[go: up one dir, main page]

WO2023011614A1 - 抗pd-l1纳米抗体及其应用 - Google Patents

抗pd-l1纳米抗体及其应用 Download PDF

Info

Publication number
WO2023011614A1
WO2023011614A1 PCT/CN2022/110423 CN2022110423W WO2023011614A1 WO 2023011614 A1 WO2023011614 A1 WO 2023011614A1 CN 2022110423 W CN2022110423 W CN 2022110423W WO 2023011614 A1 WO2023011614 A1 WO 2023011614A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
amino acid
acid sequence
sequence shown
nanobody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/110423
Other languages
English (en)
French (fr)
Inventor
徐汶新
房雪菲
徐德雨
丁列明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Betta Pharmaceuticals Co Ltd
Original Assignee
Betta Pharmaceuticals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Betta Pharmaceuticals Co Ltd filed Critical Betta Pharmaceuticals Co Ltd
Priority to US18/294,516 priority Critical patent/US20240327518A1/en
Priority to CN202280007891.6A priority patent/CN116547006B/zh
Priority to EP22852325.4A priority patent/EP4382166A4/en
Publication of WO2023011614A1 publication Critical patent/WO2023011614A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag

Definitions

  • the present invention claims the priority of the Chinese patent application with the application number 202110903293.4 submitted on August 6, 2021, and the priority of the Chinese patent application with the application number 202111162303.X submitted on September 30, 2021, and its quoted in whole or in part in this text.
  • the invention relates to the field of biomedicine, in particular to an anti-PD-L1 nanobody and its application.
  • programmed death ligand 1 plays a key role, which can bind programmed death receptor 1 (Programmed Death-1, PD-1 ), induce tumor-specific T cell apoptosis by inhibiting T cell activation, and play an immune escape and immunosuppressive role in pathological conditions such as tumors and chronic inflammation.
  • PD-L1 can restore and enhance the function of T cells by blocking the interaction of PD-1/PD-L1, so that it can play a role in tumor therapy and immune regulation. At the same time, it can also help fully activate T cell function and cytokine production by blocking the co-inhibitory function of CD80 and PD-L1.
  • Atezolizumab atezolizumab, trade name Tecentriq
  • Durvalumab durvalumab
  • Avelumab avelumab
  • Bavencio antibodies
  • various types of solid tumors such as non-small cell lung cancer, melanoma, lymphoma, head and neck cancer, and gastric cancer, etc., and have shown good tumor therapeutic effects.
  • Nanobodies are heavy chain-only antigen-binding fragments derived from Camelidae that are capable of functioning in the absence of light chains.
  • the molecular weight of nanobodies is relatively small, usually 12-16KDa, and at the same time, it retains the binding activity with antigens, and it does not require intra-chain disulfide bonds, so it is gradually emerging in immunotherapy.
  • Nanobodies are easily expressed in a variety of different expression systems, such as prokaryotic expression systems (such as in Escherichia coli), eukaryotic expression systems (such as yeast), and mammalian cell expression systems (such as CHO, HEK-293 cells), etc. expressed and easily purified.
  • prokaryotic expression systems such as in Escherichia coli
  • eukaryotic expression systems such as yeast
  • mammalian cell expression systems such as CHO, HEK-293 cells
  • Nanobodies have shown unique development and application value in cancer, autoimmune diseases, respiratory diseases, blood system diseases, etc., and more and more pharmaceutical companies are focusing on the development of nanobodies.
  • nanobodies are more common. There are still difficulties in screening nanobodies with high affinity, high specificity and industrialization prospects, which limits the research and development of nanobodies.
  • the present invention proposes an anti-PD-L1 nanobody and its use in disease treatment.
  • the present invention provides an anti-PD-L1 nanobody, a fusion protein comprising the PD-L1 nanobody, and the use of the nanobody or fusion protein in disease treatment, especially cancer treatment.
  • a first aspect of the present invention provides an anti-PD-L1 nanobody comprising CDR1, CDR2 and CDR3, wherein (a) CDR1 comprises a sequence selected from the group consisting of SEQ ID NO:1, 5 and 9, or with SEQ ID NO:1, 5 and 9 Compared with ID NO: 1, 5 and 9, there is a sequence of one or two amino acid substitutions, deletions or additions; (b) CDR2 comprises a sequence selected from the group consisting of: SEQ ID NO: 2, 6 and 10, or with SEQ ID NO: 2, 6 and 10 ID NO: 2, 6 and 10 compare, have the sequence of one or two aminoacid substitutions, deletion or addition; (c) CDR3 comprises the sequence selected from the following group: SEQ ID NO: 3, 7, 11 and 14, or Sequences having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NO: 3, 7, 11 and 14.
  • the present invention also provides an anti-PD-L1 nanobody, selected from: i) having such as SEQ ID NO: 4, or SEQ ID NO: 8, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 15, or SEQ ID NO: 28, or SEQ ID NO: 29, or SEQ ID NO: 30, or the amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO: 32; or ii) and i)
  • the homology is more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95% , more than 96%, more than 97%, more than 98%, or more than 99% of the amino acid sequences.
  • the anti-PD-L1 nanobody provided includes: (1) having CDR1 having the amino acid sequence shown in SEQ ID NO:1, CDR2 having the amino acid sequence shown in SEQ ID NO:2, and CDR3 being The amino acid sequence shown in SEQ ID NO: 3; or (2) having the amino acid sequence shown in SEQ ID NO: 5 for CDR1, the amino acid sequence shown in SEQ ID NO: 6 for CDR2, and the amino acid sequence shown in SEQ ID NO: 7 for CDR3 or (3) having the amino acid sequence shown in SEQ ID NO: 9 for CDR1, the amino acid sequence shown in SEQ ID NO: 10 for CDR2, and the amino acid sequence shown in SEQ ID NO: 11 for CDR3; or (4) CDR1 has the amino acid sequence shown in SEQ ID NO:1, CDR2 has the amino acid sequence shown in SEQ ID NO:2, and CDR3 has the amino acid sequence shown in SEQ ID NO:14.
  • the second aspect of the present invention provides a fusion protein, the fusion protein comprising: a first domain, the first domain is the anti-PD-L1 Nanobody described in the first aspect of the present invention, and a second structural domain domain, and the second domain is a functional protein different from the anti-PD-L1 Nanobody.
  • the third aspect of the present invention provides an antibody conjugate comprising the anti-PD-L1 nanobody described in the first aspect of the present invention or the fusion protein described in the second aspect of the present invention; and The anti-PD-L1 nanobody or a functional small molecule linked to the fusion protein.
  • the fourth aspect of the present invention provides an isolated polynucleotide, which encodes the anti-PD-L1 Nanobody of the first aspect or encodes the fusion protein of the second aspect.
  • the fifth aspect of the present invention provides a construct comprising the isolated polynucleotide described in the fourth aspect above.
  • the sixth aspect of the present invention provides a host cell containing the isolated polynucleotide described in the fourth aspect above or the construct described in the fifth aspect above.
  • the seventh aspect of the present invention provides a method for producing Nanobodies or fusion proteins, comprising: cultivating host cells, the host cells being the host cells described in the sixth aspect of the present invention, and collecting the Nanobodies from the culture. Antibodies or fusion proteins.
  • the eighth aspect of the present invention provides a pharmaceutical composition, comprising the anti-PD-L1 nanobody described in the first aspect above, or the fusion protein described in the second aspect above, or the antibody conjugate described in the third aspect above joint; and a pharmaceutically acceptable carrier.
  • the ninth aspect of the present invention provides a kit, comprising the anti-PD-L1 nanobody described in the first aspect above, or the fusion protein described in the second aspect above, or the antibody conjugate described in the third aspect above things.
  • the tenth aspect of the present invention provides a method for preventing and/or treating diseases, comprising: administering to a subject a preventive and/or therapeutically effective amount of the anti-PD-L1 Nanobody described in the first aspect above, or the second The fusion protein described in the aspect, or the antibody conjugate described in the third aspect.
  • the eleventh aspect of the present invention provides a use of an anti-PD-L1 nanobody or a fusion protein or an antibody conjugate in the preparation of a drug or a kit, and the anti-PD-L1 nanobody is the one described in the first aspect of the present invention
  • the anti-PD-L1 nanobody described above, the fusion protein is the fusion protein described in the second aspect of the present invention, and the antibody conjugate is the antibody conjugate described in the third aspect of the present invention.
  • Fig. 1 is the result of FACS detection of binding activity between antibody and hPD-L1 provided in Example 2 of the present invention.
  • Fig. 2 is the result of ELISA detection antibody and cynomolgus PD-L1 binding activity provided by Example 2 of the present invention.
  • Figure 3 is the results of FACS detection of the blocking activity of antibodies against PD1/PD-L1 provided in Example 3 of the present invention.
  • Fig. 4 is the result of FACS detecting the blocking activity of antibodies against CD80/PD-L1 provided in Example 3 of the present invention.
  • Fig. 5 is the result of blocking the PD-L1/PD-1 pathway by the reporter gene detection antibody provided in Example 4 of the present invention.
  • Fig. 6 is the result of the drug efficacy of the antibody provided in Example 7 of the present invention in mice.
  • the term "antibody” is used in the broadest sense to refer to a protein or polypeptide comprising an antigen-binding site or an antigen-binding fragment or antigen-binding portion, covering natural and artificial antibodies of various structures, including but not limited to intact antibodies forms or antigen-binding fragments of antibodies.
  • the antibody provided is a Nanobody.
  • Nanobodies have the same meaning as the single domain antibody, and refers to an antibody composed of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions.
  • Nanobodies comprise four conserved framework regions (FR1-FR4) and three complementarity determining regions (CDR1-CDR3) mentioned below.
  • the framework region FR1, the framework region FR2, the framework region FR3 and the framework region FR4 are separated by the complementarity determining region CDR1, the complementarity determining region CDR2 and the complementarity determining region CDR3, respectively.
  • the complementarity-determining region is also called “hypervariable region", and the complementarity-determining region determines the binding characteristics with the antigen.
  • Humanized antibody refers to a molecule based on an antigen-binding site derived from a non-human species, and an antibody based on the partial structure and sequence composition of a human immunoglobulin molecule. Compared with non-humanized antibodies, humanized antibodies retain antigen-binding activity while reducing immunogenicity.
  • Nanobodies or fusion proteins mentioned herein are usually isolatable or recombinant.
  • isolated means capable of being identified and isolated and/or recovered from a cell or cell culture expressing a polypeptide or protein.
  • an isolated polypeptide eg, an isolated antibody or an isolated fusion protein
  • isolated antibody means that it is substantially free of other antibodies or antigen-binding fragments of different antigen specificities (such as an isolated antibody that specifically binds PD-L1 is substantially free of antibodies that specifically bind antigens other than PD-L1) .
  • Recombinant means that antibodies can be produced in foreign host cells using genetic recombination techniques.
  • affinity or “binding affinity” mentioned herein is understood according to the common meaning in the art, and is used to reflect the strength and/or stability between the antigen and the binding site on the antibody or antigen-binding fragment.
  • Specifically binds to or “specifically binds to” a particular antigen or epitope, or “has specificity” for a particular antigen or epitope means to distinguish it from non-specific interactions, such specific binding can be determined by commonly used in the art measured by some methods.
  • the ability of an antibody or antigen to bind can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.
  • ELISA enzyme-linked immunosorbent assay
  • the cells carrying the antigen can be detected by flow cytometry, and the competitive binding between the antibody to be tested and the labeled antibody can be detected by measuring the positive rate index of the cells. Since the spatial structure of the antigen on the cell surface is closer to the form existing in the body, this method can better reflect the real situation.
  • the provided Nanobodies have ⁇ 100nM, ⁇ 100nM, ⁇ 50nM, ⁇ 20nM, ⁇ 10nM, ⁇ 5nM, ⁇ 1nM, ⁇ 0.5nM, ⁇ 0.1nM, ⁇ 0.09nM, ⁇ 0.08nM , ⁇ 0.07nM, ⁇ 0.06nM, ⁇ 0.05nM, ⁇ 0.04nM, ⁇ 0.03nM, ⁇ 0.02nM, ⁇ 0.01nM, ⁇ 0.001nM EC50 values.
  • the binding activity of the antibody to the antigen can also be measured by surface isoparticle resonance (SPR) or biofilm interferometry (BLI).
  • amino acid names are indicated by standard one-letter or three-letter codes commonly used in the art.
  • the "homology" of the sequences referred to herein refers to the degree of sequence identity of proteins or polypeptides or nucleic acid sequences. To determine homology, this can be done in various ways known in the art. For example, it can be obtained by using publicly available software such as BLAST, ALIGN, BLAST-2 and other software.
  • a "conservative amino acid substitution" of a sequence referred to herein refers to the replacement of another amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties. For example, conservative amino acid substitutions can be made between amino acid residues with hydrophobic side chains (e.g.
  • the CDR sequences of the antibodies shown in this paper are obtained by combining existing database analysis, for example, by combining the IMGT database (Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6:737-749 (2011)).
