4-yl} methyl) -N-ethylamino] -carbonyl} cyclohexyl) methyl] -1H-pyrrole-2,5-dione (ATAC34).

Step A: Preparation of ATAC34

To an ice-cold solution of (1r, 4r) -4 - ((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) methyl) cyclohexane-1-carboxylic acid (82 mg, 0.346 mmol) in DCM (1728 μl) was added dropwise to 1-chloro-N, N, 2-trimethylprop-1-en-1-amine (50.3 μl, 0.380 mmol). This was stirred at 0 ° C 1H and then added to an ice-cold mixture of 1- (4-amino-2 - ((ethylamino) methyl) -1H-imidazo [4,5-c] quinolin-1-yl) -2- methylpropan-2-ol (100mg, 0.319mmol) and triethylamine (133μl, 0.957mmol) in 1.6mL of DCM. The mixture became a yellow solution while being stirred overnight on room temples. The reaction was concentrated to dryness, redissolved in MeOH / CH ₂ Cl ₂ , silica gel was added, then the solvents were evaporated. Chromatography (12g gold-silica, DCM to 20% MeOH / DCM, dry load) gave a solid which was dissolved in CH ₃ CN, frozen and lyophilized to give 170 mg of N - ((4 - ((1- (dimethylamino) -2-methylprop-1-en-1-yl) amino) -1- (2 - ((1- (dimethylamino) -2-methylprop-1-en-1-yl) oxy) -2-methylpropyl) -1H-imidazo [4,5-c] quinolin-2-yl) methyl) -4 - ((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) methyl) -N-ethylcyclohexane 1-carboxamide, which was then dissolved in 50% aqueous MeCN containing 0.1% TFA and heated in a microwave reactor at 150 ° C for 60 minutes. The reaction mixture was cooled and the solvents were evaporated and chromatographed to give ATAC34 (72 mg) as a white solid. ¹ H NMR (400 MHz, (DMSO-d ⁶ ) δ 13.3 (s, 1H), 8.70-8.50 (m, 3H), 7.83-7.79 (m, 1H), 7 , 71-7.65 (m, 1H), 7.55-7.48 (m, 1H), 7.00 (s, 1H), 6.98 (s, 1H), 5.13 (bs, 1H ), 4.83 (bs, 1H), 3.65 (q, J = 7.0 Hz, 2H), 3.38 (m, 1H), 3.25 and 3.18 (d, J = 6, 5Hz, 2H), 1.69-1.52 (m, 5H), 1.45-0.88 (m, 13H) ¹⁹ F NMR (DMSO-d ⁶ ) δ -73.7, LCMS [M + H] ⁺ = 533.1.

EXAMPLE 14

Fc receptor binding to conjugates of anti-CD40 antibody and immunostimulatory compound

An anti-CD40 antibody consists of two heavy chains of SBT-040-G1WT and two light chains of an SBT-040 antibody called SBT-040-WT antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040 G1VLPLL and two light chains of an SBT-040 antibody called SBT-040 VLPLL antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1DE and two light chains of an SBT-040 antibody called SBT-040-DE antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1AAA and two light chains of an SBT-040 antibody called SBT-040-AAA antibody.

Each antibody is purified and then each of ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7 or ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20, ATAC21, ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32, ATAC33, ATAC34 or ATAC43 as described in EXAMPLE 2. Each of these conjugates is characterized by the ability of their Fc domains to bind to soluble glycosylated FcγR ectodomains from human FcγRs and their affinity therefor. This is demonstrated by performing surface plasmon resonance experiments. In these experiments, biotinylated soluble glycosylated FcγR ectodomains from all human FcγRs are immobilized on a streptavidin-coated surface. The ability of each conjugate to bind to soluble glycosylated FcγR ectodomains from all human FcγRs is then measured by surface plasmon resonance using a Biacore instrument. The data from these experiments show that the Fc domain of each of the SBT-040-WT-ATAC1-SBT-040-WT-ATAC34 or SBT-040-ATAC43 conjugates, the Fc domain of each of the SBT-040 -VLPLL-ATAC1-SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL-ATAC43 conjugates, the Fc domain of each of the SBT-040-DE-ATAC1-SBT-040-DE-ATAC34 or SBT-040- DE-ATAC43 conjugates that bind the Fc domain of each of the SBT-040-AAA-ATAC1 - the SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43 conjugates to soluble glycated FcγR ectodomains from human FcγRs becomes. Therefore, the surface plasmon resonance experiments show that the ability of the Fc domain of the antibody component of the conjugate to bind human FcγRs is not affected by the conjugation of the components of the conjugate. The affinity of each conjugate for each human FcγR is also demonstrated by the surface plasmon resonance experiments. These affinity measurements are compared to the affinity measurements for each antibody alone (as shown by EXAMPLE 1). The similarity in the affinity of each antibody alone for soluble glycosylated FcγR ectodomains from all human FcγRs with the affinity of each corresponding conjugate for soluble glycated FcγR ectodomains from all human FcγRs is shown by this comparison.

EXAMPLE 15

Affinity of anti-CD40 antibodies for CD40

An anti-CD40 antibody consists of two heavy chains of SBT-040-G1WT and two light chains of an SBT-040 antibody called SBT-040-WT antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040 G1VLPLL and two light chains of an SBT-040 antibody called SBT-040 VLPLL antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1DE and two light chains of an SBT-040 antibody called SBT-040-DE antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1AAA and two light chains of an SBT-040 antibody called SBT-040-AAA antibody.

SBT-040 WT antibodies, SBT-040 VLPLL antibodies, SBT-040-DE antibodies and SBT-040-AAA antibodies are each prepared according to standard procedures for the production of antibodies. Each antibody is purified and then characterized for the ability to bind to CD40. This characterization is demonstrated by flow cytometry experiments. For these experiments, the human Burkitt lymphoma tumor cell lines Raji and Daudi, previously shown to be CD40-positive, and the human chronic myelogenous leukemia tumor cell line K562, previously shown to be CD40- is negative, evaluated first for evaluation of their relative expression levels of CD40 by flow cytometry. This is assayed by incubating each cell line with a commercially available CD40 antibody conjugated to a fluorochrome and then running samples of incubation on a flow cytometer. The relative fluorescence intensity profiles for each cell line are shown by this data, indicating the degree of CD40 expression of each cell line. The relative fluorescence intensity profiles of the human Burkitt lymphoma tumor cell lines Raji and Daudi show that CD40 is expressed in each of these cell lines, while the relative fluorescence intensity profile of the human chronic myelogenous leukemia tumor cell line K562 shows that CD40 is not expressed in this cell line. Then, each cell line is incubated separately with purified SBT-040 WT antibody, SBT-040 VLPLL antibody, SBT-040G1DE antibody, SBT-040 AAA antibody or without antibody as a control. Each incubation is further conjugated to a secondary anti-human IgG1 antibody conjugated to FITC, which is then measured by flow cytometry for the FITC fluorescence intensity profile of each sample. The ability of each antibody to detect CD40 expression on the cell lines is indicated by its FITC fluorescence intensity profile. Specifically, the similarity between the SBT-040 WT antibody fluorescence intensity profile and each antibody with an Fc-enhanced IgG1 isotype after incubation with each of the cell lines is shown by this data. Each Fc-amplified IgG1 isotype is not altered by the ability of the antibody to bind to CD40-positive cells, is also shown by this data.

EXAMPLE 16

Affinity of anti-CD40 antibodies for CD40

An anti-CD40 antibody consists of two heavy chains of SBT-040-G1WT and two light chains of an SBT-040 antibody called SBT-040-WT antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040 G1VLPLL and two light chains of an SBT-040 antibody called SBT-040 VLPLL antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1DE and two light chains of an SBT-040 antibody called SBT-040-DE antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1AAA and two light chains of an SBT-040 antibody called SBT-040-AAA antibody.

SBT-040 WT antibodies, SBT-040 VLPLL antibodies, SBT-040-DE antibodies and SBT-040-AAA antibodies are each prepared by standard methods for the production of antibodies. Each antibody is purified and the affinity of each antibody for CD40 is measured. These affinities are measured by experiments using surface plasmon resonance. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. The ability of each antibody to bind to recombinant CD40 is then measured by surface plasmon resonance using a Biacore instrument. The data from these experiments show that SBT-040 WT antibodies, SBT-040 VLPLL antibodies, SBT-040-DE antibodies and SBT-040-AAA antibodies are each bound to recombinant CD40. Therefore, the ability of each antibody to bind to CD40 becomes binding, not disturbed by the enhanced Fc-amplified IgG1 isotypes, is demonstrated by the surface plasmon resonance data.

Furthermore, surface plasmon resonance is used to show that CD40L binding to CD40 is not blocked by these antibodies. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. Surface plasmon resonance using a Biacore instrument is then used to measure the binding affinity of CD40L in the presence of any antibody or without any antibody as a control. The binding affinity of CD40L with recombinant CD40 in the presence of each antibody is the same as the binding affinity of CD40L with recombinant CD40 in the absence of any antibody. Therefore, CD40 and CD40L binding is not altered by the presence of SBT-040 WT antibody, SBT-040 G1VLPLL antibody, SBT-040 DE antibody or SBT-040 AAA antibody.