  • IMGT database Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38:D301-D307 (2010); Ehrenmann, F., Lefranc, M.-P. Cold Spring Harbor Protoc., 6:737-749 (2011).
  • Kabat for example, you can participate in U.S.Dept.of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)
  • Chothia for example, you can refer to J.Mol.Biol.196:901-917 (1987)
  • the present invention provides an anti-PD-L1 nanobody comprising CDR1, CDR2 and CDR3, wherein (a) CDR1 comprises a sequence selected from the group consisting of SEQ ID NO:1, 5 and 9, or with SEQ ID NO:1 , 5 and 9 sequences, one or two amino acid substitutions, deletions or additions occur; (b) CDR2 comprises a sequence selected from the following group: SEQ ID NO: 2, 6 and 10, or with SEQ ID NO: Compared with 2, 6 and 10, one or two amino acid substitutions, deletions or additions occur; (c) CDR3 comprises a sequence selected from the group consisting of SEQ ID NO: 3, 7, 11 and 14, or with SEQ ID NO: 3, 7, 11 and 14 NO: Compared with sequences 3, 7, 11 and 14, one, two, three or four amino acid substitutions, deletions or additions occurred.
  • CDR1 comprises a sequence selected from the group consisting of SEQ ID NO:1, 5 and 9, or with SEQ ID NO:1 , 5 and 9 sequences, one or two amino acid
  • the mentioned amino acid deletion may be the deletion of two alanines (abbreviated as AA) or one alanine and one threonine (abbreviated as AT) at the N-terminus of the listed CDR3 sequence.
  • the amino acid increase mentioned may be sequentially connected to the C-terminus of the listed CDR1 sequence with methionine (abbreviated as M) and glycine (abbreviated as G), or sequentially connected with methionine (abbreviated as M ) and arginine (abbreviated as R) and so on.
  • the invention provides an anti-PD-L1 nanobody selected from: (1) having CDR1 as the amino acid sequence shown in SEQ ID NO:1, CDR2 as the amino acid sequence shown in SEQ ID NO:2, and CDR3 as SEQ ID The amino acid sequence of NO:3; or (2) having the amino acid sequence shown in SEQ ID NO:5 for CDR1, the amino acid sequence shown in SEQ ID NO:6 for CDR2, and the amino acid sequence shown in SEQ ID NO:7 for CDR3 or (3) having CDR1 as the amino acid sequence shown in SEQ ID NO:9, CDR2 as the amino acid sequence shown in SEQ ID NO:10, and CDR3 as the amino acid sequence shown in SEQ ID NO:11; or (4) having CDR1 is the amino acid sequence shown in SEQ ID NO:1, CDR2 is the amino acid sequence shown in SEQ ID NO:2, and CDR3 is the amino acid sequence shown in SEQ ID NO:14.
  • the provided Nanobody has a sequence with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:1; has a sequence compared to the sequence shown in SEQ ID NO:2 , a sequence with one or two amino acid substitutions, deletions or additions; and a sequence with one, two, three or four amino acid substitutions, deletions or additions compared with the sequence shown in SEQ ID NO:3.
  • the provided Nanobody has a sequence with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:5; has a sequence compared to the sequence shown in SEQ ID NO:6 , a sequence with one or two amino acid substitutions, deletions or additions; and a sequence with one, two, three or four amino acid substitutions, deletions or additions compared with the sequence shown in SEQ ID NO:7.
  • the provided Nanobody has a sequence with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO: 9; has a sequence compared to the sequence shown in SEQ ID NO: 10 , a sequence with one or two amino acid substitutions, deletions or additions; and a sequence with one, two, three or four amino acid substitutions, deletions or additions compared with the sequence shown in SEQ ID NO:11.
  • the provided Nanobody has a sequence with one or two amino acid substitutions, deletions or additions compared to the sequence shown in SEQ ID NO:1; has a sequence compared to the sequence shown in SEQ ID NO:2 , a sequence with one or two amino acid substitutions, deletions or additions; and a sequence with one, two, three or four amino acid substitutions, deletions or additions compared with the sequence shown in SEQ ID NO:14.
  • amino acid sequences of the mentioned CDR regions are shown in Table 1 below:
  • the present invention provides an anti-PD-L1 nanobody, comprising a variable region domain composed of a framework region FR and a complementarity determining region CDR.
  • the provided Nanobodies comprise, in addition to the mentioned CDR sequences, the framework regions FR.
  • the framework region FR includes FR1, FR2, FR3 and FR4, and the framework region FR and CDR sequences are separated to form a Nanobody.
  • FR1 is selected from the amino acid sequence shown in SEQ ID NO:16, or SEQ ID NO:20, or SEQ ID NO:23, or SEQ ID NO:26.
  • FR1 is selected from sequences having at least one, two, three, four or five conservative amino acid substitutions compared to SEQ ID NO: 16, 20, 23, 26.
  • FR2 is selected from the amino acid sequence shown in SEQ ID NO:17, or SEQ ID NO:21, or SEQ ID NO:24.
  • FR2 is selected from sequences having at least one, two, three or four conservative amino acid substitutions compared to SEQ ID NO: 17, 21, 24.
  • FR3 is selected from the amino acid sequence shown in SEQ ID NO:18, or SEQ ID NO:22, or SEQ ID NO:25, or SEQ ID NO:27.
  • FR3 is selected from sequences having at least one, two, three, four, five or six conservative amino acid substitutions compared to SEQ ID NO: 18, 22, 25 or 27.
  • FR4 is selected from the amino acid sequence shown in SEQ ID NO: 19, or compared with the sequence shown in SEQ ID NO: 19, a sequence with one, two or three conservative amino acid substitutions. Conservative amino acid substitutions mentioned in the FR regions are preferentially replaced by human framework region sequences.
  • amino acid sequences of each framework region shown are shown in Table 2 below:
  • provided Nanobodies comprise, in addition to the CDR regions, framework regions FR.
  • the framework region FR is selected from: (1) FR1 is the amino acid sequence shown in SEQ ID NO:16, FR2 is the amino acid sequence shown in SEQ ID NO:17, FR3 is the amino acid sequence shown in SEQ ID NO:18 and FR4 is the amino acid sequence shown in SEQ ID NO:19; or (2) FR1 is the amino acid sequence shown in SEQ ID NO:20, FR2 is the amino acid sequence shown in SEQ ID NO:21, and FR3 is SEQ ID NO:22 The amino acid sequence shown and FR4 are the amino acid sequence shown in SEQ ID NO:19; or (3) FR1 is the amino acid sequence shown in SEQ ID NO:23, FR2 is the amino acid sequence shown in SEQ ID NO:24, FR3 The amino acid sequence shown in SEQ ID NO:25 and FR4 are the amino acid sequence shown in SEQ ID NO:19; or (4) FR1 is the amino acid sequence shown in SEQ ID
  • the present invention provides an anti-PD-L1 nanobody comprising the amino acid sequence shown in SEQ ID NO:4.
  • an anti-PD-L1 Nanobody is provided, comprising the amino acid sequence shown in SEQ ID NO:8.
  • an anti-PD-L1 Nanobody is provided, comprising the amino acid sequence shown in SEQ ID NO:12.
  • an anti-PD-L1 Nanobody is provided, comprising the amino acid sequence shown in SEQ ID NO:13.
  • an anti-PD-L1 Nanobody is provided, comprising the amino acid sequence shown in SEQ ID NO:15.
  • amino acid sequences of the provided anti-PD-L1 Nanobodies are shown in Table 3 below.
  • the anti-PD-L1 nanobody has and SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 15 respectively Compared with the amino acid sequence, the homology is more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequences.
  • the provided anti-PD-L1 nanobody has at least One conservative amino acid substitution, for example having 1 conservative amino acid substitution, 2 conservative amino acid substitutions, 3 conservative amino acid substitutions, 4 conservative amino acid substitutions, 5 conservative amino acid substitutions, 6 conservative amino acid substitutions, 7 conservative amino acid substitutions, 8 conservative amino acid substitutions, Conservative amino acid substitutions, 9 conservative amino acid substitutions or 10 conservative amino acid substitutions.
  • the mentioned conservative amino acid substitutions occur preferentially in the framework regions.
  • the provided anti-PD-L1 Nanobodies can also be humanized Nanobodies.
  • Humanized Nanobodies are less immunogenic while retaining the binding activity of Nanobodies to PD-L1.
  • the provided humanized Nanobodies retain the amino acid sequences of the CDR regions and replace the FR framework regions with humanized framework region sequences.
  • the FR framework region sequences of the provided humanized Nanobodies comprise partially humanized framework region sequences.
  • the mentioned partially humanized framework region sequence can be obtained by replacing the framework region sequence with a humanized framework region sequence, and performing back mutations on key amino acids that affect the structure and function of the antibody.
  • Humanized Nanobodies can be obtained by methods commonly used in the art (eg CDR grafting).
  • Human framework region sequences can be obtained from published human sequences. For example, through database comparison and computer homology modeling, the human FR region template with the greatest homology is found, and the key residues in the FR region that need to be back-mutated are determined comprehensively to obtain high-affinity humanized antibodies. These sequences are usually recorded in commonly used databases, such as PDB protein structure database, IMGT, Genebank and other databases. Humanized nanobodies can be further back-mutated to retain some properties of the original sequence, such as affinity and physiological activity. Most of these back mutations occurred in the FR2 framework region and FR3 framework region. For example, a back mutation occurs at position 2, position 10, position 11, or position 12 of the FR2 region. Another example is a back mutation at position 10, position 12, position 13, position 21, position 29, or position 32 of the FR3 region.
  • the humanized Nanobody is set forth in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.
  • the provided humanized nanobodies have low immunogenicity and high affinity to PD-L1.
  • the present invention also provides an anti-PD-L1 nanobody, selected from: i) having such as SEQ ID NO: 4, or SEQ ID NO: 8, or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 15, or SEQ ID NO: 28, or SEQ ID NO: 29, or SEQ ID NO: 30, or the amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO: 32; or ii) and i)
  • the homology is more than 85%, more than 86%, more than 87%, more than 88%, more than 89%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95% , more than 96%, more than 97%, more than 98%, or more than 99% of the amino acid sequences.
  • the present invention also provides a fusion protein, which includes: a first domain, the first domain is the above-mentioned anti-PD-L1 nanobody; and a second domain, the second structure Domains are functional proteins distinct from anti-PD-L1 Nanobodies.
  • the first domain and the second domain may be connected directly or via a linker.
  • the fusion protein has the characteristics of anti-PD-L1 antibody and the activity of functional protein. It can not only be used to prepare immune targeting drugs, but also can be widely used in immunodiagnosis, antibody purification, and quantitative analysis of antibodies and antigens.
  • linkers are linkers commonly used in the art, which may be some oligopeptides or polypeptides. These oligopeptides or polypeptides can be any amino acid sequence that provides flexibility. ⁇ :GS ⁇ SG ⁇ GGS ⁇ GSG ⁇ SGG ⁇ GGG ⁇ GGGS ⁇ SGGG ⁇ GGGGS ⁇ GGGGGSGS ⁇ GGGGS ⁇ GGGGSGGGGS ⁇ GGGGSGGGGSGGGGGGSGGGGSGGGGGGS ⁇ GGGGSGGGGSGGGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGS ⁇ ; GSGGGSGGGSGGGSGGGSGGGSGGGS ⁇ ; GSGGGSGGGSGGGSGGGSGGGS ⁇ ; ⁇ :
  • the second domain can be linked to the C-terminal (carboxy-terminal) amino acid of the first domain.
  • the second structural domain can be one or more functional proteins.
  • the C-terminal of the first structural domain When the C-terminal of the first structural domain is connected to a functional protein molecule, it can also bind to one or several functional protein molecules as needed, and can pass through the C-terminal An amino acid or an N-terminal amino acid (amino terminal) binds to one of the functional protein molecules.
  • the second domain can be an immunoglobulin Fc region, for example, it can be a human immunoglobulin Fc region.
  • Nanobodies lack the Fc domain and do not have ADCC or CDC activity. Protein fusion of nanobody and immunoglobulin Fc region can enhance the ADCC or CDC function of nanobody and prolong the half-life of the drug in vivo.
  • the second domain can also be a cytokine.
  • Cytokines mentioned include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-14, IL-18, IL-21, IFN- ⁇ , TNF, TGF- ⁇ , GM-CSF, etc., one or more of these cytokines can be connected to the first domain , to form a fusion protein.
  • the cytokine is linked to the C-terminal amino acid of the first domain.
  • the second domain can also be an antigen targeting protein other than PD-L1.
  • Nanobodies are small in size and high in stability, and can easily form bispecific antibodies or multispecific antibodies with other antigen-targeting proteins, such as anti-PD-1, anti-PD-L1, anti-EGFR, anti-c-Met , Anti-TIM-3, Anti-LAG-3, Anti-CD-47, Anti-TIGIT, Anti-CD-39, Anti-CD-73, Anti-41BB, Anti-OX40, Anti-CD-3, Anti-CD-40, Anti-GARP-TGF- ⁇ Complex, anti-TGF- ⁇ , anti-GARP, anti-Her2, anti-Her3, anti-CTLA-4, anti-GARP-TGF- ⁇ complex and other antigen-targeting proteins to prepare bi- and multi-specific antibodies to prepare more antigen-targeting proteins functional antibodies.