EXAMPLE 17

Fc receptor binding to conjugates of anti-CD40 antibody and immunostimulatory compound

An anti-CD40 antibody consists of two heavy chains of SBT-040-G1WT and two light chains of an SBT-040 antibody called SBT-040-WT antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040 G1VLPLL and two light chains of an SBT-040 antibody called SBT-040 VLPLL antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1DE and two light chains of an SBT-040 antibody called SBT-040-DE antibody. An anti-CD40 antibody consists of two heavy chains of SBT-040-G1AAA and two light chains of an SBT-040 antibody called SBT-040-AAA antibody.

SBT-040 WT antibodies, SBT-040 VLPLL antibodies, SBT-040-DE antibodies and SBT-040-AAA antibodies are each prepared by standard methods for the production of antibodies. Each antibody is purified and then each of ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7 or ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32, ATAC33, ATAC34 or ATAC43 as described in EXAMPLE 2. The affinity of each conjugate for CD40 is then measured by experiments using surface plasmon resonance. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. The ability of each conjugate to bind to recombinant CD40 is then measured by surface plasmon resonance using a Biacore instrument. The data from these experiments show that each of the SBT-040-WT-ATAC1-SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugates, each of the SBT-040-VLPLL-ATACI-SBT-040 -VLPLL-ATAC34 or SBT-040-VLPLL-ATAC43 conjugates, each of the SBT-040-DE-ATAC1-SBT-040-DE- ATAC34 or SBT-040-DE-ATAC43 conjugates or any of the SBT-040-AAA-ATAC1 - which binds SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43 conjugates to recombinant CD40. Therefore, the surface plasmon resonance experiments show that the ability of each component antibody to bind to CD40 is not due to the enhanced Fc-enhanced IgG1 isotypes or antibody conjugation to ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7 or ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32, ATAC33, ATAC34 or ATAC43.

Furthermore, surface plasmon resonance is used to show that CD40L binding to CD40 is not blocked in the presence of any conjugate. In these experiments, biotinylated recombinant CD40 is immobilized on a streptavidin-coated surface. Surface plasmon resonance using a Biacore instrument is then used to measure the binding affinity of CD40L in the presence of each conjugate or without conjugate as a control. It is demonstrated by these experiments that the binding affinity of CD40L for recombinant CD40 in the presence of each conjugate is the same as the binding affinity of CD40L for recombinant CD40 in the absence of any conjugate. Therefore, CD40 and CD40L binding is mediated by the presence of any of the SBT-040-WT-ATAC1-SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugates, any of the SBT-040 VLPLLs. ATAC1-SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL-ATAC43 conjugates, any of the SBT-040-DE-ATAC1-SBT-040-DE-ATAC34 or SBT-040-DE-ATAC43 conjugates or any of SBT-040-AAA-ATAC1 - the SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43 conjugates not affected.

EXAMPLE 18

Cytokine production is enhanced by conjugates of anti-CD40 antibody and immunostimulatory compound

This example demonstrates that cytokine production by dendritic cells is enhanced upon administration of conjugates of antibody and immunostimulatory compound in culture. In this experiment, dendritic cells (DCs) are obtained from peripheral blood mononuclear cells (PBMCs). DCs are obtained by placing human PBMCs in a culture dish. The resulting adherent cells are washed with RPMI containing 10% fetal calf serum and then incubated for 7 days in a complete medium containing 10 ng / ml IL-4 and 100 ng / ml GM-CSF. The non-adherent cells are isolated and washed. These isolated cells are passed through a flow cytometer to ensure CD11c expression, confirming the identity of the DCs as DCs through CD11c expression. The DCs are then incubated with either the antibodies described in Example 1 or the conjugates described in Example 3. Specifically, the DCs are amplified with any of SBT-040 WT antibody, SBT-040 VLPLL antibody, SBT-040-DE antibody, SBT-040-AAA antibody, the SBT-040-WT-ATAC1 conjugate. SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugate, the SBT-040-VLPLL-ATAC1 conjugate-SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL-ATAC43 conjugate, the SBT 040-DE-ATAC1 conjugate-SBT-040-DE-ATAC34 or SBT-040-DE-ATAC43 conjugate, the SBT-040-AAA-ATAC1 - the SBT-040-AAA-ATAC34 or SBT-040-AAA- ATAC43 conjugate or a non-binding isotype control antibody. Each culture is then incubated for 24 hours and the supernatant from each culture is analyzed using a cytokine bead assembly assay. Cytokine expression levels of IFNγ, IL-8, IL-12 and IL-2 are measured by the cytokine bead assembly assay. The supernatant of the culture containing the non-binding isotype control shows that the level of cytokine expression compared to the supernatant of the cultures, the SBT-040-WT, SBT-040-VLPLL, SBT-040-DE or SBT -040-AAA is reduced. In addition, the level of cytokine expression in the supernatant from cultures containing SBT-040-WT, SBT-040-VLPLL, SBT-040-DE, or SBT-040-AAA, compared to the supernatant from cultures containing any from the SBT-040-WT-ATAC1 conjugate-SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugate, any of the SBT-040-VLPLL-ATACI conjugate-SBT-040-VLPLL-ATAC34 or SBT Any of the SBT-040-DE-ATAC1 conjugate SBT-040-DE-ATAC34 or SBT-040-DE-ATAC43 conjugate or any of the SBT-040-AAA-ATAC1- Conjugate-SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43 conjugate.

EXAMPLE 19

Cytokine production by multiple donor dendritic cells was enhanced by conjugates of anti-CD40 antibody and immunostimulatory compound

Antibody and immunostimulatory compound conjugates enhanced immunostimulatory cytokines produced by human dendritic cells in a concentration-dependent manner when added to and incubated with the cells. Human donor dendritic cells (DCs) were derived from CD14 ⁺ monocytes isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using a commercially available kit. The monocytes were cultured in RPMI containing 10% fetal calf serum for seven days in complete medium supplemented with 25 ng / ml IL-4 and 10 ng / ml GM-CSF. The media was replaced on the third day with fresh media plus cytokines. On day six, conjugates of anti-CD40 antibody and immunostimulatory compound SBT-040-G1-ATAC23 and SBT-040-G1-ATAC17 and control antibody were added to individual vessels containing the dendritic cells. After 24 hours of further incubation, the supernatants were collected and the cytokines IL-6, TNFα, IL-12p70 and IL-10 produced by the dendritic cells were quantified by multiplex ELISA using commercial reagents and plate readers of Meso Scale Discovery via electrochemiluminescent signal. The results are shown for the immunostimulatory cytokines IL-12p70 and TNFα for donor-derived dendritic cells. 31A Figure 12 shows the concentration of IL-12p70 produced by DCs from donor 358 after incubation with SBT-040-G1-ATAC23 or SBT-040-G1-ATAC17 compared to SBT-050-WT. 31B Figure 12 shows the concentration of IL-12p70 produced by DCs from donor 363 after incubation with SBT-040-G1-ATAC23 or SBT-040-G1-ATAC17 compared to SBT-050-WT. 31C shows the concentration of TNFα produced by DCs from donor 358 after incubation with SBT-040-G1-ATAC23 or SBT-040-G1-ATAC17 compared to a Anti-HER2 antibody was generated. 31D Figure 10 shows the concentration of TNFα produced by DCs from donor 363 after incubation with SBT-040-G1-ATAC23 or SBT-040-G1-ATAC17 compared to an anti-HER2 antibody.

EXAMPLE 20

Secretion of immunostimulatory cytokines is enhanced by conjugates of anti-CD40 antibody and immunostimulatory compound with different linkers and FcγR binding

Antibody and immunostimulatory compound conjugates enhanced the production of immunostimulatory cytokines by human dendritic cells in a concentration-dependent manner when added to and incubated with the cells. Human dendritic cells (DCs) from two donors were derived from CD4 ⁺ monocytes isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using a commercially available kit. The monocytes were cultured in RPMI containing 10% fetal calf serum for seven days in complete medium supplemented with 25 ng / ml IL-4 and 10 ng / ml GM-CSF. The media was replaced on the third day with fresh media plus cytokines. On day six, conjugates of anti-CD40 antibody and immunostimulatory compound SBT-50 G1 and commercially available soluble CD40L were added to individual vessels containing the dendritic cells. Specifically, the DCs were amplified with any of SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-040-AAA-ATAC29, SBT-040 -VLPLL-ATAC29, SBT-040-WT-ATAC1, SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30, SBT-040-AAA -ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC30, and SBT-040-AAA-ATAC30. After 24 hours, further incubation supernatants were collected and the cytokines IL-6, TNFα, IL-12p70 and IL-10 produced by the dendritic cells were quantitated by multiplex ELISA using commercial reagents and plate readers of Meso Scale Discovery via electrochemiluminescent signal. 32A shows the concentration of IL-12p70 produced by DCs after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-040- AAA-ATAC29, SBT-040-VLPLL-ATAC29, SBT-040-WT-ATAC1, SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT ATAC30, SBT-040-AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC30, and SBT-040-AAA-ATAC30 compared to SBT-050-G2 antibodies or CD40 ligands. 32B Figure 4 shows the concentration of IL-6 produced by DCs from donor 2 after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT 040 AAA ATAC29, SBT040 VLPLL ATAC29, SBT040 WT ATAC1, SBT040 G2 ATAC1, SBT040 WT ATAC12, SBT040 G2 ATAC12, SBT040 -WT-ATAC30, SBT-040-AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC30, and SBT-040-AAA ATAC30 was generated compared to SBT-050-G2 or CD40 ligands. The results are shown for the immunostimulatory cytokines IL-12p70 and IL-6. The treatment concentrations for each molecule, shown from right to left on the x-axis, were 0.08 μg / ml, 0.310 μg / ml, 1.25 μg / ml, and 5.00 μg / ml.