  • Nanobodies can be coupled with diagnostic reagents (such as radionuclides or optical tracers, etc.) to achieve specific tumor imaging.
  • Fusion proteins can be prepared by methods commonly used in the art. For example, fusion proteins can be prepared by means of genetic engineering techniques. The gene encoding the first structural domain and the second structural domain are connected through a linker sequence, and the obtained connection product is cloned into a vector, and expressed through a prokaryotic or eukaryotic expression system.
  • the anti-PD-L1 Nanobodies or fusion proteins mentioned herein are substantially pure.
  • the said substantially pure means that the purity of the produced anti-PD-L1 nanobody can reach more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, More than 97%, more than 98%, more than 99%, more than 99.5% and so on.
  • the present invention also provides an antibody conjugate, the antibody conjugate comprising the anti-PD-L1 nanobody or fusion protein; and the anti-PD-L1 nanobody or the fusion protein linked functional small molecules.
  • the mentioned functional small molecules may be developed or undeveloped small molecule drugs.
  • Nanobodies have good solubility and high stability, and are suitable for chemical coupling with small molecule drugs.
  • Nanobodies and small molecule drugs are coupled to form nanobody drug conjugates, which can enhance the therapeutic effect of antitumor drugs and reduce adverse reactions.
  • small molecules such as toxins, chemotherapeutic drugs, and photosensitizers can be coupled to Nanobodies through linkers.
  • antibody Protac conjugates can also be obtained by coupling antibodies with protac molecules, such as linking antibodies and protease targeting ligands (such as E3 ligase ligands) through linkers, to bring the target protein and intracellular
  • protac molecules such as linking antibodies and protease targeting ligands (such as E3 ligase ligands) through linkers.
  • protease targeting ligands such as E3 ligase ligands
  • the distance between E3 ubiquitin ligases utilizes the ubiquitin-proteasome pathway to specifically degrade target proteins.
  • the present invention also provides an isolated polynucleotide, which encodes the above-mentioned anti-PD-L1 nanobody or encodes the fusion protein.
  • An isolated polynucleotide can be DNA, RNA, or cDNA, among others.
  • the polynucleotide sequence encoding anti-PD-L1 nanobody or fusion protein can be obtained according to the routine methods of those skilled in the art.
  • the present invention also provides a construct comprising the isolated polynucleotide mentioned herein.
  • a variety of methods commonly used in the art can be used to obtain constructs, for example, through in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc., for example, polynucleotides can be inserted into the multiple cloning sites of expression vectors to form constructs .
  • the construct may contain various operators such as promoters, terminators, and marker genes as necessary, and these operators are operably linked to the polynucleotide.
  • the promoter is usually used to provide a signal to start transcription.
  • the promoter can choose lactose promoter (Lac), Trp promoter, Tac promoter, phage PL and PR promoter according to the needs; the terminator provides the signal for transcription termination during transcription. Signaling, marker genes on constructs are often used for screening. Of course, there can also be an enhancer as needed to enhance the expression of the protein.
  • the expression vector is not particularly limited, and may be some commercially available expression vectors, or artificially modified expression vectors such as plasmids, phages, viruses, etc.
  • the virus may be a plant cell virus, a mammalian cell virus, or the like.
  • the constructs can express antibodies or proteins in vitro, and can also be transferred into cells to express antibodies or proteins.
  • the present invention also provides a host cell containing the above-mentioned isolated polynucleotide or the above-mentioned construct.
  • Any cell suitable for expression of a polynucleotide or construct for antibody or protein can be used as a host cell.
  • the host cells may be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as yeast cells, mammalian cells, etc. Commonly used host cells can be yeast cells, CHO, HEK-293, COS cells, insect cells of Drosophila S2 or Sf9.
  • Host cells containing polynucleotides or constructs can be obtained by methods commonly used in the art, such as microinjection, electroporation, chemical transfection, virus-mediated transformation, and the like.
  • the present invention also provides a method for producing a nanobody or a fusion protein, comprising: cultivating the host cell mentioned in the present invention; and collecting the nanobody or fusion protein. After the collected Nanobody or fusion protein is purified, a substantially pure Nanobody or fusion protein product can be obtained.
  • the said substantially pure means that the purity of the produced anti-PD-L1 nanobody can reach more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, More than 97%, more than 98%, more than 99%, more than 99.5% and so on.
  • the present invention also provides a pharmaceutical composition, comprising the anti-PD-L1 nanobody, or the fusion protein, or antibody conjugate; and a pharmaceutically acceptable carrier.
  • the mentioned pharmaceutically acceptable carrier is acceptable to the subject at the dose or concentration used.
  • Pharmaceutically acceptable carriers include, but are not limited to: buffers or salts, such as Tris, phosphate, citrate, sodium acetate, citric acid, sodium citrate, and other organic acids; antioxidants, such as ascorbic acid; preservatives For example benzalkonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine.
  • the pharmaceutical preparations used in the body of the subject are generally sterile, and the sterile pharmaceutical preparations can be obtained by using methods commonly used in the art, for example, they can be obtained by filtering through sterile membranes.
  • Those skilled in the art can select appropriate pharmaceutically acceptable carriers according to the prepared dosage form of the pharmaceutical composition to prepare different dosage forms, for example, various dosage forms such as injections, freeze-dried preparations, tablets, etc. can be prepared.
  • the present invention also provides a kit, which includes the above-mentioned Nanobody, or the above-mentioned fusion protein, or antibody conjugate.
  • the kit may also include containers, buffer reagents, controls such as positive controls and negative controls as needed. Those skilled in the art can make corresponding selections as needed.
  • the kit may also include instructions for use, so as to facilitate the operation and use of those skilled in the art.
  • the present invention further provides a method for preventing and/or treating diseases, comprising: administering to a subject a preventive and/or therapeutically effective amount of the above-mentioned anti-PD-L1 nanobody, or fusion protein, or antibody conjugate.
  • references to a “therapeutically effective amount” is capable of resulting in a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or a reduction in the prevention of suffering due to the disease.
  • a “prophylactically effective amount” will generally be lower than a therapeutically effective amount.
  • the provided anti-PD-L1 nanobody has an inhibitory rate of more than 10%, more than 15%, more than 20%, and 25% More than 30%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, even 90% % or more or 95% or more.
  • the subject referred to may be either an animal or a human. For example, it may be mammals, including cattle, sheep, mice, horses and the like.
  • anti-PD-L1 nanobody or fusion protein or antibody conjugate can treat various diseases, especially cancer, including but not limited to non-small cell lung cancer (NSCLC), melanoma, urothelial carcinoma, hepatocellular carcinoma, renal Cell carcinoma, classical Hodgkin's lymphoma, cervical cancer, head and neck squamous cell carcinoma, bladder cancer, lung cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, breast cancer, ovarian cancer, etc.
  • NSCLC non-small cell lung cancer
  • melanoma melanoma
  • urothelial carcinoma hepatocellular carcinoma
  • renal Cell carcinoma classical Hodgkin's lymphoma
  • cervical cancer cervical cancer
  • bladder cancer lung cancer
  • nasopharyngeal cancer gastric cancer
  • esophageal cancer colorectal cancer
  • breast cancer breast cancer
  • ovarian cancer etc.
  • PD-1 antibody CTLA-4 antibody, EGFR antibody, c-Met antibody, CD-40 antibody, CD-3 antibody, CD70 antibody, TIGIT antibody, CD-39 antibody, OX40 antibody, TGF ⁇ antibody, GARP antibody , Her2 antibody, c-Met antibody, LAG-3 antibody, Tim-3 antibody, 4-1BB antibody, etc. in combination to enhance the therapeutic effect on cancer.
  • the provided antibodies can be administered at a therapeutically effective dose of about 0.01 mg/kg to about 100 mg/kg, for example, 0.1 mg/kg to 100 mg/kg, 1 mg/kg to 100 mg/kg, 5 mg/kg to 90 mg/kg, 10 mg/kg kg to 80mg/kg, 10mg/kg to 70mg/kg, 10mg/kg to 60mg/kg, 10mg/kg to 50mg/kg, 10mg/kg to 40mg/kg, 10mg/kg to 35mg/kg, 15mg/kg to 30mg/kg, 20mg/kg to 30mg/kg, 15mg/kg to 25mg/kg.
  • the dosage administered may vary over the course of the treatment.
  • the dosage administered can be varied in light of the subject's response during the course of treatment.
  • the provided anti-PD-L1 nanobody or fusion protein can also be combined with a variety of clinical treatment options, such as chemotherapy, radiotherapy, anti-angiogenic therapy, surgery, etc., to improve the healing effect on tumors.
  • clinical treatment options such as chemotherapy, radiotherapy, anti-angiogenic therapy, surgery, etc.
  • radiotherapy can be used to destroy tumors with a larger burden, combined with immunotherapy to achieve the pursuit and interception of tiny tumors, so that the killing of tumor cells can be maximized.
  • the present invention also provides the use of anti-PD-L1 nanobody or fusion protein or antibody conjugate in the preparation of medicine or kit.
  • the provided anti-PD-L1 nanobody or fusion protein or antibody conjugate can be prepared into a medicine for preventing or treating various diseases. It can also be used to prepare kits and used as reagent components in kits for the diagnosis of specific antigens.
  • the anti-PD-L1 nano library was obtained by alpaca immunization, and screening and identification were carried out at the same time to obtain candidate nanobodies.
  • fusion protein composed of human PD-L1 protein ectodomain sequence and human immunoglobulin Fc region sequence with Freund's adjuvant, emulsify, immunize healthy alpaca, and stimulate B cells to express antigen-specific nanobodies. Then alpaca blood was collected, lymphocytes were separated, total RNA was extracted by Trizol method, cDNA was obtained by reverse transcription, and VHH antibody gene fragment was obtained by PCR amplification from the reverse transcribed cDNA.
  • yeast homologous recombinase will connect the fragments to the vectors to form a complete plasmid, and establish a library of yeast transformants with high insertion rate and excellent diversity, and culture them under auxotrophic conditions. Maintain a stable passage in the base.
  • the yeast cells expressing the antibody were co-incubated with the magnetic beads enriched with the target protein antigen, and after multiple enrichment cultures, the enriched products of the magnetic beads were identified by flow cytometry and sorted. After multiple rounds of screening, five highly expressed positive clones were screened and named Antibody 1 to Antibody 5, respectively.
  • the amino acid sequence of antibody 1 is shown in SEQ ID NO: 4, and analyzed by IMGT/DomainGapAlign tool, its CDR regions are SEQ ID NO: 1, 2 and 3; the amino acid sequence of antibody 2 is shown in SEQ ID NO: 8, its CDR regions are respectively SEQ ID NO: 5, 6 and 7; the amino acid sequence of antibody 3 is shown in SEQ ID NO: 12, and its CDR regions are respectively SEQ ID NO: 9, 10 and 11; antibody 4
  • the amino acid sequence of antibody 5 is shown in SEQ ID NO:13, and its CDR regions are respectively SEQ ID NO:9, 10 and 11; the amino acid sequence of antibody 5 is shown in SEQ ID NO:15, and its CDR regions are respectively SEQ ID NO: 1, 2 and 14.
  • the obtained different antibodies are expressed and purified using a mammalian cell system to obtain antibodies with a purity of at least 90%.
  • the binding activity of different antibodies and different antigens was then determined.
  • Roche's atezolizumab (Tecentriq) was used as a positive control, and the positive controls mentioned in the following examples all used atezolizumab.
  • MDA-MB-231 cells ATCC
  • PD-L1 antigen endogenously expressing PD-L1 antigen were added to 96-well plates at 1 ⁇ 105 cells/well.
  • Antibody samples of different concentrations were then added (initial concentration was 50 nM, 5-fold serial dilution), and incubated at 4°C for 30 minutes.
  • a fluorescently labeled goat anti-human IgG secondary antibody manufactured by the manufacturer's
  • the antibody-antigen binding dose-response curve was generated using geometric values, and the four-parameter raw data was plotted using Graphpad Prism V6.0 software to determine the EC50 value of antibody binding antigen. The results are shown in Table 5.
  • the mean fluorescence intensity (MFI) values obtained as a function of antibody concentration are shown in Figure 1.
  • the experimental results showed that: the binding activity of antibody 1 to antibody 5 at the cell level was similar to that of the positive control, showing high binding activity.
  • Cynomolgus/Rhesus macaque PD-L1-hFc protein (manufacturer: ACRO Biosystems) was coated on a 96-well microtiter plate and incubated overnight at 4°C. Wash with washing buffer, and then add 2% BSA in PBS solution to block for 60 minutes. After washing with washing buffer, different concentrations of the antibody to be tested (initial concentration 100 nM, 4-fold serial dilution) were added, and incubated at 37°C for 120 minutes. Wash with washing buffer, then add HRP-labeled goat anti-human secondary antibody (1:10000 dilution, manufacturer: Jackson), and incubate at 37°C for 60 minutes.
  • Antibody 5 0.034 hIgG1 not combined positive control 0.094
  • antibodies 1 to 5 can bind to Cyno PD-L1, showing high binding activity.
  • 1ug/ml mouse PD-L1-hFc protein (manufacturer: ACRO Biosystems) was coated on a 96-well microtiter plate and incubated overnight at 4°C. Wash with washing buffer, add PBS solution containing 2% BSA to block for 60 minutes. After washing with washing buffer, different concentrations of the antibody to be tested (initial concentration 100 nM, 4-fold serial dilution) were added, and incubated at 37° C. for 120 minutes. Wash with washing buffer, then add HRP-labeled goat anti-human secondary antibody (1:10000 dilution), and incubate at 37°C for 60 minutes. Wash with washing buffer, add 100ul of TMB chromogenic solution to each well, react at room temperature for 10-15 minutes, add 100ul of 2M hydrochloric acid stop solution to stop the reaction, and read the OD450 value with a microplate reader.
  • Example 3 measured the blocking activity of the antibody against PD-1/PD-L1 or the blocking activity against CD-80/PD-L1.
  • the blocking effect of the antibody on PD-1 and its ligand PD-L1 was detected by a method based on competitive flow cytometry. Resuscitate the 293T-PD-1 cell line overexpressing PD-1 (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.), spread the cells in a 96-well plate at 1 ⁇ 10 5 cells/well, and mix different concentrations of Antibody dilution solution (starting working concentration is 200nM, 4-fold dilution) and PD-L1-Biotin dilution solution (2ug/ml, 50ul/well) were mixed and incubated at room temperature for 30 minutes.
  • starting working concentration is 200nM, 4-fold dilution
  • PD-L1-Biotin dilution solution 2ug/ml, 50ul/well
  • the antibody-antigen blocking reaction curve was generated using geometric values, and a four-parameter graph was drawn using Graphpad Prism V6.0 software to determine the IC50 value, as shown in Table 7 below.
  • Figure 3 shows the mean fluorescence intensity (MFI) results obtained as a function of antibody concentration.
  • the blocking effect of the antibody on PD-L1 and CD-80 was detected by a method based on competitive flow cytometry. Resuscitate the 293T-PD-L1 cell line overexpressing PD-L1 (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.), spread the cells in a 96-well plate at 1 ⁇ 10 5 per well, and mix different concentrations of Antibody diluent (initial working concentration: 200nM, 4-fold dilution) was added to the cells, and incubated at 4°C for 60 minutes. After incubation, wash with PBS containing 2% FBS, centrifuge at 1200 rpm for 4 minutes, and discard the supernatant.
  • Antibody diluent initial working concentration: 200nM, 4-fold dilution
  • the antibody-antigen blocking reaction curve was generated using geometric values, and a four-parameter graph was drawn using Graphpad Prism V6.0 software to determine the IC50 value, as shown in Table 8 below.
  • Figure 4 shows the mean fluorescence intensity (MFI) results obtained as a function of antibody concentration.
  • Example 4 Study antibody blocking of PD-1/PD-L1 pathway by reporter gene experiment.
  • the experiment consists of two genetically engineered cell lines: Jurkat-PD1-CD3zeta-NFAT-Luc2 is the effector cell (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.), which stably expresses PD-1ECD and CD in Jurkat cells
  • the fusion protein composed of -3zeta is inserted with the luciferase reporter gene driven by T cell activation factor (NFAT); 293T-hPD-L1 cells (manufacturer: Kangyuan Biotech (Beijing) Co., Ltd.), 293T cells expressing human PD-L1.
  • the interaction of PD-1/PD-L1 acts as the first signal, and the intracellular CD3zeta chain acts as the second signal to transmit the activation signal inwardly, and the NFAT-driven luciferase reporter gene is expressed and emits fluorescence ;
  • the combination of PD-1/PD-L1 is blocked, the transmission of activation signal is inhibited, and the luciferase reporter gene cannot be expressed.
  • 293T-hPD-L1 cells were plated in 96-well plates at 2 ⁇ 10 4 cells/well, and Jurkat-PD1-CD3zeta-NFAT-Luc2 effector cells were added to 96-well plates at 2 ⁇ 10 4 cells/well; Then different concentrations of the antibody to be tested (initial concentration 60ug/ml, 4-fold serial dilution) were added and incubated overnight at 37°C. Add 100 ul luciferase substrate ONE-Glo TM Luciferase Assay system detection reagent (Promega) to each well, and incubate for 5 minutes in the dark. A microplate reader reads the fluorescent signal in the 96-well plate.
  • the CDR-Grafting Method is used to obtain humanized antibodies. This method divides the amino acid sequences of the antibody and the template into FR regions and CDR regions, and then grafts the CDR regions to the FR regions of the template, so that The CDR region that determines the specificity of the antibody and the framework region of the human antibody are combined to achieve the purpose of humanization. At the same time, in order to avoid the decrease of antibody affinity, the key amino acid residue positions are calculated by computer simulation and other technologies, and the method of reverse mutation is used to ensure the affinity of humanized antibodies.
  • the humanized sequence number of Antibody 1 was Antibody 1hu, and the amino acid sequence was shown in SEQ ID NO:28.
  • the humanized sequence number of Antibody 2 is Antibody 2hu, and the amino acid sequence is shown in SEQ ID NO:29.
  • the humanized sequence number of Antibody 3 is Antibody 3hu, and the amino acid sequence is shown in SEQ ID NO:30.
  • a humanized sequence of antibody 4 is numbered as antibody 4hu1, and its amino acid sequence is shown in SEQ ID NO:31; another sequence of humanized antibody 4 is numbered as antibody 4hu2, and its amino acid sequence is shown in SEQ ID NO:32.
  • Example 6 the activity of the humanized antibody prepared in Example 5 was determined.
  • the binding activity of the humanized antibody to PD-L1 was detected by flow cytometry.
  • the MC38-hPD-L1 cell line overexpressing human PD-L1 (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.) was revived, and the cells were spread in a 96-well plate at 1 ⁇ 10 5 per well.
  • Different concentrations of the antibody to be tested were added (the initial concentration was 100 nM, and a 5-fold serial dilution was performed), and incubated at 4° C. for 30 minutes.
  • a fluorescently labeled goat anti-human IgG secondary antibody (manufacturer: abcam, catalog number: 98596) was added to detect the antibody bound to the cell surface.