EXAMPLE 21

Conjugates of anti-CD40 antibody and immunostimulatory compound enhanced cell surface expression of immunoactivating proteins

Human dendritic cells showed increased expression of CD83, CD86 and MHC class II cell surface proteins after treatment with conjugates of anti-CD40 antibody and immunostimulatory compound. The increased expression of these surface proteins was dose-dependent.

Human dendritic cells (DCs) from two donors were derived from CD14 ⁺ monocytes isolated from peripheral blood mononuclear cells (PBMCs) by negative selection using a commercially available kit. The monocytes were cultured in RPMI containing 10% fetal calf serum for seven days in complete medium supplemented with 25 ng / ml IL-4 and 10 ng / ml GM-CSF. The media was replaced on the third day with fresh media plus cytokines. On day six, SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-AAA-ATAC30 or a control SBT-050-WT were added to separate aliquots of dendritic cells , After another 24 hr incubation, the cells were collected and washed by centrifugation, then stored on ice for 30 minutes using manufacturer recommended concentrations of commercially available monoclonal anti-CD83, anti-CD86 and anti-MHC class monoclonal antibodies. II antibodies conjugated to laser-sensitive fluorene. A separate aliquot for each treatment was made with IgG-matched isotype control antibody conjugate for the Anti-CD86 antibody, the anti-CD83 antibody and the anti-MHC class II antibody stained. After washing to remove unbound antibody-fluorine molecules, the stained cells were subjected to FACS analysis using a Celesta flow cytometer (BD Biosciences) gated on living cells. The output was analyzed by the FlowJo v10.2 software (FlowJo LLC) and adapted with the Prism 7.01 software (GraphPad Software, Inc.). 33A shows a dose-dependent increase in CD86 expression on dendritic cells after treatment with SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-AAA-ATAC30 compared to treatment a control SBT-050-WT or staining with an isotype control. 33B shows a dose-dependent increase in CD83 expression on dendritic cells after treatment SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-AAA-ATAC30 in comparison to treatment Control SBT-050-WT or staining with an isotype control. 33C shows a dose-dependent increase in MHC class II expression on dendritic cells after treatment with SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-AAA-ATAC30 compared to the treatment of a control SBT-050-WT or staining with an isotype control. The graph shows plots of treatment protein concentration on the x-axis versus mean fluorescence intensity for the cell surface protein on the y-axis.

EXAMPLE 22

Treatment of cancer by administering a conjugate

This example describes the treatment of cancer with a conjugate. A human patient is diagnosed with a cancer. A conjugate, as in the scheme 8th is administered to the patient with a pharmaceutically acceptable carrier. 8th is a conjugate comprising an antibody construct and an immunostimulatory compound. The antibody construct is an antibody containing two heavy chains, as shown in gray, and two light chains, as shown in light gray. The antibody comprises two antigen binding sites ( 810 and 815 ), and part of the heavy chains contains Fc domains ( 805 and 820 ). The immunostimulatory compounds ( 830 and 840 ) are linked to the antibody by linkers ( 860 and 870 ) conjugated.

As another example, a human patient is diagnosed with a cancer. A conjugate, as in the scheme 9 is administered to the patient with a pharmaceutically acceptable carrier. 9 is a conjugate comprising one antibody construct, two targeting binding domains, and two immunostimulatory compounds. The antibody construct is an antibody containing two heavy chains, as shown in gray, and two light chains, as shown in light gray. The antibody comprises two antigen binding sites ( 910 and 915 ), and part of the heavy chains contains Fc domains ( 905 and 920 ). The immunostimulatory compounds ( 930 and 940 ) are linked to the antibody by linkers ( 960 and 970 ) conjugated. The targeting binding domains are conjugated to the antibody ( 980 and 985 ).

As an additional example, a human patient is diagnosed with a cancer. A conjugate, as in the scheme 10 is administered to the patient with a pharmaceutically acceptable carrier. 10 is a conjugate comprising an antibody construct and two immunostimulatory compounds. The antibody construct contains the Fc region of an antibody with heavy chains shown in gray and two scaffolds shown in light gray. The antibody construct comprises two antigen binding sites ( 1010 and 1015 ) in the scaffolds, and part of the heavy chains contains Fc domains ( 1005 and 1020 ). The immunostimulatory compounds ( 1030 and 1040 ) are linked to the antibody construct by linkers ( 1060 and 1070 ) conjugated.

As another example, a human patient is diagnosed with a cancer. A conjugate, as in the scheme 11 is administered to the patient with a pharmaceutically acceptable carrier. 11 is a conjugate that contains one antibody construct, two targeting domains and two immunostimulatory Compounds includes. The antibody construct contains the Fc region of an antibody with heavy chains shown in gray and two scaffolds shown in light gray. The antibody construct comprises two antigen binding sites ( 1110 and 1115 ) in the scaffolds, and part of the heavy chains contains Fc domains ( 1105 and 1120 ). The immunostimulatory compounds ( 1130 and 1140 ) are linked to the antibody construct by linkers ( 1,160 and 1170 ) conjugated. The targeting binding domains are conjugated to the antibody construct ( 1180 and 1185 ).

As another example, a human patient is diagnosed with a cancer. A conjugate, as in the scheme 12 is administered to the patient with a pharmaceutically acceptable carrier. 12 is a conjugate comprising an antibody construct and two immunostimulatory compounds. The antibody construct contains the F (ab ') ₂ region of an antibody with heavy chains shown in gray and light chains shown in light gray and two scaffolds shown in dark gray. The antibody construct comprises two antigen binding sites ( 1210 and 1215 ), and part of two scaffolds contain Fc domains ( 1220 and 1245 ). The immunostimulatory compounds ( 1230 and 1240 ) are linked to the antibody construct by linkers ( 1260 and 1270 ) conjugated.

As another example, a human patient is diagnosed with a cancer. A conjugate, as in the scheme 13 is administered to the patient with a pharmaceutically acceptable carrier. 13 is a conjugate comprising one antibody construct, two targeting binding domains, and two immunostimulatory compounds. The antibody construct contains the F (ab ') ₂ region of an antibody with heavy chains shown in gray and light chains shown in light gray and two scaffolds shown in dark gray. The antibody construct comprises two antigen binding sites ( 1310 and 1315 ), and part of two scaffolds contain Fc domains ( 1320 and 1345 ). The immunostimulatory compounds ( 1330 and 1340 ) are linked to the antibody construct by linkers ( 1360 and 1370 ) conjugated. The targeting binding domains are conjugated to the antibody construct ( 1380 and 1385 ).

As another example, a human patient is diagnosed with a cancer. A conjugate, as in the scheme 14 is administered to the patient with a pharmaceutically acceptable carrier. 14 is a conjugate comprising an antibody construct and two immunostimulatory compounds. The antibody construct contains two scaffolds shown in light gray and two scaffolds shown in dark gray. The antibody construct comprises two antigen binding sites ( 1410 and 1415 ) and part of the two dark gray scaffolds contain Fc domains ( 1420 and 1445 ). The immunostimulatory compounds ( 1430 and 1440 ) are linked to the antibody construct by linkers ( 1460 and 1470 ) conjugated.

As another example, a human patient is diagnosed with a cancer. A conjugate, as in the scheme 15 is administered to the patient with a pharmaceutically acceptable carrier. 15 is a conjugate comprising one antibody construct, two targeting binding domains, and two immunostimulatory compounds. The antibody construct contains two scaffolds shown in light gray and two scaffolds shown in dark gray. The antibody construct comprises two antigen binding sites ( 1510 and 1515 ) and part of the two dark gray scaffolds contain Fc domains ( 1520 and 1545 ). The immunostimulatory compounds ( 1530 and 1540 ) are linked to the antibody construct by linkers ( 1560 and 1570 ) conjugated. The targeting binding domains are conjugated to the antibody construct ( 1580 and 1585 ).

EXAMPLE 23

Determination of K _d values

Kd _is measured by a radioactively labeled antigen binding assay (RIA) performed on the Fab version of an antibody of interest and its antigen, as described by the following test.

Solution binding affinity of Fabs for antigen is measured by equilibrating the Fab with a minimum concentration of ( ¹²⁵ 1) -labeled antigen in the presence of a titer series of unlabelled antigen, then capturing bound antigen with an anti-Fab antibody coated plate (see e.g. B. Chen et al., J. Mol. Biol. 293: 865-881 (1999) ). To establish conditions for the assay, multi-well plates are coated overnight with 5 μg / ml of an anti-Fab capture antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) followed by 2% (w / o). v) Block bovine serum albumin in PBS for two to five hours at room temperature (about 23 ° C). In a non-adsorbent plate (Nunc # 269620), 100 pM or 26 pM [ ¹²⁵ I] antigen is mixed with serial dilutions of a Fab of interest (e.g., in accordance with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57: 4593-4599 (1997) ). The Fab of interest is then incubated overnight; however, the incubation may be continued for a longer period of time (eg, about 65 hours) to ensure equilibrium is reached. Thereafter, the mixtures are transferred to the trapping plate for incubation at room temperature (eg, over one hour). The solution is then removed and the plate (TWEEN-20 ^®) in PBS eight times with 0.1% polysorbate 20th When the plates are dried, 150 μl / well scintillant is added and the plates counted on a TOPCOUNT ^™ gamma counter (Packard) for ten minutes. Concentrations of each Fab that yield less than or equal to 20% of the maximum binding are selected for use in competitive binding assays.