  • the geometric values were used to generate antibody-antigen binding dose-response curves, and the original data of four parameters were drawn using Graphpad Prism V6.0 software to determine the EC50 value of antibody binding antigen, as shown in Table 10
  • the experimental results showed that the humanized antibody showed good binding activity to the antigen.
  • the humanized antibody was tested to block the binding effect of PD-L1 and its ligand PD-1.
  • 293T-hPD1 cells were plated in 96-well plates at 1 ⁇ 10 5 per well, and different concentrations of the antibody to be tested (the initial working concentration was 200nM, diluted 4 times) and PD-L1-Biotin dilution (2ug/ml ,50ul/well) mixed and incubated at room temperature for 30 minutes. Then the mixture was added to the cells at 100ul per well, mixed evenly, and incubated at 4°C for 60 minutes, and the blocking activity of the antibody was detected with a PE fluorescent-labeled streptavidin flow-type antibody. Measure its mean fluorescence value. Next, the antibody-antigen blocking reaction curve was generated using geometric values, and a four-parameter graph was drawn using Graphpad Prism V6.0 software to determine the IC50 value, as shown in Table 11 below.
  • the experimental results show that the humanized antibody can block the binding activity of PD-1/PD-L1.
  • the humanized antibody can better block the binding of the antigen to CD-80.
  • mice When the tumor volume reached 75-100mm 3 , the mice were divided into groups, 6 mice in each group, and given different drug treatments. They were divided into antibody 1 experimental group, antibody 2 experimental group, antibody 3 experimental group, antibody 4 experimental group, antibody 5 experimental group and vehicle group (PBS); correspondingly, mice in each treatment group were given different drugs.
  • the dosage is 3 mg/kg
  • the volume of administration is 5 ⁇ L/g
  • the route of administration is intraperitoneal injection (ip)
  • the frequency of administration is twice a week (BIW). Tumor and body weight changes of the mice were recorded.
  • the results shown in FIG. 6 show the effects of the antibody 1 to antibody 5 experimental groups and the vehicle group on the tumor volume of mice.
  • plural means at least two, such as two, three, etc., unless otherwise specifically defined.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

提供了一种抗PD-L1纳米抗体及其应用。抗PD-L1纳米抗体包含CDR1、CDR2和CDR3，CDR1包含选自SEQ ID NO:1、5和9的序列，CDR2包含选自SEQ ID NO:2、6和10的序列，CDR3包含选自SEQ ID NO:3、7、11和14的序列。该抗体能够阻断PD-1/PD-L1、CD-80/PD-L1信号通路，抑制肿瘤细胞的生长，表现出肿瘤治疗作用。

Description

抗PD-L1纳米抗体及其应用
相关申请的交叉引用
本发明要求于2021年08月06日递交的申请号为202110903293.4中国专利申请的优先权，以及于2021年09月30日递交的申请号为202111162303.X的中国专利申请的优先权，并将其全部或者部分引用在本文中。
技术领域
本发明涉及生物医药领域，具体涉及一种抗PD-L1纳米抗体及其应用。
背景技术
作为抗肿瘤免疫的负调节因子，程序性死亡受体配体(programmed death ligand 1，PD-L1)发挥着关键的作用，其能够结合程序性死亡受体1(Programmed Death-1，PD-1)，通过抑制T细胞活化来诱导肿瘤特异性T细胞凋亡，在肿瘤、慢性炎症等病理情况下起到免疫逃逸、免疫抑制等作用。PD-L1通过阻断PD-1/PD-L1的相互作用，可以恢复和增强T细胞的功能，从而可以发挥肿瘤治疗以及免疫调节的作用。同时，还可以通过阻断CD80和PD-L1的共抑制功能，有利于全面激活T细胞功能和细胞因子产生。随着研究的进展，针对PD-L1的单克隆抗体，例如Atezolizumab(阿替利珠单抗，商品名为Tecentriq)、Durvalumab(度伐利尤单抗)、Avelumab(阿维单抗，商品名为Bavencio)等多种抗体被应用于治疗多种类型的实体瘤中，例如非小细胞肺癌、黑色素瘤、淋巴癌、头颈癌和胃癌等等，并表现出良好的肿瘤治疗效果。
单克隆抗体作为免疫检查点抑制剂，已经成为治疗多种癌症的有效手段。然而，传统的单克隆抗体分子量通常比较大(大约在150kDa左右)，其给药后组织渗透性相对较差，且具有比较高的免疫原性，限制了其对于目标肿瘤的靶向功能。纳米抗体是源自于骆驼科的仅含有重链的抗原结合片段，其能够在没有轻链的情况下发挥作用。纳米抗体的分子量比较小，通常在12～16KDa，同时还保留了与抗原的结合活性，而且其不需要链内二硫键，逐渐在免疫治疗中崭露头角。纳米抗体容易在多种不同的表达体系中表达，例如可以通过原核表达系统(例如在大肠杆菌)、真核表达系统(例如酵母)及哺乳动物细胞表达系统(例如CHO、HEK-293细胞)等表达，并易于纯化。2018年9月全球首个纳米抗体Caplacizumab在欧盟获批上市，用于治疗成人获得性血栓性血小板减少性紫癫(aTTP)患者。纳米抗体在癌症、自身免疫病、呼吸系统疾病、血液系统疾病等多个方面表现出独特的开发应用价值，也有越来越多的医药企业着眼于纳米抗体的开发。
然而纳米抗体不像传统的单克隆抗体的开发更为普遍，筛选高亲和力、高特异性且具有产业化前景的纳米抗体尚存在困难，使得纳米抗体的研究和开发受到了一定的限制。
发明内容
本发明提出了一种抗PD-L1纳米抗体及其在疾病治疗中的用途。本发明提供了抗PD-L1纳米抗体、包含PD-L1纳米抗体的融合蛋白以及该纳米抗体或者融合蛋白在疾病治疗，尤其是在癌症治疗中的用途。
本发明的第一方面提供了一种抗PD-L1纳米抗体，包含CDR1、CDR2和CDR3，其中(a)CDR1包含选自下组的序列：SEQ ID NO:1、5和9，或者与SEQ ID NO:1、5和9相比，具有一个或者两个氨基酸取代、缺失或者增加的序列；(b)CDR2包含选自下组的序列：SEQ ID NO:2、6和10，或者与SEQ ID NO:2、6和10相比，具有一个或者两个氨基酸取代、缺失或者增加的序列；(c)CDR3包含选自下组的序列：SEQ ID NO:3、7、11和14，或者与SEQ ID NO:3、7、11和14相比，具有一个、两个或者三个氨基酸取代、缺失 或者增加的序列。
本发明还提供了一种抗PD-L1纳米抗体，选自：i)具有如SEQ ID NO:4、或SEQ ID NO:8、或SEQ ID NO:12、或SEQ ID NO:13、或SEQ ID NO:15、或SEQ ID NO:28、或SEQ ID NO:29、或SEQ ID NO:30、或SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列；或者ii)与i)相比，同源性在85％以上、86％以上、87％以上、88％以上、89％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、或者99％以上的氨基酸序列。
根据本发明的实施例，所提供的抗PD-L1纳米抗体包括：(1)具有CDR1为SEQ ID NO:1所示的氨基酸序列、CDR2为SEQ ID NO:2所示的氨基酸序列、CDR3为SEQ ID NO:3所示的氨基酸序列；或者(2)具有CDR1为SEQ ID NO:5所示的氨基酸序列、CDR2为SEQ ID NO:6所示的氨基酸序列、CDR3为SEQ ID NO:7所示的氨基酸序列；或者(3)具有CDR1为SEQ ID NO:9所示的氨基酸序列、CDR2为SEQ ID NO:10所示的氨基酸序列、CDR3为SEQ ID NO:11所示的氨基酸序列；或者(4)具有CDR1为SEQ ID NO:1所示的氨基酸序列、CDR2为SEQ ID NO:2所示的氨基酸序列、CDR3为SEQ ID NO:14所示的氨基酸序列。
本发明的第二方面提供了一种融合蛋白，所述融合蛋白包括：第一结构域，所述第一结构域为本发明第一方面所述的抗PD-L1纳米抗体，以及第二结构域，所述第二结构域为不同于抗PD-L1纳米抗体的功能蛋白。
本发明的第三方面提供了一种抗体偶联物，所述抗体偶联物包括本发明第一方面所述的抗PD-L1纳米抗体或者本发明第二方面所述的融合蛋白；以及与所述抗PD-L1纳米抗体或者与所述融合蛋白连接的功能小分子。
本发明的第四方面提供了一种分离的多核苷酸，所述分离的多核苷酸编码第一方面所述的抗PD-L1纳米抗体或者编码第二方面所述的融合蛋白。
本发明的第五方面提供了一种构建体，所述构建体包括上述第四方面所述的分离的多核苷酸。
本发明的第六方面提供了一种宿主细胞，所述宿主细胞含有上述第四方面所述的分离的多核苷酸或者上述第五方面所述的构建体。
本发明的第七方面提供了一种生产纳米抗体或者融合蛋白的方法，包括：培养宿主细胞，所述宿主细胞为本发明第六方面所述的宿主细胞，以及从培养物中收集所述纳米抗体或者融合蛋白。
本发明的第八方面提供了一种药物组合物，包括上述第一方面所述的抗PD-L1纳米抗体、或上述第二方面所述的融合蛋白、或上述第三方面所述的抗体偶联物；以及药学上可接受的载体。
本发明的第九方面提供了一种试剂盒，包括上述第一方面所述的抗PD-L1纳米抗体、或上述第二方面所述的融合蛋白、或上述第三方面所述的抗体偶联物。
本发明的第十方面提供了一种预防和/或治疗疾病的方法，包括：给予受试者预防和/或治疗有效量的上述第一方面所述的抗PD-L1纳米抗体，或第二方面所述的融合蛋白，或第三方面所述的抗体偶联物。
本发明的第十一方面提供了一种抗PD-L1纳米抗体或者融合蛋白或抗体偶联物在制备药物或者试剂盒中的用途，所述抗PD-L1纳米抗体为本发明第一方面所述的抗PD-L1纳米抗体，所述融合蛋白为本发明第二方面所述的融合蛋白，所述抗体偶联物为本发明第三方面所述的抗体偶联物。
附图说明
图1为本发明的实施例2提供的FACS检测抗体和hPD-L1的结合活性结果。
图2为本发明的实施例2提供的ELISA检测抗体和食蟹猴PD-L1的结合活性结果。
图3为本发明的实施例3提供的FACS检测抗体对于PD1/PD-L1的阻断活性结果。
图4为本发明的实施例3提供的FACS检测抗体对于CD80/PD-L1的阻断活性结果。
图5为本发明的实施例4提供的报告基因检测抗体对于PD-L1/PD-1通路的阻断结果。
图6为本发明的实施例7提供的抗体在小鼠体内的药效结果。
具体实施方式
下面对本文中的一些术语进行解释和说明，这些解释和说明仅用于方便本领域技术人员理解，不应看作是对本发明保护范围的限制。
本文中，术语“抗体”作最广泛意义使用，指包含抗原结合位点或抗原结合片段或抗原结合部分的蛋白质或多肽，涵盖各种结构的天然抗体和人工抗体，包括但不限于完整的抗体形式或抗体的抗原结合片段。根据具体实施方式，所提供到的抗体为纳米抗体。
本文中，所提到的纳米抗体与单域抗体具有相同的含义，是指仅由一个重链可变区组成的抗体，它是具有完整功能的最小的抗原结合片段。纳米抗体包括下文所提到的四个保守的框架区(FR1-FR4)和三个互补决定区(CDR1-CDR3)。框架区FR1、框架区FR2、框架区FR3和框架区FR4，分别被互补决定区CDR1、互补决定区CDR2和互补决定区CDR3隔开。其中互补决定区也称为“高变区”，互补决定区决定了与抗原的结合特性。
“人源化抗体”是指基于来源于非人物种的抗原结合位点的分子，和基于人的免疫球蛋白分子的部分结构和序列组成的抗体。人源化抗体相较于非人源化的抗体，保留了与抗原的结合活性，同时降低了免疫原性。
本文中所提到的纳米抗体或者融合蛋白，通常是可分离的或者重组的。“可分离的”是指能够从表达多肽或者蛋白的细胞或者细胞培养物中鉴定并且分离和/或回收。通常，可以通过至少一个纯化步骤来制备分离的多肽(例如分离的抗体或者分离的融合蛋白)。“分离的抗体”是指基本上不含有不同抗原特异性的其他抗体或抗原结合片段(如特异性结合PD-L1的分离抗体基本上不含有特异性结合PD-L1之外的抗原的抗体)。“重组”意味着可以使用基因重组技术在外源宿主细胞中产生抗体。