EXAMPLE 24

Determination of K _d values

K _d is determined using surface plasmon resonance assays using a BIACORE ^® -2000 or a BIACORE ^® -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ° C with immobilized antigen CM5 chips at ~ 10 response units (RU) measured. Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.) are activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions , The antigen is diluted with 10mM sodium acetate, pH 4.8, to 5μg / ml (~ 0.2μM), prior to injection at a flow rate of 5μl / min, to about 10 response units (RU) of the coupled protein receive. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) in PBS are injected with 0.05% polysorbate 20 (TWEEN-20 ^™ ) surfactant (PEST) at 25 ° C at a flow rate of approximately 25 μl / min , Association rates (k _on) and dissociation rates (k _off) are obtained using a simple one-to-one Langmuir binding model (BIACORE ^® Evaluation Software version 3.2) is calculated by simultaneous adjustment of the association and dissociation sensorgrams. The equilibrium dissociation constant (K _d ) is calculated as the ratio k _off / k _on . See, for example, B. Biol et al., J. Mol. Biol. 293: 865-881 (1999). , When the on-rate exceeds 106 M-1 s-1 in the surface plasmon resonance test described above, the on-rate can be determined using a fluorescence quenching technique that increases or decreases the fluorescence emission intensity (excitation = 295 nm emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) at 25 ° C in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a Stop Float Spectrophotometer (Aviv Instruments) or an SLM-AMINCO ^™ 8000 Series Spectrophotometer (ThermoSpectronic) with a stirred cuvette.

EXAMPLE 25

Lysine-based bioconjugation

The antibody construct is changed to a suitable buffer, for example, phosphate, borate, PBS, Tris-acetate at a concentration of about 2 mg / ml to about 10 mg / ml. An appropriate number of equivalents of the construct of immunostimulatory compound and linker (ATAC) were added as a solution with stirring. Depending on the physical properties of the immunostimulatory compound and linker construct, a co-solvent was introduced prior to the addition of the immunostimulatory compound-linker construct to facilitate solubility. The reaction was stirred at room temperature for 2 hours to about 12 hours, depending on the reactivity observed. The progress of the reaction was monitored by LC-MS. Once the reaction was considered complete, the remaining immunostimulatory compound-linker constructs were removed by applicable methods and the conjugate of antibody construct and immunostimulatory compound was changed to the desired formulation buffer. Lysine-linked conjugates were synthesized with 10 mg of antibody (mAb) and 10 equivalents of ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, ATAC8, ATAC9 or ATAC10 using the conditions described in Scheme 34 below (ADC = Antibody Conjugate Conjugate and immunostimulatory compound). Monomer content and ratios of immunostimulatory compound to antibody construct (molar ratios) were determined according to the methods described in Examples 27-31. Scheme 34:

EXAMPLE 26

Cysteine-based bioconjugation

The antibody construct was changed to an appropriate buffer, for example, phosphate, borate, PBS, Tris-acetate at a concentration of about 2 mg / ml to about 10 mg / ml with an appropriate number of equivalents of a reducing agent, e.g. B. dithiothreitol or tris (2-carboxyethyl) phosphine. The resulting solution was stirred for an appropriate amount of time and temperature to effect the desired reduction. The construct of immunostimulatory compound and linker was added as a solution with stirring. Depending on the physical properties of the immunostimulatory compound and linker construct, a co-solvent was introduced prior to the addition of the immunostimulatory compound-linker construct to facilitate solubility. The reaction was stirred at room temperature for about 1 hour to about 12 hours, depending on the reactivity observed. The progress of the reaction was monitored by liquid chromatography mass spectrometry (LC-MS). Once the reaction was considered complete, the remaining free construct of immunostimulatory compound and linker was removed by applicable methods and the conjugate of antibody construct and immunostimulatory compound was changed to the desired formulation buffer. Such cysteine-based conjugates were synthesized starting from 10 mg of antibody (mAb) and 7 equivalents of ATAC11-ATAC45 using the conditions described in Scheme 35 below (ADC = antibody construct-immunostimulatory compound conjugate). The monomer content and the drug-antibody ratios can be determined by the methods described in Examples 27-31. Scheme 35:

EXAMPLE 27

Determination of the molar ratio

This example illustrates a method by which the molar ratio is determined. One microgram of the antibody construct-immunostimulatory compound conjugate is injected into an LC / MS, such as an Agilent 6550 iFunnel Q-TOF equipped with an Agilent Dual Jet Stream ESI source coupled to the Agilent 1290 Infinity UHPLC system , Raw data is obtained and deconvolved with software such as Agilent MassHunter Qualitative Analysis Software with BioConfirm using the deconvolution algorithm Maximum Entropy. The average mass of intact conjugate of antibody construct and immunostimulatory compound is calculated by the software, which can use the peak peak height at 25% for the calculation. These data are then imported into another program to calculate the molar ratio of the antibody construct-immunostimulatory compound conjugate, such as the Agilent molar ratio calculator.

EXAMPLE 28

Determination of molar ratio for SBT-040-G1WT conjugated to a Cys-directed compound

28 Figure 7 shows the HPLC analysis of SBT-040-G1WT conjugated to a Cys-directed drug-linker tool compound. First, 10 μl of a 5 mg / ml solution of the antibody-drug conjugate was placed in a HPLC system set-up with a TOSOH TSKgel Butyl-NPR ™ Hydrophobic Interaction Chromatography (HIC) column (2.5 μM particle size, 4.6 mm × 35 mm). Then, a procedure was carried out over 18 minutes in which the mobile phase gradient ran from 100% of mobile phase A to 100% of mobile phase B within 12 minutes, followed by a six minute re-equilibration at 100% mobile phase A. The flow rate was 0.8 ml / min and the detector was set to 280 nM. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B was 25% isopropanol in 25 mM sodium phosphate (pH 7). After the run, the chromatogram was integrated and the molar ratio was determined by summation of the weighted peak area. The molar ratio was calculated to be about 4.56 with 7% unconjugated antibody.

EXAMPLE 29

Determination of molar ratio for SBT-040-G1WT conjugated to ATAC11

29 Figure 4 shows the HPLC analysis of SBT-040-G1WT conjugated to ATAC11, which is a cleavable maleimide-Val-Ala-PABA-Gardiquimod linker. First, 10 μl of a 5 mg / ml solution of the antibody-drug conjugate was placed in a HPLC system set-up with a TOSOH TSKgel Butyl-NPR ™ Hydrophobic Interaction Chromatography (HIC) column (2.5 μM particle size, 4.6 mm × 35 mm). Then, a procedure was carried out over 18 minutes in which the mobile phase gradient ran from 100% of mobile phase A to 100% of mobile phase B within 12 minutes, followed by a six minute re-equilibration at 100% mobile phase A. The flow rate was 0.8 ml / min and the detector was set to 280 nM. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B was 25% isopropanol in 25 mM sodium phosphate (pH 7). After the run, the chromatogram was integrated and the molar ratio was determined by summation of the weighted peak area. The molar ratio was calculated to be about 4.5.

EXAMPLE 30

Determination of molar ratio for SBT-040-G2WT conjugated to ATAC11

30 Figure 4 shows the HPLC analysis of SBT-040-G2WT conjugated to ATAC11, which is a cleavable maleimide-Val-Ala-PABA-Gardiquimod linker. First, 10 μl of a 5 mg / ml solution of the antibody-drug conjugate was placed in a HPLC system set-up with a TOSOH TSKgel Butyl-NPR ™ Hydrophobic Interaction Chromatography (HIC) column (2.5 μM particle size, 4.6 mm × 35 mm). Then, a procedure was carried out over 18 minutes in which the mobile phase gradient ran from 100% of mobile phase A to 100% of mobile phase B within 12 minutes, followed by a six minute re-equilibration at 100% mobile phase A. The flow rate was 0.8 ml / min and the detector was set to 280 nM. Mobile phase A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B was 25% isopropanol in 25 mM sodium phosphate (pH 7). After the run, the chromatogram was integrated and the molar ratio was determined by summation of the weighted peak area. The molar ratio was calculated to be about 4.0.

EXAMPLE 31

Additional method for determining the molar ratio

Another method for determining the molar ratio is as follows. First, 10 μl of a 5 mg / ml solution of a conjugate of antibody construct and immunostimulatory compound in a HPLC system setup with a TOSOH TSKgel Butyl-NPR ™ Hydrophobic Interaction Chromatography (HIC) column attached thereto (2.5 μM particle size, 4.6 mm × 35 mm). Then, in the course of 18 minutes, a procedure is performed in which the mobile phase gradient runs from 100% of mobile phase A to 100% of mobile phase B within 12 minutes, followed by a six minute re-equilibration at 100% mobile phase A. The flow rate is 0.8 ml / min and the detector is set to 280 nM. Mobile phase A is 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B is 25% isopropanol in 25 mM sodium phosphate (pH 7). After the run, the chromatogram is integrated and the molar ratio is determined by summation of the weighted peak area.