本文中，“包含”或者“包括”意指包括所提到的要素或者步骤，但是不排除其他要素或者步骤。当然，除非另有说明，包含或者包括也涵盖了由所提及的要素或者步骤组成的情形。例如，当提及包含某个具体序列时，也旨在涵盖由该具体序列组成的抗体。
本文所提到的“亲和力”或者“结合亲和力”按照本领域的通常含义来理解，用来反映抗原和抗体或者抗原结合片段上的结合位点之间的强度和/或稳定性。
“特异性结合”或“特异性结合于”特定抗原或表位，或对特定抗原或表位“具有特异性”意味着与非特异性相互作用相区分，这种特异性结合可以通过本领域常用的一些方法测得。抗体或抗原结合的能力可以通过酶联免疫吸附测定(ELISA)或者本领域技术人员熟悉的其他技术来测量。例如可以通过流式细胞仪测定对携带有抗原的细胞进行检测，通过测定细胞的阳性率指标来检测待测抗体与标记抗体的竞争结合情况。由于细胞表面的抗原空间结构更接近于体内存在的形式，所以通过该方法更能反映真实的情况。结合本发明的具体实施方式，所提供的纳米抗体具有≤100nM，≤100nM，≤50nM，≤20nM，≤10nM，≤5nM，≤1nM，≤0.5nM，≤0.1nM，≤0.09nM，≤0.08nM，≤0.07nM，≤0.06nM，≤0.05nM，≤0.04nM，≤0.03nM，≤0.02nM，≤0.01nM，≤0.001nM的EC50值。除此之外，还可以通过表面等粒子共振技术(SPR)或生物薄膜干涉技术(BLI)测定抗体与抗原的结合活性。
本文所用到的氨基酸的名称以本领域通用的标准的单字母或者三字母代码表示。本文中所提到的序列的“同源性”是指蛋白质或者多肽或者核酸序列比较，序列相同的程度。为了确定同源性，可以通过本领域已知的多种方式实现。例如可以使用公开的可以获得的软件如BLAST、ALIGN、BLAST-2等软件获得。本文所提到的序列的“保 守氨基酸取代”是指用具有相似生理化学特性的侧链的不同氨基酸残基置换另一氨基酸残基。例如可以在具有疏水性侧链的氨基酸残基之间(例如Met、Ala、Val、Leu和Ile)发生保守氨基酸取代，在具有中性亲水性侧链的残基之间(例如Cys、Ser、Thr、Asn和Gln)发生保守氨基酸取代，在具有酸性侧链的残基之间(例如Asp、Glu)发生保守氨基酸取代，在具有碱性侧链的氨基酸之间(例如His、Lys和Arg)发生保守氨基酸取代，或者在具有芳香族侧链的残基之间(例如Trp、Tyr和Phe)进行保守氨基酸取代。如本领域已知，保守氨基酸取代通常不会引起蛋白质构象结构的显著变化，因此可以保留蛋白质的生物活性。
本文中所示出的抗体的CDR序列示结合已有的数据库分析获得的，例如是结合IMGT数据库(Ehrenmann F.,Kaas Q.and Lefranc M.-P.Nucleic Acids Res.,38:D301-D307(2010)；Ehrenmann,F.,Lefranc,M.-P.Cold Spring Harbor Protoc.,6:737-749(2011))获得的。当然还可以通过Kabat(例如可以参加U.S.Dept.of Health and Human Servies,“Sequences of Proteins of Immunological Interest”(1983))、Chothia(例如可以参见J.Mol.Biol.196:901-917(1987))等获得。本领域技术人员可知的是，由于数据库分析方法的差异所带来的CDR序列的差异，也都包含在本发明的保护范围之内。
抗PD-L1纳米抗体
本发明提供了一种抗PD-L1纳米抗体，包含CDR1、CDR2和CDR3，其中(a)CDR1包含选自下组的序列：SEQ ID NO:1、5和9，或者与SEQ ID NO:1、5和9序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；(b)CDR2包含选自下组的序列：SEQ ID NO:2、6和10，或者与SEQ ID NO:2、6和10相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；(c)CDR3包含选自下组的序列：SEQ ID NO:3、7、11和14，或者与SEQ ID NO:3、7、11和14序列相比，发生一个、两个、三个或者四个氨基酸取代、缺失或者增加的序列。例如，所提到的氨基酸缺失可以是在所列出的CDR3序列的N端缺失两个丙氨酸(缩写为AA)或者一个丙氨酸和一个苏氨酸(缩写为AT)。例如，所提到的氨基酸增加可以是在所列出的CDR1序列的C端依次连接有甲硫氨酸(缩写为M)和甘氨酸(缩写为G)，或者依次连接有甲硫氨酸(M)和精氨酸(缩写为R)等等。
本发明提供了一种抗PD-L1纳米抗体，选自：(1)具有CDR1为SEQ ID NO:1所示的氨基酸序列、CDR2为SEQ ID NO:2所示的氨基酸序列、CDR3为SEQ ID NO:3的氨基酸序列；或者(2)具有CDR1为SEQ ID NO:5所示的氨基酸序列、CDR2为SEQ ID NO:6所示的氨基酸序列、CDR3为SEQ ID NO:7所示的氨基酸序列；或者(3)具有CDR1为SEQ ID NO:9所示的氨基酸序列、CDR2为SEQ ID NO:10所示的氨基酸序列、CDR3为SEQ ID NO:11所示的氨基酸序列；或者(4)具有CDR1为SEQ ID NO:1所示的氨基酸序列、CDR2为SEQ ID NO:2所示的氨基酸序列、CDR3为SEQ ID NO:14所示的氨基酸序列。在一些实施方式中，所提供的纳米抗体具有与SEQ ID NO:1所示序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；具有与SEQ ID NO:2所示序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；和具有与SEQ ID NO:3所示序列相比，发生一个、两个、三个或者四个氨基酸取代、缺失或者增加的序列。在一些实施方式中，所提供的纳米抗体具有与SEQ ID NO:5所示序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；具有与SEQ ID NO:6所示序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；和具有与SEQ ID NO:7所示序列相比，发生一个、两个、三个或者四个氨基酸取代、缺失或者增加的序列。在一些实施方式中，所提供的纳米抗体具有与SEQ ID NO:9所示序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；具有与SEQ ID NO:10所示序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；和具有与SEQ ID NO:11所示序列相比，发生一个、两个、三个或者四个氨基酸取代、缺失或者增加的序列。在一些实施方式中，所提供的纳米抗体具有与SEQ ID NO:1所示序列相比，发生一个或者 两个氨基酸取代、缺失或者增加的序列；具有与SEQ ID NO:2所示序列相比，发生一个或者两个氨基酸取代、缺失或者增加的序列；和具有与SEQ ID NO:14所示序列相比，发生一个、两个、三个或者四个氨基酸取代、缺失或者增加的序列。
所提到的CDR区的氨基酸序列分别如下表1所示：
表1不同抗体的CDR序列
序列编号 氨基酸序列
SEQ ID NO:1 GRSFSSSG
SEQ ID NO:2 INSSGGDT
SEQ ID NO:3 AAKEGGGPSSIPAIYDY
SEQ ID NO:5 GFTFSSYA
SEQ ID NO:6 INTGSEIV
SEQ ID NO:7 ATGLVSAEHDGI
SEQ ID NO:9 GRDFLTYG
SEQ ID NO:10 INWSGSMT
SEQ ID NO:11 AARRGAVTYASSNEYEH
SEQ ID NO:14 AAKEGGGPSSIPAIFDY
本发明提供了一种抗PD-L1纳米抗体，包括框架区FR和互补决定区CDR组成的可变区结构域。所提供的纳米抗体除了所提到的CDR序列，还包括框架区FR。框架区FR包括FR1、FR2、FR3和FR4，框架区FR和CDR序列间隔构成纳米抗体。在至少一些实施方式中，FR1选自SEQ ID NO:16、或SEQ ID NO:20、或SEQ ID NO:23、或SEQ ID NO:26所示的氨基酸序列。在一些实施方式中，FR1选自与SEQ ID NO:16、20、23、26相比，至少具有一个、两个、三个、四个或者五个保守氨基酸取代的序列。在至少一些实施方式中，FR2选自SEQ ID NO:17、或SEQ ID NO:21、或SEQ ID NO:24所示的氨基酸序列。在一些实施方式中，FR2选自与SEQ ID NO:17、21、24相比，至少具有一个、两个、三个或者四个保守氨基酸取代的序列。在至少一些实施方式中，FR3选自SEQ ID NO:18、或SEQ ID NO:22、或SEQ ID NO:25、或SEQ ID NO:27所示的氨基酸序列。在一些实施方式中，FR3选自与SEQ ID NO:18、22、25或者27相比，至少具有一个、两个、三个、四个、五个或者六个保守氨基酸取代的序列。在至少一些实施方式中，FR4选自SEQ ID NO:19所示的氨基酸序列，或者与SEQ ID NO:19所示序列相比，具有一个、两个或者三个保守氨基酸取代的序列。FR区所提到的保守氨基酸取代，以取代为人源框架区序列为优先。
所示出的各框架区氨基酸序列分别如下表2所示：
表2框架区序列
序列编号 氨基酸序列
SEQ ID NO:16 QVQLVESGGGLVQAGGSLRLSCAAS
SEQ ID NO:17 MGWFRQAPGKDREFVAT
SEQ ID NO:18 YYRDSVEGRFTTSRDNAKNTMSLQMNDLKPEDTAVYYC
SEQ ID NO:19 WGQGTQVTVSS
SEQ ID NO:20 QVQLVESGGGLVQPGGSLSLSCVAS
SEQ ID NO:21 MRWVRQAPGKEPEWVAG
SEQ ID NO:22 VYANSVRGRFAISRDNARDTLFLQMNSLKPEDTAVYYC
SEQ ID NO:23 EVQLVESGGGSVQTGGSLRLSCVAS
SEQ ID NO:24 MGWFRQAPGKEREFVAS
SEQ ID NO:25 YYADSVKGRFTISRDNAKNTVYLQMNSLKPGDTAVYIC
SEQ ID NO:26 EVQLVESGGGLVQAGDSLRLSCTAS
SEQ ID NO:27 YYADSVEGRFTTSRDNAKNTMSLQMNDLKPEDTAVYYC
在至少一些实施方式中，所提供的纳米抗体除了CDR区之外，还包括框架区FR。所述框架区FR选自：(1)FR1为SEQ ID NO:16所示的氨基酸序列、FR2为SEQ ID NO:17所示的氨基酸序列、FR3为SEQ ID NO:18所示的氨基酸序列和FR4为SEQ ID NO:19所示 的氨基酸序列；或者(2)FR1为SEQ ID NO:20所示的氨基酸序列、FR2为SEQ ID NO:21所示的氨基酸序列、FR3为SEQ ID NO:22所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列；或者(3)FR1为SEQ ID NO:23所示的氨基酸序列、FR2为SEQ ID NO:24所示的氨基酸序列、FR3为SEQ ID NO:25所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列；或者(4)FR1为SEQ ID NO:26所示的氨基酸序列、FR2为SEQ ID NO:24所示的氨基酸序列、FR3为SEQ ID NO:25所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列；或者(5)FR1为SEQ ID NO:16所示的氨基酸序列、FR2为SEQ ID NO:17所示的氨基酸序列、FR3为SEQ ID NO:27所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列。
在至少一些实施方式中，本发明提供了一种抗PD-L1纳米抗体，包括SEQ ID NO:4所示的氨基酸序列。在至少一些实施方式中，提供了一种抗PD-L1纳米抗体，包括SEQ ID NO:8所示的氨基酸序列。在至少一些实施方式中，提供了一种抗PD-L1纳米抗体，包括SEQ ID NO:12所示的氨基酸序列。在至少一些实施方式中，提供了一种抗PD-L1纳米抗体，包括SEQ ID NO:13所示的氨基酸序列。在至少一些实施方式中，提供了一种抗PD-L1纳米抗体，包括SEQ ID NO:15所示的氨基酸序列。
所提供的抗PD-L1纳米抗体的氨基酸序列如下表3所示。
表3氨基酸序列
Figure PCTCN2022110423-appb-000001
在至少一些实施方式中，所述抗PD-L1纳米抗体分别具有和SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:13、或者SEQ ID NO:15所示的氨基酸序列相比，同源性在85％以上、86％以上、87％以上、88％以上、89％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、或者99％以上的氨基酸序列。所提供的抗PD-L1纳米抗体和SEQ ID NO:4、SEQ ID NO:8、SEQ ID NO:12、SEQ ID NO:13、或者SEQ ID NO:15所示的氨基酸序列相比，具有至少一个保守氨基酸取代，例如具有1个保守氨基酸取代、2个保守氨基酸取代、3个保守氨基酸取代、4个保守氨基酸取代、5个保守氨基酸取代、6个保守氨基酸取代、7个保守氨基酸取代、8个保守氨基酸取代、9个保守氨基酸取代或者10个保守氨基酸取代。所提到的保守氨基酸取代优先发生在框架区。
所提供的抗PD-L1纳米抗体还可以是人源化的纳米抗体。人源化的纳米抗体的免疫原性更低，同时保留了纳米抗体与PD-L1的结合活性。在至少一些实施方式中，所提供的人源化的纳米抗体保留了CDR区氨基酸序列，在FR框架区替换为人源化的框架区序列。在至少一些实施方式中，所提供的人源化的纳米抗体的FR框架区序列含有部分人源化的框 架区序列。根据具体实施方式，所提到的部分人源化的框架区序列可以通过框架区序列替换为人源化的框架区序列后，对于影响抗体结构和功能的关键氨基酸进行回复突变获得。可以通过本领域常用的方法(例如互补决定区移植法)获得人源化的纳米抗体。人源的框架区序列可以通过已经发表的人源的序列获得。例如通过数据库比对和计算机同源建模，寻找出具有最大同源性的人的FR区模板，综合考虑确定FR区需要进行回复突变的关键残基，获得高亲和力的人源化抗体。这些序列通常记载在常用的数据库中，例如PDB蛋白质结构数据库、IMGT、Genebank等数据库。人源化的纳米抗体可以进一步进行回复突变，保留原始序列的一些性质，例如亲和力和生理活性等。这些回复突变多发生在FR2框架区和FR3框架区。例如在FR2区域的第2位、第10位、第11位、或第12位等发生回复突变。再例如在FR3区域的第10位、第12位、第13位、第21位、第29位、或第32位等发生回复突变。
在至少一些实施方式中，人源化的纳米抗体如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31或SEQ ID NO:32所示。所提供的人源化的纳米抗体具有低的免疫原性，而且和PD-L1具有高亲和力。
表4人源化氨基酸序列
Figure PCTCN2022110423-appb-000002
本发明还提供了一种抗PD-L1纳米抗体，选自：i)具有如SEQ ID NO:4、或SEQ ID NO:8、或SEQ ID NO:12、或SEQ ID NO:13、或SEQ ID NO:15、或SEQ ID NO:28、或SEQ ID NO:29、或SEQ ID NO:30、或SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列；或者ii)与i)相比，同源性在85％以上、86％以上、87％以上、88％以上、89％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、或者99％以上的氨基酸序列。
融合蛋白
本发明还提供了一种融合蛋白，所述融合蛋白包括：第一结构域，所述第一结构域为上述所述的抗PD-L1纳米抗体；以及第二结构域，所述第二结构域为不同于抗PD-L1纳米抗体的功能蛋白。第一结构域和第二结构域可以直接相连，也可以通过连接子连接。融合蛋白具有抗PD-L1的抗体特性以及功能蛋白的活性，不仅可以用来制备免疫靶向药物，还可以广泛应用于免疫诊断、抗体纯化及抗体和抗原的定量分析等。
本文中，可用的连接子为本领域常用的连接子，可以为一些寡肽或者多肽。这些寡肽 或者多肽可以是任何能够提供柔性的氨基酸序列。连接子包括但不限于以下组成的组：GS、SG、GGS、GSG、SGG、GGG、GGGS、SGGG、GGGGS、GGGGGSGS、GGGGSGS、GGGGSGGS、GGGGSGGGGSGGGGS、GGGGSGGGGS、GGGGSGGGGSGGGGSGGGGS、GGGGSGGGGSGGGGSGGGGSGGGGS、GGGGSGGGGSGGGGSGGGGSGGGSGGGS等等。
在至少一些实施方式中，第二结构域可以和第一结构域的C端(羧基端)氨基酸连接。第二结构域可以是一种或者多种功能蛋白，当第一结构域的C端连接上一种功能蛋白分子之后，还可以根据需要结合一种或者几种功能蛋白分子，并可以通过C端氨基酸或者N端氨基酸(氨基端)与其中的一种功能蛋白分子结合。
所述第二结构域可以为免疫球蛋白Fc区，例如可以为人免疫球蛋白Fc区。纳米抗体缺少Fc结构域，不具有ADCC或者CDC活性。将纳米抗体和免疫球蛋白Fc区进行蛋白融合，可以增强纳米抗体的ADCC或者CDC功能，并可以延长药物在体内的半衰期。第二结构域还可以为细胞因子。所提到的细胞因子包括但不限于IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、IL-13、IL-15、IL-14、IL-18、IL-21、IFN-γ、TNF、TGF-β、GM-CSF等，这些细胞因子的一种或者多种可以第一结构域连接，组成融合蛋白。在至少一些实施方式中，细胞因子和第一结构域的C端氨基酸连接。第二结构域还可以为不同于PD-L1抗原靶向蛋白。纳米抗体体积小而且稳定性高，可以方便地和其他的抗原靶向蛋白形成双特异性抗体或者多特异性抗体，例如可以与抗PD-1、抗PD-L1、抗EGFR、抗c-Met、抗TIM-3、抗LAG-3、抗CD-47、抗TIGIT、抗CD-39、抗CD-73、抗41BB、抗OX40、抗CD-3、抗CD-40、抗GARP-TGF-βComplex、抗TGF-β、抗GARP、抗Her2、抗Her3、抗CTLA-4、抗GARP-TGF-β复合物等抗原靶向蛋白制备双、多特异性抗体，以制备具有更多抗原靶向功能的抗体。纳米抗体可以与诊断试剂(如放射性核素或者光学示踪剂等)偶联，实现特定的肿瘤成像。
可以通过本领域常用的方法制备融合蛋白。例如可以借助于基因工程的技术来制备融合蛋白。将编码第一结构域和第二结构域的基因通过一段接头序列进行连接，所获得的连接产物克隆至载体中，并通过原核或者真核表达系统进行表达。
本文中所提到的抗PD-L1纳米抗体或者融合蛋白基本上是纯的。所述基本上是纯的是指，生产的抗PD-L1纳米抗体的纯度能够达到90％以上，91％以上，92％以上，93％以上，94％以上，95％以上，96％以上，97％以上，98％以上，99％以上，99.5％以上等等。
抗体偶联物
本发明还提供了一种抗体偶联物，所述抗体偶联物包括所述的抗PD-L1纳米抗体或者融合蛋白；以及与所述抗PD-L1纳米抗体或者与所述融合蛋白连接的功能小分子。所提到的功能小分子可以为已开发的或者未开发的小分子药物。纳米抗体的溶解性好、稳定性高，适合于和小分子药物进行化学偶联。纳米抗体和小分子药物偶联形成纳米抗体药物偶联物，可以增强抗肿瘤药物的治疗效果，并可以减少不良反应。例如可以通过连接子将毒素、化疗药物、光敏剂等小分子偶联到纳米抗体上。当然还可以通过将抗体与protac分子等偶联，获得抗体Protac偶联物，如通过连接子将抗体和蛋白酶靶向配体(如E3连接酶配体)连接，拉近目标蛋白和细胞内的E3泛素连接酶的距离，利用泛素-蛋白酶体途径特异性的降解靶蛋白。
分离的多核苷酸
本发明还提供了一种分离的多核苷酸，所述分离的多核苷酸编码上述所述的抗PD-L1纳米抗体或者编码所述的融合蛋白。分离的多核苷酸可以为DNA、RNA、或者cDNA等。编码抗PD-L1纳米抗体或者融合蛋白的多核苷酸序列可以根据本领域技术人员的常规方法获得。
构建体
本发明还提供了一种构建体，其包括本文中所提到的分离的多核苷酸。本领域常用的 多种方法都可以用来获得构建体，例如可以通过体外重组DNA技术、DNA合成技术、体内重组技术等等，例如可以将多核苷酸插入到表达载体多克隆位点形成构建体。构建体中可以根据需要含有启动子、终止子、标记基因等多种操纵因子，这些操纵因子可操作地与多核苷酸进行连接。启动子通常用来提供开始转录的信号，启动子可以根据需要选择乳糖启动子(Lac)、Trp启动子、Tac启动子、噬菌体的PL和PR启动子；终止子在转录过程中提供转录终止的信号，构建体上的标记基因常用作筛选。当然还可以根据需要还有增强子，增强蛋白的表达。表达载体不做特殊限制，可以是市售的一些表达载体，也可以是根据需要人工改造后的表达载体，例如质粒、噬菌体、病毒等。病毒可以为植物细胞病毒、哺乳动物细胞病毒等。构建体可以在体外表达抗体或者蛋白，也可以被转入到细胞中表达抗体或者蛋白。
宿主细胞
本发明还提供了一种宿主细胞，其含有上述分离的多核苷酸或者上述所述的构建体。任何适用于多核苷酸或者构建体进行抗体或蛋白表达的细胞都可以作为宿主细胞。宿主细胞可以是原核细胞，如细菌细胞；也可以是真核细胞，例如酵母细胞、哺乳动物细胞等。常用的宿主细胞可以是酵母细胞、CHO、HEK-293、COS细胞、果蝇S2或Sf9的昆虫细胞。采用本领域常用的方法可以获得含有多核苷酸或者构建体的宿主细胞，如显微注射法、电穿孔法、化学转染法、病毒介导的转化法等。
生产纳米抗体或者融合蛋白的方法
本发明又提供了一种生产纳米抗体或者融合蛋白的方法，包括：培养本发明所提到的宿主细胞；以及收集所述纳米抗体或者融合蛋白。收集到的纳米抗体或者融合蛋白经过纯化后，可以获得基本上纯的纳米抗体或者融合蛋白产物。所述基本上是纯的是指，生产的抗PD-L1纳米抗体的纯度能够达到90％以上，91％以上，92％以上，93％以上，94％以上，95％以上，96％以上，97％以上，98％以上，99％以上，99.5％以上等等。
药物组合物
本发明还提供了一种药物组合物，包括所述的抗PD-L1纳米抗体、或所述的融合蛋白、或抗体偶联物；以及药学上可接受的载体。
所提到的药学上可接受的载体在采用的剂量或者浓度下是可以被受试者接受的。药学上可接受的载体包括但不限于：缓冲剂或盐，例如Tris、磷酸盐、柠檬酸盐、醋酸钠、枸橼酸、枸橼酸钠和其它有机酸；抗氧化剂，例如抗坏血酸；防腐剂例如苯扎氯铵；氨基酸，例如组氨酸、盐酸组氨酸、甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸。用于受试者体内的药物制剂一般是无菌的，可以采用本领域常用的方法来获得无菌的药物制剂，例如可以通过无菌滤膜过滤的方法来获得。本领域技术人员可以根据药物组合物所制备的剂型选择合适的药学上可接受的载体，来制备成不同的剂型，例如可以制备成注射剂、冻干剂、片剂等多种剂型。
试剂盒
本发明还提供了一种试剂盒，所述试剂盒包括上述所述的纳米抗体、或者上述所述的融合蛋白、或者抗体偶联物。所述试剂盒还可以根据需要包括容器、缓冲试剂、对照物如阳性对照物和阴性对照物。本领域技术人员可以根据需要进行相应的选择。相应地，试剂盒中还可以包括使用说明书，以便于本领域技术人员的操作和使用。
预防和/或治疗疾病的方法
本发明又提供了一种预防和/或治疗疾病的方法，包括：给予受试者预防和/或治疗有效量的上述抗PD-L1纳米抗体，或融合蛋白，或抗体偶联物。
所提到的“治疗有效量”能够导致疾病症状的严重性降低，疾病无症状期的频率和持续时间增加，或者防止因疾病引起的痛苦降低。“预防有效量”通常会低于治疗有效量。所提供的抗PD-L1纳米抗体相较于未经过抗PD-L1纳米抗体治疗的受试者来说，受试者体内 细胞的抑制率达到10％以上，15％以上，20％以上，25％以上，30％以上，40％以上，45％以上，50％以上，55％以上，60％以上，65％以上，70％以上，75％以上，80％以上，85％以上，甚至是90％以上或者95％以上。所提到的受试者可以是动物也可以是人。例如可以是哺乳动物，包括牛、羊、鼠、马等。
应用抗PD-L1纳米抗体或者融合蛋白或者抗体偶联物可以治疗多种疾病，尤其是癌症，包括但不限于非小细胞肺癌(NSCLC)、黑色素瘤、尿路上皮癌、肝细胞癌、肾细胞癌、经典霍奇金淋巴瘤、宫颈癌、头颈部鳞状细胞癌、膀胱癌、肺癌、鼻咽癌、胃癌、食管癌、结直肠癌、乳腺癌、卵巢癌等。当然，所提供的抗PD-L1纳米抗体或者融合蛋白还可以和多种其他抗体联用，达到预防或者治疗疾病的目的。例如可以和PD-1抗体、CTLA-4抗体、EGFR抗体、c-Met抗体、CD-40抗体、CD-3抗体、CD70抗体、TIGIT抗体、CD-39抗体、OX40抗体、TGFβ抗体、GARP抗体、Her2抗体、c-Met抗体、LAG-3抗体、Tim-3抗体、4-1BB抗体等联用，从而增强对于癌症的治疗效果。所提供的抗体可以按照约0.01mg/kg至约100mg/kg的治疗有效剂量施用，例如0.1mg/kg至100mg/kg，1mg/kg至100mg/kg，5mg/kg至90mg/kg，10mg/kg至80mg/kg，10mg/kg至70mg/kg，10mg/kg至60mg/kg，10mg/kg至50mg/kg，10mg/kg至40mg/kg，10mg/kg至35mg/kg，15mg/kg至30mg/kg，20mg/kg至30mg/kg，15mg/kg至25mg/kg。在某些实施方式中，施用剂量可以结合治疗过程改变。在一些实施方式中，施用剂量可以结合治疗过程中受试者的反应而改变。
所提供的抗PD-L1纳米抗体或者融合蛋白还可以与多种临床治疗方案结合，例如可以结合化疗、放疗、抗血管生成治疗、手术等，提高对于肿瘤的治愈效果。例如可以通过放疗击碎负荷更大的肿瘤，再结合免疫治疗实现对于微小的肿瘤的围追堵截，从而可以将杀伤肿瘤细胞放至最大化。
本发明还提供了抗PD-L1纳米抗体或者融合蛋白或者抗体偶联物在制备药物或者试剂盒中的用途。应用所提供的抗PD-L1纳米抗体或者融合蛋白或者抗体偶联物可以制备成药物，用于预防或者治疗多种疾病。还可以用来制备试剂盒，作为试剂盒中的试剂成分使用，用作特异性抗原的诊断。
下面参考附图详细描述本发明的实施例，需要说明的是，这些实施例旨在解释本发明，并方便本领域技术人员理解，而不能理解为对本发明保护范围的限制。除非另外说明，本发明中所公开的实验方法、检测方法、制备方法等均采用本技术领域常规的分子生物学、生物化学、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中均有完善的记载和说明。
实施例1
通过羊驼免疫的方法获得抗PD-L1纳米文库，同时进行筛选和鉴定，获得候选纳米抗体。
将人源的PD-L1蛋白胞外域序列和人免疫球蛋白Fc区序列构成的融合蛋白和弗氏佐剂混合，乳化，免疫健康羊驼，刺激B细胞表达抗原特异性的纳米抗体。然后采集羊驼血，分离淋巴细胞，采用Trizol法提取总RNA，反转录获得cDNA，并从反转录的cDNA中PCR扩增获得VHH抗体基因片段。VHH基因片段与酵母展示载体共同电转化感受态酵母菌中，酵母同源重组酶将片段与载体连接形成完整质粒，建立库插入率高和多样性优越的酵母转化子文库，并在营养缺陷培养基中保持传代稳定。
将表达抗体的酵母细胞和富集了靶蛋白抗原的磁珠共孵育，并多次的富集培养后，用流式细胞仪鉴定磁珠富集产物，并进行分选。经过多轮筛选，筛选获得5个高表达的阳性克隆，分别命名为抗体1～抗体5。经过序列检测，抗体1的氨基酸序列如SEQ ID NO:4所示，通过IMGT/DomainGapAlign工具分析，其CDR区分别为SEQ ID NO:1、2和3；抗体2的氨基酸序列如SEQ ID NO:8所示，其CDR区分别为SEQ ID NO:5、6和7；抗体3的氨基酸序列如SEQ ID NO:12所示，其CDR区分别为SEQ ID NO:9、10和11；抗体4的氨 基酸序列如SEQ ID NO:13所示，其CDR区分别为SEQ ID NO:9、10和11；抗体5的氨基酸序列如SEQ ID NO:15所示，其CDR区分别为SEQ ID NO:1、2和14。
实施例2
利用哺乳动物细胞体系对所获得的不同抗体进行表达纯化，获得纯度至少在90％以上的抗体。然后测定了不同抗体和不同抗原的结合活性。并以罗氏阿替利珠单抗(Tecentriq)作为阳性对照，以下实施例中提到的阳性对照均采用阿替利珠单抗。
(1)FACS检测抗体和hPD-L1的结合活性
采用流式细胞仪对不同抗体与抗原的亲和力进行检测。将内源性表达PD-L1抗原的MDA-MB-231细胞(ATCC)按1×10 5个/孔加入到96孔板中。然后加入不同浓度的抗体样品(初始浓度为50nM，5倍梯度稀释)，4摄氏度条件下孵育30分钟。然后加入荧光标记的羊抗人IgG二抗(厂家：Abcam)，以检测结合到细胞表面的抗体。利用几何值生成抗体-抗原结合剂量反应曲线，并利用Graphpad Prism V6.0软件绘制四参数的原始数据，确定抗体结合抗原的EC50值，其结果如表5所示。随抗体浓度的变化所获得的平均荧光强度(MFI)值如图1所示。
表5
  EC50(nM)
抗体1 0.089
抗体2 0.031
抗体3 0.042
抗体4 0.039
抗体5 0.053