EXAMPLE 32

Affinity measurement of unconjugated anti-CD40 antibody or conjugate of anti-CD40 antibody and immunostimulatory compound for recombinant CD40 ectodomain Fcγ receptors using BioLayer interferometry

These examples show affinity measurements of unconjugated anti-C40 antibody or conjugate of anti-CD40 antibody and immunostimulatory compound for Fc receptors (FcRs). Antibody affinity for its antigen (such as CD40) and Fcγ receptors (such as FcγRI, FcγRII, FcγRIII) was quantitated using BioLayer interferometry (BLI). The anti-CD40 antibodies with different Fc (SBT-040-WT, SBT-040-VLPLL, SBT-040-AAA and SBT-040-G2) were first prepared as described in EXAMPLE 1 and then conjugated with an immunostimulatory compound ( any of ATAC1-ATAC34 and ATAC 43) as described in EXAMPLE 2. After successful conjugation, their molecular Intercellular domain (ECD) interactions of CD40 and various human FcγRs were quantified using BLI.

Analysis of the CD40-ECD interaction was done with the Octet Red 96 instrument (ForteBio). The Octet systems use the company's own BLI to analyze the biomolecular interaction. Unconjugated anti-CD40 antibodies (SBT-040-WT, SBT-040-VLPLL, SBT-040-AAA, SBT-040-G2) and conjugates of anti-CD40 antibody and immunostimulatory compound were tested on anti-human Fc biosensors immobilized and incubated with varying concentrations of monomeric human or rhesus CD40 ranging from 1.2 nM to 300 nM in PBS. The experiments included 5 steps: (1) baseline acquisition (60 s); (2) loading antibodies and conjugates of antibody construct and immunostimulatory compound to anti-human Fc biosensor (120 s); (3) second baseline detection (60 sec); (4) Association of interacting monomeric CD40 ECD protein for k _on measurement (120 s); (5) Dissociation of interacting monomeric CD40-ECD for k _off measurement (360 s). The interacting monomeric CD40-ECD was used at 5-6 concentrations in the 3-fold concentration series. The data was analyzed with Octet Data Analysis Software 9.0 (ForteBio) and adapted to the 1: 1 binding model. Equilibrium dissociation constants (K _D ) were calculated by the ratio of k _on to k _off . Selected data are shown in TABLE 9. All conjugates of anti-CD40 antibody and immunostimulatory compound had a similar binding as unconjugated anti-CD40 antibody to monomeric human or rhesus CD40 ECD.

Interaction analysis of human FcγR was also performed with the Octet Red 96 instrument. For interactions of human FcγRI and FcγRIIA, unconjugated anti-CD40 antibodies or conjugates of anti-CD40 antibody and immunostimulatory compound were immobilized on anti-human Fc biosensors and with a varying concentration of monomeric FcγR ranging from 1.2 nM to 1 μM incubated in PBS. The experiments included 5 steps: (1) baseline acquisition (60 s); (2) loading of anti-CD-40 antibodies or conjugates of antibody construct and immunostimulatory compound on anti-human Fc biosensor (120 s); (3) second baseline detection (60 sec); (4) Association of interacting monomeric CD40 ECD protein for k _on measurement (120 s); (5) Dissociation of interacting monomeric CD40-ECD for k _off measurement (360 s). The interacting monomeric CD40-ECD was used at 5-6 concentrations in the 3-fold concentration series. The data was analyzed with Octet Data Analysis Software 9.0 (ForteBio) and adapted to the 1: 1 binding model. Equilibrium dissociation constants (K _D ) were calculated by the ratio of k _on to k _off . Selected data are shown in TABLE 10. There is very little to no change in the interaction of anti-CD40 antibody and immunostimulatory compounds with human FcγRI and FcγRIIA compared to interactions of unconjugated anti-CD40 antibody with the respective monomeric FcγR protein.

For interaction studies with human FcγRIIB / C, FcγRIIIA F158, FcγRIIIA V158 and FcγRIIIB, the proteins were immobilized on anti-His-tag biosensors and with different concentrations of anti-CD40 anti-CD40 antibodies or anti-CD40 antibody and conjugates immunostimulatory compound in the range of 0.04 μM to 8 μM incubated. This format was chosen because of the weak interactions when antibodies were first captured and FcγR was added afterwards. The experiment involved 5 steps: (1) baseline acquisition (60 s); (2) loading of anti-CD-40 antibodies or conjugates of antibody construct and immunostimulatory compound on anti-human Fc biosensor (120 s); (3) second baseline detection (60 sec); (4) association of interacting protein for k _on measurement (120 s); (5) Dissociation of interacting FcγR for k _off measurement (300 s). The interacting anti-CD40 antibodies or conjugates of anti-CD40 antibody and immunostimulatory compound were used at 4 concentrations of a 2-fold concentration series. The data was analyzed using Octet Data Analysis Software 9.0 (ForteBio) and adapted to the avidity-binding model. Equilibrium dissociation constants (K _D ) were calculated by the ratio of k _on to k _off . Selected data are shown in TABLE 10. In most cases, there were no changes with the conjugates of anti-CD40 antibody and immunostimulatory compound with human FcγRIIB / C, FcγRIIIA F158, FcγRIIIA V158 and FcγRIIIB compared to the parental unconjugated antibodies. In some cases, there were small changes, usually within 2-fold, such as SBT040-G2 ATAC11 interaction with FcγRIIIA F158 compared to unconjugated SBT040-G2.

EXAMPLE 33

Stability of conjugates of anti-CD40 antibody and immunostimulatory compound in IgG-depleted human serum

The stability of the conjugates of anti-CD40 antibody and immunostimulatory compound in human serum (IgG-depleted) was determined at 37 ° C for 96 hours using a direct HIC-UV analysis approach (Method A) or an affinity capture approach (Method B) measured. SBT-040-G1-ATAC12, SBT-040-G2-ATAC12 or SBT-040-G1-ATAC30 were added to IgG-depleted human serum (BBI solutions # SF142-2) in sterile tubes (75% final serum concentration) and the samples were divided into 4 aliquots of the same size, then transferred to a 37 ° C incubator. One of the aliquots of each sample was removed from the incubator at each time point (T = 0 h, 24 h, 48 h, 96 h) and average drug-antibody ratios (DAR) were recorded.

Method A: Direct HIC-UV analysis

At 0, 24, 48, and 96 hours after the start of the incubation, the conjugates of anti-CD40 antibody and immunostimulatory compound added to IgG-depleted human serum were purified by analytical hydrophobic interaction chromatography (HIC) using a TOSOH TSKgel butyl-NPR 4 , 6mm x 35mm HIC column (TOSOH Bioscience, # 14947), coupled to a Dionex Ultimate 3000RS HPLC system (ThermoFisher Scientific, Hemel Hemstead, UK). The results are described below in TABLE 11. TABLE 11 Time (h) Average DAR SBT-040-G1 ATAC12 SBT-040-G2-ATAC12 SBT-040-G1 ATAC30 0 4.3 3.5 3.7 24 4.1 3.5 3.6 48 3.9 3.5 3.4 96 3.8 3.4 3.0

Method B: affinity capture, deglycosylation, and RP-ESI-MS analysis

ADCs were immunocaptured from the IgG-depleted human serum using an anti-human IgG (Fc-specific) biotin antibody immobilized on streptavidin beads at 0, 24, 48 and 96 hours after the start of incubation. After elution from the beads, the beads were deglycosylated using agarose-immobilized EndoS (Genovis Inc, USA). The deglycosylated ADCs were coupled to electrospray ionization mass spectrometry (RP-ESI-MS) by reverse-phase chromatography coupled to an Acquity Nano-UPLC in conjunction with a Xevo G2S Q-TOF (Waters, Elstree, UK) was, analyzed. The separation was done with an Acquity UPLC online coupled with an ESI-MS mass spectrometer. Mass spectrometric analysis was performed in the positive ion mode with sampling from 1000 to 4000 m / z in high mass mode of operation. The inner sheath produced by each sample was deconvoluted using the MaxEnt1 algorithm provided in the MassLynx software (Waters, Elstree, UK). The results are given in the table below. Time (h) Average DAR SBT-040-G1 ATAC4 SBT-040-G1 ATAC3 0 4.4 3.8 24 4.7 3.8 48 4.6 3.7 96 4.5 3.7

EXAMPLE 34

Synthesis of ATAC18

Schritt A: Herstellung von Int ATAC18-1

This example demonstrates the synthesis of (1R, 6R, 8R, 9S, 10S, 15R, 17R, 18S) -8,17-bis (2-amino-6-oxo-1,9-dihydropurin-9-yl) -18 - (3- {2- [2- (2- {2- [3- (2,5-dioxo-1H-pyrrol-1-yl) propionylamino] ethoxy} ethoxy) ethoxy] ethoxy} propionylamino) -9- hydroxy-3,12-dioxy-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadecae-3,12-dione triethylammonium salt (ATAC18).

Step A: Preparation of Int ATAC18-1

To a solution containing 100 mg (0.106 mmol) of compound 21 in 5 mL of DMSO and 39 mg (0.106 mmol) of 3- [2- (2- {2- [2- (tert-butoxycarbonylamino) ethoxy] ethoxy} ethoxy) ethoxy] propionic acid, 22 mg (0.16 mmol) HOBT and 27 mg (0.212 mmol) DIC were added. The reaction mixture was stirred at room temperature for 16 hours and then purified by reverse phase chromatography without work-up. The resulting product fractions were lyophilized to give 70 mg of product which was covered with 7 ml of a 1: 2 mixture of TFA and CH ₂ Cl ₂ . The mixture was stirred for 2 h at ambient temperature before the solvent was removed. The resulting residue Int ATAC18-1 was used directly in the next step without purification. Step B: Preparation of Int ATAC 18-2

A solution containing Int ATAC18-1 (50 mg, 0.04 mmol) and 3.0 mL of methylamine (33% in anhydrous ethanol) was stirred for 16 h at room temperature. The reaction mixture was concentrated to afford Int ATAC18-2 as a white foam which was used directly in the next step. LCMS (ESI, m / z): 1051 [M + H]. Step C: Preparation of Int ATAC18-3

The above crude Int ATAC18-2 was azeotroped three times with 3: 1 pyridine / triethylamine and then dissolved in 0.8 ml of pyridine. 2 ml of triethylamine and 1 ml of triethylamine trihydrofluride were added simultaneously to this solution at 55 ° C. After stirring for 1 h, the bath was removed and anhydrous acetone was added immediately. The mixture was stirred for 20 minutes and the white solid was collected by filtration. The precipitate was washed with anhydrous acetone. The product was purified by preparative flash chromatography. The resulting solution was lyophilized to give 3 mg Int ATAC18-3 as a white solid. LCMS: (ESI, m / z): 937.6 [M + H] ⁺ . ¹ H NMR (DMSO-d ⁶ ): δ 7.99 (s, 1H), 7.89 (s, 1H), 5.73 (m, 2H), 5.06 (m, 1H); 4.84-4.74 (m, 2H); 4.53 (t, J = 5.1 Hz, 1H), 4.21 (m, 1H), 2.96 (m, 2H), 3.73-3.29 (m, 16H), 2.87 (q, J = 7.2Hz, 8H), 2.35 (m, 2H), 1.10 (t, J = 7.2Hz, 12H). ³¹ P-NMR (DMSO-d ⁶ ) δ 0.25, -1.32. Step D: Preparation of ATAC18

To a solution containing 2.0 mg (0.002 mmol) of Int ATAC18-3 in 0.2 ml of DCM was added 0.5 mg (0.004 mmol) of DIPEA, then 1.06 mg (0.004 mmol) of 2,5-dioxo -1-pyrrolidinyl-3- (2,5-dioxo-1H-pyrrol-1-yl) -propionate. The resulting solution was stirred overnight, then the solvent was evaporated. Reverse-phase column chromatography gave the desired compound ATAC18 as a white solid. LCMS: (ESI, m / z): 1088 [M + H] ⁺ , ¹ H-NMR (D ₂ O) δ 8.15 (s, 1H), 8.05 (s, 1H), 7.07 ( s, 2H), 5.88-5.84 (m, 2H), 5.26 (m, 1H), 5.05-4.91 (m, 2H), 4.76 (m, 1H), 4 , 35 (m, 1H), 4.29-4.20 (m, 5H), 3.77 (t, 2H), 3.7-3.5 (m, 16H), 3.33 (m, 2H ), 3.15 (q, J = 7.2 Hz, 15H), 2.52 (m, 1H), 1.25 (t, J = 7.2 Hz, 21H). ³¹ P-NMR (DMSO-d ⁶ ) δ 1.51, 0.62.

EXAMPLE 35

Synthesis of ATAC43

Schritt A: Herstellung von Int ATAC 43-1

This example demonstrates the synthesis of 2,3,4,5,6-pentafluorophenyl-5 - {(1S, 6R, 8R, 9S, 10R, 15R, 17R, 18S) -8,17-bis (2-amino-6 oxo-1,9-dihydro-9-yl) -18-hydroxy-3,12-dioxo-3,12-dioxy-2.4.7.11.13.16-hexaoxa-3λ5.12λ5-diphosphatricyclo [13.3.0.06,10] octadec-9-ylamino} -5-oxovalerate triethylammonium salt (ATAC43)

Step A: Preparation of Int ATAC 43-1

To a solution containing 50 mg (0.072 mmol) of compound 2 in 2.5 ml of DMSO was added 57 mg (0.72 mmol) of pyridine and 5 mg of DMAP. The resulting solution was stirred for 10 minutes, then treated with 82 mg (0.72 mmol) glutaric anhydride. The reaction mixture was stirred at room temperature for 16 hours and then purified by reverse phase chromatography without work-up. Fractions containing product were lyophilized for 48 hours to give 10 mg Int ATAC 43-1 as a white solid. LCMS (ESI, m / z): 804 [M + H]. ^{1 H} NMR (DMSO-d ⁶ ) δ 10.6 (bs, 2H), 9.20 (bs, 1H), 7.99 (s, 1H), 7.92 (s, 1H), 7.56 (bs , 1H), 6.56 (m, 4H), 5.76 (dd, J = 8.1, 17.7 Hz, 2H), 4.94 (m, 1H), 4.76-4.68 ( m, 2H), 4.58 (m, 1H), 4.22 (m, 1H), 4.03-3.91 (m, 6H), 3.97 (q, J = 7.0 Hz, 12H ), 2.21 (t, J = 7.2 Hz, 2H), 2.09-1.99 m, 2H), 1.67-1.60 (m, 2H), 1.20 (t, J = 7.2 Hz, 18H). ³¹ P NMR (DMSO-d ⁶ ) d 0.44, -1.3. Step B: Preparation of ATAC43

To a solution containing 7.0 mg (0.009 mmol) of Int ATAC 43-1 and 2.6 mg (0.013 mmol, 1.5 eq.) Of pentafluorophenol in 300 μL DMSO was added 1.8 mg (0.012 mmol, 1 , 3 eq.) EDC and the reaction mixture was stirred for 16 h at room temperature and then purified by reverse phase chromatography without workup. Fractions containing product were lyophilized to give ATAC 43 as a white solid. LCMS (ESI, m / z): 970 [M + H].

EXAMPLE 36

STING agonist screening test

Biological materials and general procedures. The following reporter cell lines, reagents and ligands were obtained from InvivoGen: THP-1 dual cells (thpd-nfis); THP-1 Dual KO-STING cells (thpd-kostg); Quanti-Blue (rep -qb1); Quanti-Luc (rep -qlc1); Normocin (ant-nr-1); Zeocin (ant-zn-1); Blasticidin (ant-bl-1); PMA (tlrl-pma); 2 ', 3'-cGAMP (tlrl-nacga); and 2'3'-c-di-AM (PS) 2 (Rp, Rp) (tlrl-nacda2r). 3 ', 3'-cGAMP (SML 1232) and 3', 5'-cyclic-di-GMP (SML 1228) were purchased from Sigma. THP-1 dual cells were cultured in RPMI 1640 (Lonza) supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 μg / ml penicillin, 50 U / ml streptomycin (all from Gibco). The cells were passaged at 0.7x10 6 cells / ml every 2-3 days. 100 μg / ml normocin, 100 μg / ml zeocin and 10 μg / ml blasticidin were added every other run to maintain reporter expression according to the manufacturer's instructions.

General Procedure for In Vitro Screening of CDNs for Cytokine Induction Activity.

THP-1 dual or THP-1 dual KO-STING cells were plated at 50,000 cells per tube in 200 μl culture medium in 96-well plates and ripened with 150 nM PMA for 16-18 hours. The cells were washed the next day in culture medium and the supernatants were removed. The cells were incubated for 30 min at 37 ° C in a 5% CO ₂ incubator with 150 μl CDNs prepared in permeabilization buffer with 2 μg / ml digitonin (CalBiochem) in four different concentrations (1, 0.1, 0.01 and 0.001 μM). After incubation, the permeabilization buffer and CDNs were removed and replaced with 150 μl of culture media. The cells were additionally incubated in a 5% CO ₂ incubator for 23.5 h at 37 ° C. Before harvesting the supernatant, the cells were rotated at 300 x g for 5 minutes to remove cell debris. The ISG54 activity was quantified indirectly from the supernatants using QUANTI-Luc, which was prepared and used according to the manufacturer's instructions. Activity of the NF-kB-secreted alkaline phosphatase signaling pathway was quantified indirectly from supernatants using QUANTI-Blue, which was prepared and used according to the manufacturer's instructions. CellTiterGlo (Promega) was used to lyse and evaluate viability according to the manufacturer's instructions. Plates were analyzed on Envision (Perkin Elmer) or Synergy (BioTek) plate readers.

EXAMPLE 37

Tumor size in a mouse tumor model is reduced by conjugates of anti-CD40 antibody and immunostimulatory compound

The example demonstrates that the tumor size in a mouse tumor model is reduced after the administration of a conjugate of anti-CD40 antibody and immunostimulatory compound. The immunocompromised mouse strain NSG (NOD.Cg-PrkdcscidIL-2rgtm1Wj1 / SzJ) into which human tumor cells are injected with human T cells and myeloid dendritic cells (mDC) is used as a mouse tumor model. The immune-mediated activity of the conjugate of anti-CD40 antibody and immunostimulatory compound in vivo is assessed by examining human mDCs in this model.

Conjugates of anti-CD40 antibody and immunostimulatory compound or anti-CD40 antibody (control) are injected intraperitoneally into mice. Immediately after this injection, Raji cells (a B cell lymphoma tumor cell line), human T cells, and mDCs from the same human donor are injected into the mice. The Raji cells are injected subcutaneously at a concentration of 1 x 10 ^ 7 cells / mouse. The human T cells are injected at a concentration of 1 x 10 ^ 6 cells / mouse and mDCs are injected at a concentration of 5 x 10 ^ 5 cells / mouse. Tumor growth is measured twice per week using calipers, starting seven days after tumor cell transfer and ending at study end, about 3 weeks after tumor cell inoculation. Tumor size is reduced in mice receiving the conjugates of anti-CD40 antibody and immunostimulatory compound compared to mice receiving the anti-CD40 antibody.

EXAMPLE 38

Priming of T cells by dendritic cells is enhanced by conjugates of anti-CD40 antibody and immunostimulatory compound

This example demonstrates that priming of T cells by dendritic cells is enhanced by conjugates of anti-CD40 antibody and immunostimulatory compound. After conjugates of anti-CD40 antibody and immunostimulatory compound is administered, upregulation of costimulatory molecules and cytokine production is induced by dendritic cells. The priming of a T cell response is therefore enhanced by the stimulation of dendritic cells in this way. A co-culture test of human mDC and T cells in vitro is used to demonstrate this.

Myeloid dendritic cells (mDCs) and allogeneic T cells are isolated from peripheral blood mononuclear cells (PBMCs), labeled with a dye to monitor cell division, and co-cultured for 5 days in the presence of anti-CD40 antibody and immunostimulatory compound or isotype control conjugates. T cell activation is assessed as a percentage of dividing cells as measured by flow cytometry using the indicator dye. The percentage of dividing T cells is in T cells cocultured with the conjugates of anti-CD40 antibody and immunostimulatory compound compared to the percentage of dividing T cells co-cultivated with the isotype control , elevated.

While aspects of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It is understood that various alternatives to the aspects of the disclosure described herein may be used in the practice of the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 5846514 [0137, 0137]
• US 6334997 [0137, 0137]

Cited non-patent literature

• Dean, Dennis C .; Editor. Recent Advances in the Synthesis and Applications of Radiolabeled Compounds for Drug Discovery and Development. [In: Curr. Pharm. Des., 2000; 6 (10)] 2000, 110 ff. [0140]
• George W .; Varma, Rajender S. The Synthesis of Radiolabeled Compounds via Organometallic Intermediates, Tetrahedron, 1989, 45 (21), 6601-21 [0140]
• Evans, E. Anthony. Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64 (1-2), 9-32 [0140]
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• Biol. 299: 865-881 (1999) [0488]

Claims (57)

A conjugate comprising: a) an immunostimulatory compound; b) an antibody construct comprising an antigen-binding domain and an Fc domain, wherein i) a Kd for the binding of said antigen binding domain to said first antigen in the presence of said immunostimulatory compound is less than about 100 nM and not greater than about 100 times a Kd for the binding of said antigen binding domain to that first antigen in the absence of said immunostimulatory compound; ii) a Kd for binding of said Fc domain to an Fc receptor in the presence of said immunostimulatory compound is not greater than about 100 times a Kd for binding of said Fc domain to said Fc receptor in the absence of said immunostimulatory compound; and c) a linker which attaches said antibody construct to said immunostimulatory compound, said linker being covalently linked to said immunostimulatory compound, and wherein a molar ratio of said immunostimulatory compound to said antibody construct is less than 8. The conjugate of claim 1, wherein said linker is attached to said antibody construct at a cysteine or lysine residue. The conjugate of any of claims 1-2, wherein said Fc receptor is an Fc receptor found on a dendritic cell. The conjugate of any of claims 1-3, wherein said immunostimulatory compound linked via a linker to said antibody construct does not interfere with the binding of said immunostimulatory compound to its receptor. The conjugate of any of claims 1-4, wherein said antibody construct further comprises a targeting binding domain. The conjugate of claim 5, wherein said targeting binding domain specifically binds a second antigen. The conjugate of any one of claims 5-6, wherein said targeting binding domain is conjugated to the antibody construct at a C-terminal end of that Fc domain. The conjugate of any of claims 1-7, wherein said antigen binding domain is an antibody or non-antibody framework. The conjugate of any of claims 1-8, wherein said antigen binding domain is at least 80% homologous to an antigen binding domain from an antibody or non-antibody framework. The conjugate of any one of claims 1-9, wherein a complementarity determining region of the antigen binding domain is a light chain sequence that is at least 80% homologous to SEQ ID NO: 27, a light chain sequence that is at least 80% homologous SEQ ID NO: 28, a light chain sequence at least 80% homologous to SEQ ID NO: 29, a heavy chain sequence at least 80% homologous to SEQ ID NO: 23, a heavy chain sequence, which is at least 80% homologous to SEQ ID NO: 24, or a heavy chain sequence that is at least 80% homologous to SEQ ID NO: 25. The conjugate of any one of claims 1-9, wherein a complementarity determining region of the antigen binding domain is a light chain sequence that is at least 80% homologous to SEQ ID NO: 35, a light chain sequence that is at least 80% homologous SEQ ID NO: 36, a light chain sequence at least 80% homologous to SEQ ID NO: 37, a heavy chain sequence at least 80% homologous to SEQ ID NO: 31, a heavy chain sequence, which is at least 80% homologous to SEQ ID NO: 32, or a heavy chain sequence which is at least 80% homologous to SEQ ID NO: 33. The conjugate of any of claims 1-9, wherein said antibody binding domain comprises at least 90% or 100% homology to: a) HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 23, HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 24, an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 25, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 27, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 28, and LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 29; or b) HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 31, HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 32, an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 33, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 35, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 36, and LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 37. The conjugate of any one of claims 5-12, wherein said targeting binding domain is an scFv. The conjugate of any one of claims 5-9 and 11-13, wherein said targeting binding domain is a CD40 agonist. The conjugate of any one of claims 5-9 and 11-14, wherein a complementarity determining region of said targeting binding domain is a light chain sequence at least 83% homologous to SEQ ID NO: 27, a light chain sequence, the SEQ ID NO: 28 is a light chain sequence that is at least 88% homologous to SEQ ID NO: 29, a heavy chain sequence that is at least 87% homologous to SEQ ID NO: 23, a heavy chain sequence which is at least 87% homologous to SEQ ID NO: 24, or a heavy chain sequence which is at least 94% homologous to SEQ ID NO: 25. The conjugate of any of claims 5-9 and 11-14, wherein said targeting binding domain comprises at least 90% or 100% homology to HC-CDR1 comprising an amino acid sequence of SEQ ID NO: 23, HC-CDR2 comprising an amino acid sequence of SEQ ID NO: 24, an HC-CDR3 comprising an amino acid sequence of SEQ ID NO: 25, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 27, LC-CDR1 comprising an amino acid sequence of SEQ ID NO: 28, and LC-CDR3 comprising an amino acid sequence of SEQ ID NO: 29, The conjugate of any of claims 1-9 and 11-16, wherein said first antigen is a tumor antigen. The conjugate of any of claims 1-9 and 11-17, wherein said first antigen is CD5, CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7- H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate binding protein, A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Le ^y , CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2 receptor, de2-7 EGFR, fibroblast activating protein, tenascin, metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2 / neu, idiotype, MAGE A3, non-mutant p53, NY-ESO-1, PMSA, GD2, CEA, MelanA / MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyrosinase, survivin, PSA, hTERT, sarcoma translocations Breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin B1, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE A1, sLe (animal ), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AM L, NY-BR-1, RGS5, SART3, STn, carbonic anhydrase IX, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE1, B7H3, Legumain, Tie3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAIL1, MUC16, MAGE A4, MAGE C2, GAGE, EGFR, CMET, HER3, MUC15, MSLN, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, or Fos-related antigen is 1. The conjugate of any one of claims 1-10, 12 and 13, wherein said first antigen is expressed on an immune cell. The conjugate of any of claims 1-10, 12, 13 and 19, wherein said first antigen is CD40. The conjugate of any of claims 1-10, 12, 13, 19 and 20, wherein said antigen binding domain is a CD40 agonist. The conjugate of any of claims 1-21, wherein said antibody construct is a human antibody or a humanized antibody. The conjugate of any of claims 1-22, wherein said antibody construct comprises a light chain sequence that is at least 90% homologous to SEQ ID NO: 4, a variable domain sequence that is at least 90% homologous to SEQ ID NO: 6 a heavy chain sequence at least 90% homologous to SEQ ID NO: 15, a variable domain sequence at least 90% homologous to SEQ ID NO: 20, a heavy chain sequence at least 90% homologous to SEQ ID NO: 16, a heavy chain sequence that is at least 90% homologous to SEQ ID NO: 17, or a heavy chain sequence that is at least 90% homologous to SEQ ID NO: 18, is one sequence a light chain that is at least 90% homologous to SEQ ID NO: 34, or a heavy chain sequence that is at least 90% homologous to SEQ ID NO: 30. The conjugate of any of claims 1-23, wherein is the Fc domain variant having at least one amino acid residue change compared to a wild-type sequence of that Fc domain. The conjugate of any of claims 1-24, wherein the linker is a peptide. The conjugate of any one of claims 1-25, wherein a Kd for the binding of said antigen binding domain to said antigen in the presence of said immunostimulatory compound is less than about 100 nM and not greater than about 100 times a Kd for the binding of said antigen binding domain to that antigen in the absence of that immunostimulatory compound; and that Kd for binding of said Fc domain to said Fc receptor in the presence of said immunostimulatory compound is not greater than about 100 times a Kd for binding of said Fc domain to said Fc receptor in the absence of said immunostimulatory compound. The conjugate of any of claims 1-26, wherein said molar ratio of immunostimulatory compound to antibody construct is less than 5. The conjugate of any of claims 1-27, wherein said conjugate is in a pharmaceutical formulation. The conjugate of any one of claims 1-28, wherein said linker at an amino acid residue is attached to said antibody construct that does not interfere with the binding of said Fc domain to said Fc receptor. The conjugate of any of claims 1-29, wherein said linker is not attached to an amino acid residue of said Fc domain selected from the group consisting of: 221, 224, 227, 228, 230, 231, 223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 249, 250, 258, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 275, 276, 278, 280, 281, 283, 285, 286, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 305, 313, 318, 323, 324, 325, 327, 328, 329, 330, 331, 332, 333, 335, 336, 396 or 428, wherein the numbering of the amino acid residues in that Fc domain is according to the EU index as in Kabat. The conjugate of any one of claims 1-30, wherein said Fc domain is selected from the group consisting of a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, and a human IgG4 domain. Fc domain. The conjugate of any of claims 1-31, wherein said conjugate induces secretion of a cytokine by an antigen presenting cell. The conjugate of any one of claims 1-32, wherein said immunostimulatory compound is a toll-like receptor agonist, STING agonist or RIG-I agonist. The conjugate of any one of claims 1-33, wherein said immunostimulatory compound is a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist TLR8 agonist, a TLR9 agonist or a TLR10 agonist. The conjugate of any one of claims 1-34, wherein said immunostimulatory compound is selected from the group consisting of: S-27609, CL307, UC-IV150, imiquimod, gardiquimod, resiquimod, motolimod, VTS-1463GS-9620, GSK2245035, TMX 101, TMX-201, TMX-202, isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240, KU34B and CL663. A compound represented by the structure of formula (I):

or pharmaceutically acceptable salts thereof, wherein: X ^{1 is} selected from -OR ² and -SR ² ; X ^{2 is} selected from -OR ³ and -SR ³ ; B ¹ and B ^{2 are} independently selected from optionally substituted nitrogenous bases; Y is selected from -OR ⁴ , NR ⁴ R ⁴ and halogen; R ¹ , R ² , R ³ and R ⁴ at each occurrence are independently selected from hydrogen, -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each of which is optionally independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle, and _3-10 -linked heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carboxylic cycle and 3- to 10-membered heterocycle in R ¹ , R ² , R ³ and R ^{4 is} optionally independently substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ -C (O) R ¹⁰⁰ , -C (O ) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl; and R ^{100 is} independently selected at each occurrence from hydrogen; and C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbo cycle, and _3-10 membered heterocycle, each of which is optionally independently substituted on each occurrence with one or more more substituents selected from halogen, -CN, -NO ₂ , = O, = S, and haloalkyl. The compound or salt of claim 36, wherein the compound of formula (I) is represented by formula (IA):

or pharmaceutically acceptable salts thereof. The compound or salt of claim 36, wherein the compound of formula (I) is represented by formula (IB):

or a pharmaceutically acceptable salt thereof. The compound or salt of any one of claims 36-38, wherein B ¹ and B ^{2 are} each independently optionally substituted with one or more substituents, wherein the optional substituents on B ¹ and B ² are independently selected on each occurrence from halogen, = O, = S, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ and -CN; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each independently independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle, and _3-10 -linked heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carbo-cycle and 3- to 10-membered heterocycle in R ¹ , R ² , R ³ and R ^{4 is} independently optionally substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ -C (O) R ¹⁰⁰ , -C ( O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl, and C _2-6 alkynyl. The compound or salt of any one of claims 36-39, wherein X ^{1 is} selected from -OH and -SH. The compound or salt of any one of claims 36-40, wherein X ^{2 is} selected from -OH and -SH. The compound or salt of any of claims 36-41, wherein Y is selected from -OH, -OC _1-10 alkyl, -NH (C _1-10 alkyl) and -NH ₂ . The compound or salt of any of claims 36-42, wherein the compound of formula (I) is represented by formula (IC):

or a pharmaceutically acceptable salt thereof. The compound or salt of claim 43, wherein the compound of formula (IC) is represented by formula (ID):

or a pharmaceutically acceptable salt thereof. An antibody construct / immunostimulatory compound conjugate comprising a compound or salt of any one of claims 36-44, an antibody and a linker group, wherein the compound or salt is linked to the antibody through the linker group. A compound represented by the structure of formula (II):

or a pharmaceutically acceptable salt thereof, wherein: X ^{1 is} selected from -OR ² and -SR ² ; X ^{2 is} selected from -OR ³ and -SR ³ ; B ¹ and B ^{2 are} independently selected from optionally substituted nitrogenous bases, wherein each optional substituent is independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN , R ⁶ , and -X ³ ; Y is selected from -OR ⁴ , -SR ⁴ , -NR ⁴ R ⁴ , and halogen; Z is selected from -OR ⁵ , -SR ⁵ , and NR ⁵ R ⁵ ; R ¹ , R ² , R ³ , R ⁴ , and R ^{5 are} independently selected from -X ³ ; Hydrogen, -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each on each occurrence is independently optionally substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O ) R ^100, -C (O) OR ^100, -OC (O) R ^100, -NO _2, = O, = S, = N (R ^(100), -P O) (OR ¹⁰⁰⁾ _2, -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _3-10 carboxylic cycle and 3- to 10-membered heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carbo-cycle and 3- to 10-membered heterocycle are optionally substituted in R ¹ , R ² , R ³ , R ⁴ , and R ⁵ is one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ) -P (O) (ORR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 alkyl, C _2-6 alkenyl _, C _2-6 alkynyl; R ^{6 is} independently selected from -C (= O) R ¹⁰⁰ , -C (= O) OR ¹⁰⁰ and -C (= O) NR ¹⁰⁰ ; C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, each independently independently substituted on each occurrence with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , N ( R ¹⁰⁰ ) ₂ , -S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ^100), -P (O) (OR ¹⁰⁰⁾ _2, -OP (O) (OR ¹⁰⁰⁾ _2, -CN, C _3-10 -Carbozyklus and 3- to 10- membered heterocycle; and C _3-10 carboxylic cycle and 3- to 10-membered heterocycle, wherein each C _3-10 carboxylic cycle and 3- to 10-membered heterocycle in R ^{6 is} optionally substituted with one or more substituents selected from halogen, -OR ¹⁰⁰ , -SR ^100, -N (R ¹⁰⁰⁾ _2, -S (O) R ^100, -S (O) ₂ R ^100, -C (O) R ^100, -C (O) OR ^100, -OC (O ) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -P (O) (OR ¹⁰⁰ ) ₂ , -OP (O) (OR ¹⁰⁰ ) ₂ , -CN, C _1-6 Alkyl, C _2-6 alkenyl, C _2-6 alkynyl; R ^{100 is} independently selected at each occurrence from hydrogen; and C _1-10 alkyl, C _2-10 alkenyl, C _2-10 alkynyl, C _3-10 carbo cycle, and _3-10 membered heterocycle, each of which is independently independently optionally substituted with one or more on each occurrence more substituents selected from halo, -CN, -NO ₂ , = O, = S, and haloalkyl; and X ^{3 is} a linker group, wherein at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , X ¹ , X ² , a B ¹ substituent and a B ² substituent is -X ³ . The compound or salt of claim 46, wherein the compound of formula (II) is represented by a structure of formula (IIA):

or pharmaceutically acceptable salts thereof. The compound or salt of claim 46, wherein the compound of formula (II) is represented by a structure of formula (IIB):

or a pharmaceutically acceptable salt thereof. The compound or salt of any of claims 46-48, wherein B ¹ and B ^{2 are} independently selected from optionally substituted purines. The compound or salt of any one of claims 46-49, wherein B ^{1 is} substituted with X ³ and optionally one or more additional substituents independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , - S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN, and R ⁶ . The compound or salt of claim 49, wherein B ^{1 is} represented by:

and wherein B ^{1 is} optionally substituted with one or more substituents. The compound or salt of any of claims 46-51 wherein B ^{2 is} substituted with X ³ and optionally one or more additional substituents independently selected from halogen, -OR ¹⁰⁰ , -SR ¹⁰⁰ , -N (R ¹⁰⁰ ) ₂ , - S (O) R ¹⁰⁰ , -S (O) ₂ R ¹⁰⁰ , -C (O) R ¹⁰⁰ , -C (O) OR ¹⁰⁰ , -OC (O) R ¹⁰⁰ , -NO ₂ , = O, = S, = N (R ¹⁰⁰ ), -CN, and R ⁶ . The compound or salt of claim 49, wherein B ^{2 is} represented by:

and wherein B ^{2 is} optionally substituted with one or more substituents. 54. The compound or salt of any of claims 46-51, wherein -X ^{3 is} represented by the formula:

The compound or salt of any of claims 46-53, wherein -X ^{3 is} represented by the formula:

wherein RX comprises a reactive group. The compound or salt of any of claims 46-54, wherein -X ^{3 is} represented by the formula:

wherein RX * is a reactive group that has reacted with a group on an antibody to form an antibody-drug conjugate. The compound or salt of any of claims 46-54, wherein -X ^{3 is} represented by the formula:

wherein RX comprises a reactive group. The compound or salt of any of claims 45-53, wherein -X ^{3 is} represented by the formula:

wherein RX * is a reactive group that has reacted with a group on an antibody to form an antibody-drug conjugate.

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