hIgG1 不结合
阳性对照 0.059
实验结果表明：抗体1至抗体5在细胞水平上的结合活性和阳性对照相似，表现出高的结合活性。
(2)ELISA检测抗体和食蟹猴PD-L1(Cyno PD-L1)的结合活性
将1ug/ml的Cynomolgus/Rhesus macaque PD-L1-hFc蛋白(厂家：ACRO Biosystems)包被于96孔酶标板中，4℃孵育过夜。用洗涤缓冲液洗涤，然后加入含2％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗涤后加入不同浓度的待测抗体(初始浓度为100nM,4倍梯度稀释)，37℃孵育120分钟。用洗涤缓冲液洗涤，然后加入HRP标记的羊抗人二抗(1：10000稀释，厂家：Jackson)，37℃孵育60分钟。用洗涤缓冲液洗涤，每孔加入100ul的TMB显色液，室温反应10-15分钟后，加入100ul 2M的盐酸终止液终止反应。用酶标仪读取OD450值，并计算EC50值，如表6所示。图2示出了不同抗体浓度下检测到的OD450值。
表6
  EC50(nM)
抗体1 0.133
抗体2 0.136
抗体3 0.040
抗体4 0.034
抗体5 0.034
hIgG1 不结合
阳性对照 0.094
从结果可以看出抗体1到抗体5都能和Cyno PD-L1结合，表现出高的结合活性。
(3)ELISA检测抗体和鼠PD-L1的结合活性
将1ug/ml的鼠PD-L1-hFc蛋白(厂家：ACRO Biosystems)包被于96孔酶标板，4℃孵育过夜。用洗涤缓冲液洗涤，加入含2％BSA的PBS溶液封闭60分钟。用洗涤缓冲液洗涤后加入不同浓度的待测抗体(初始浓度为100nM，4倍梯度稀释)，于37℃孵育120分钟。用洗涤缓冲液洗涤，然后加入HRP标记的羊抗人二抗(1：10000稀释),37℃孵育60分钟。用洗涤缓冲液洗涤，每孔加入100ul的TMB显色液，室温反应10-15分钟后，加入100ul 2M的盐酸终止液终止反应，用酶标仪读取OD450值。
实验结果表明：所制备的所有抗体均没有表现出与鼠PD-L1的结合活性。
实施例3
实施例3测定了抗体对于PD-1/PD-L1的阻断活性或者对于CD-80/PD-L1的阻断活性。
(1)FACS测定抗体对于PD-1/PD-L1的阻断活性
采用基于竞争性流式细胞的方法，检测抗体对于PD-1与其配体PD-L1的阻断作用。复苏过表达PD-1的293T-PD-1细胞系(厂家：康源博创生物科技(北京)有限公司)，将细胞按1×10 5个/孔铺在96孔板中，将不同浓度的抗体稀释液(起始工作浓度为200nM,4倍稀释)和PD-L1-Biotin稀释液(2ug/ml,50ul/well)混合，室温孵育30分钟。然后将混合液按100ul每孔加入细胞中，混匀，4摄氏度孵育60分钟，然后用含2％FBS的PBS洗涤，在1200rpm下离心4分钟，弃掉上清。加入100ul每孔的PE标记的链霉亲和素，避光孵育30分钟，孵育结束后用含2％FBS的PBS洗涤。加入120ul的FACS缓冲液重悬细胞，上机检测。根据FSC/SSC对活细胞进行射门，并测量其平均荧光值。用几何值生成抗体-抗原阻断反应曲线，并利用Graphpad Prism V6.0软件绘制四参数图形，确定IC50值，如下表7所示。图3示出了随抗体浓度的变化所获得的平均荧光强度(MFI)结果。
表7
  IC50(nM)
抗体1 1.024
抗体2 2.077
抗体3 1.678
抗体4 2.103
抗体5 2.536
hIgG1 无阻断
阳性对照 1.01
实验结果表明，所制备的抗体都能阻断抗原和其配体的结合。
(2)FACS测定抗体对于CD-80/PD-L1的阻断活性
采用基于竞争性流式细胞的方法，检测抗体对于PD-L1与CD-80的阻断作用。复苏过表达PD-L1的293T-PD-L1细胞系(厂家：康源博创生物科技(北京)有限公司)，将细胞按1×10 5每孔铺在96孔板中，将不同浓度的抗体稀释液(起始工作浓度为200nM,4倍稀释)加入细胞中，4℃孵育60分钟。孵育结束后用含2％FBS的PBS洗涤，在1200rpm条件下离心4分钟，弃掉上清。然后加入100ul每孔的human-CD80-mFc(2ug/ml)，4℃避光孵育30分钟。孵育结束后用含2％FBS的PBS洗涤2遍。每孔加入100ul 1:200稀释的荧 光标记的羊抗鼠二抗(厂家：Abcam)，4℃避光孵育30分钟。孵育结束后用含2％FBS的PBS洗涤2遍，流式上机检测。测量其平均荧光值。用几何值生成抗体-抗原阻断反应曲线，并利用Graphpad Prism V6.0软件绘制四参数图形，确定IC50值，如下表8所示。图4示出了随抗体浓度的变化所获得的平均荧光强度(MFI)结果。
表8
样品编号 IC50(nM)
抗体1 3.954
抗体2 3.355
抗体3 4.146
抗体4 4.757
抗体5 2.979
阳性对照 2.676
hIgG1 NA
实验结果表明所提供的抗体都能较好的阻断抗原-human CD-80的结合。
实施例4
实施例4通过报告基因实验研究抗体对于PD-1/PD-L1通路的阻断。该实验由两种基因工程细胞系组成：Jurkat-PD1-CD3zeta-NFAT-Luc2为效应细胞(厂家：康源博创生物技术(北京)有限公司)，在Jurkat细胞中稳定表达PD-1ECD以及CD-3zeta组成的融合蛋白，同时插入了活化T细胞的因子(NFAT)所驱动的荧光素酶报告基因；293T-hPD-L1细胞(厂家：康源博创生物技术(北京)有限公司)，即表达人PD-L1的293T细胞。当两种细胞共培养时，PD-1/PD-L1相互作用作为第一信号，胞内的CD3zeta链作第二信号向内传递活化信号，NFAT驱动的荧光素酶报告基因得以表达，发出荧光；当加入PD-L1抗体时，阻断PD-1/PD-L1的结合，抑制活化信号的传递，荧光素酶报告基因不能表达。
将293T-hPD-L1细胞按2×10 4个/孔铺在96孔板中，将Jurkat-PD1-CD3zeta-NFAT-Luc2效应细胞按照2×10 4个/孔继续加到96孔板中；然后加入不同浓度的待测抗体(起始浓度为60ug/ml，4倍梯度稀释)，在37℃下孵育过夜。每孔加入100ul的荧光素酶底物ONE-Glo TM Luciferase Assay system检测试剂(Promega)，避光孵育5分钟。酶标仪读取96孔板中的荧光信号。以相对荧光值作为y-轴，抗体样品的浓度作为x-轴，画出四参数曲线。使用GraphPad Prism软件分析该曲线并得出抗体样品的IC50值。图5示出了随抗体浓度变化所获得的平均荧光强度结果。
表9
抗体编号 IC50(nM)
抗体1 0.378
抗体2 0.200
抗体3 0.211
抗体4 0.260
抗体5 0.305
阳性对照 0.489
hIgG1 NA
从结果可以看出，抗体1至抗体5均表现出阻断活性。
实施例5
采用互补决定区移植法(CDR-Grafting Method)获得人源化抗体，该方法通过将抗体 和模板的氨基酸序列统一划分为FR区和CDR区，然后将CDR区移植到模板的FR区上，使得决定抗体特异性的CDR区和人源抗体的框架区相互组合，达到人源化的目的。同时为了避免抗体亲和力下降，通过计算机模拟等技术计算出关键氨基酸残基位点，利用回复突变的方法保证人源化抗体的亲和力。
最终确定，抗体1人源化后的序列编号为抗体1hu，氨基酸序列如SEQ ID NO:28所示。抗体2人源化的序列编号为抗体2hu，氨基酸序列如SEQ ID NO:29所示。抗体3人源化的序列编号为抗体3hu，氨基酸序列如SEQ ID NO:30所示。抗体4人源化的一条序列编号为抗体4hu1，氨基酸序列如SEQ ID NO:31所示；抗体4人源化的另一条序列编号为抗体4hu2，氨基酸序列如SEQ ID NO:32所示。
实施例6
实施例6对于实施例5制备的人源化抗体进行了活性测定。
1、FACS测定人源化抗体与人源PD-L1的结合活性
利用流式细胞仪的方法检测人源化抗体与PD-L1的结合活性。复苏过表达人PD-L1的MC38-hPD-L1细胞系(厂家：康源博创生物科技(北京)有限公司)，将该细胞按1×10 5每孔铺在96孔板中。加入不同浓度的待测抗体(起始浓度为100nM，进行5倍梯度稀释)，于4℃条件下孵育30分钟。然后加入荧光标记的羊抗人IgG二抗(厂家：abcam，货号：98596)，用于检测结合到细胞表面的抗体。利用几何值生成抗体-抗原结合剂量反应曲线，并利用Graphpad Prism V6.0软件绘制四参数的原始数据，确定抗体结合抗原的EC50值，如下表10所示。
表10
样品编号 EC50(nM)
抗体1hu 0.051
抗体1 0.041
抗体2hu 0.18
抗体2 0.14
抗体3hu 0.025
抗体3 0.025
抗体4hu1 0.18
抗体4hu2 0.29
抗体4 0.16
阳性对照 0.21
hIgG1 未结合
实验结果表明，人源化后的抗体与抗原表现出良好的结合活性。
2、FACS测定抗体对于PD-1/PD-L1的阻断活性
基于竞争性流式细胞的方法，检测了人源化抗体阻断PD-L1与其配体PD-1的结合作用。将293T-hPD1细胞按1×10 5每孔铺在96孔板中，将不同浓度的待测抗体(起始工作浓度为200nM，4倍稀释)和PD-L1-Biotin稀释液(2ug/ml,50ul/well)混合，室温孵育30分钟。然后将混合液按100ul每孔加入细胞中，混匀，4℃孵育60分钟，用PE荧光标记的链霉亲和素流式抗体检测抗体的阻断活性。测量其平均荧光值。接着用几何值生成抗体-抗原阻断反应曲线，并利用Graphpad Prism V6.0软件绘制四参数图形，确定IC50值，如下表11所示。
表11
样品编号 IC50(nM)
抗体1hu 4.0
抗体1 4.2
抗体2hu 3.4
抗体2 2.7
抗体3hu 1.0
抗体3 1.1
抗体4hu1 2.6
抗体4hu2 12
抗体4 2.8
阳性对照 2.4
hIgG1 无阻断
实验结果表明人源化的抗体能够阻断PD-1/PD-L1的结合活性。
3、FACS测定人源化抗体对于CD-80/PD-L1的阻断活性
采用竞争性流式细胞的方法，检测抗体阻断PD-L1与CD-80的结合作用。将293T-PD-L1细胞按1×10 5每孔铺在96孔板中，将不同浓度的抗体稀释液(起始工作浓度为200nM,4倍稀释)加入细胞中，4℃孵育30分钟。孵育结束后用含2％FBS的PBS洗涤，在1200rpm下离心4分钟，弃上清。然后加入100ul每孔的human-CD80-mFc(2ug/ml，厂家：ACRO)，4℃避光孵育30分钟。孵育结束后用含2％FBS的PBS洗涤。加入100ul每孔1：200稀释的荧光标记的羊抗鼠IgG二抗，4℃避光孵育30分钟。孵育结束后用含2％FBS的PBS洗涤，流式上机检测。测量其平均荧光值。
表12
样品编号 IC50(nM)
抗体1hu 1.8
抗体1 2.6
抗体2hu 1.7
抗体2 1.5
抗体3hu 3.2
抗体3 5.2
抗体4hu1 1.5
抗体4 1.5
hIgG1 无阻断
阳性对照 3.0
从结果可以看出人源化后的抗体能较好的阻断抗原与CD-80的结合。
实施例7
准备PD-L1人源化转基因小鼠模型hPD-L1-C57BL/6雌性小鼠(厂家：江苏集萃药康生物科技有限公司，能够正常表达人源化的PD-L1)。将人源化PD-L1肿瘤细胞系MC-38-hPD-L1 KL3#细胞(厂家：康源博创生物科技(北京)有限公司)接种在小鼠右侧肩甲部位皮下，每只小鼠接种体积为0.1ml，每只小鼠接种细胞数目为1x 10 6。当肿瘤体积达到75-100mm 3时，对小鼠进行分组，每组小鼠6只，并给予不同的药物处理。分别分为分为抗体1实验组、抗体2实验组、抗体3实验组、抗体4实验组、抗体5实验组和vehicle组(PBS)；相应地，各处理组小鼠给予不同的药物。给药剂量为3mg/kg，给药体积为5μL/g，给药途径为腹腔注射(i.p.)，给药频次为每周两次(BIW)。记录小鼠的肿瘤和体重变化。
图6给出的结果示出了抗体1～抗体5实验组以及vehicle组对于小鼠肿瘤体积的影响。 实验结果发现：经过不同的抗体药物处理，各实验小鼠的肿瘤体积得到明显的抑制；各抗体药物表现出良好的肿瘤抑制效果。
在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体实施方式”、或“一些实施方式”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一种抗PD-L1纳米抗体，其特征在于，包含：
    (a)CDR1，所述CDR1包含选自下组的序列：SEQ ID NO:1、5和9，或者与SEQ ID NO:1、5和9相比，具有一个或者两个氨基酸取代、缺失或者增加的序列；
    (b)CDR2，所述CDR2包含选自下组的序列：SEQ ID NO:2、6和10，或者与SEQ ID NO:2、6和10相比，具有一个或者两个氨基酸取代、缺失或者增加的序列；
    以及
    (c)CDR3，所述CDR3包含选自下组的序列：SEQ ID NO:3、7、11和14，或者与SEQ ID NO:3、7、11和14相比，具有一个、两个或者三个氨基酸取代、缺失或者增加的序列。
  2. 根据权利要求1所述的抗PD-L1纳米抗体，其特征在于，包含：
    (1)具有CDR1为SEQ ID NO:1所示的氨基酸序列、CDR2为SEQ ID NO:2所示的氨基酸序列、CDR3为SEQ ID NO:3所示的氨基酸序列；或者
    (2)具有CDR1为SEQ ID NO:5所示的氨基酸序列、CDR2为SEQ ID NO:6所示的氨基酸序列、CDR3为SEQ ID NO:7所示的氨基酸序列；或者
    (3)具有CDR1为SEQ ID NO:9所示的氨基酸序列、CDR2为SEQ ID NO:10所示的氨基酸序列、CDR3为SEQ ID NO:11所示的氨基酸序列；或者
    (4)具有CDR1为SEQ ID NO:1所示的氨基酸序列、CDR2为SEQ ID NO:2所示的氨基酸序列、CDR3为SEQ ID NO:14所示的氨基酸序列。
  3. 根据权利要求1或2所述的抗PD-L1纳米抗体，其特征在于，还包括框架区FR，所述框架区FR包括FR1、FR2、FR3和FR4，所述FR1、FR2、FR3和FR4分别包括如下所示氨基酸序列：
    (1)FR1包括选自SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:23、SEQ ID NO:26，或者与SEQ ID NO:16、20、23或者26相比，具有一个、两个或者三个保守氨基酸取代的序列；
    (2)FR2包括选自SEQ ID NO:17、SEQ ID NO:21、SEQ ID NO:24，或者与SEQ ID NO:17、21或者24相比，具有一个、两个或者三个保守氨基酸取代的序列；
    (3)FR3包括选自SEQ ID NO:18、SEQ ID NO:22、SEQ ID NO:25、SEQ ID NO:27，者与SEQ ID NO:18、22、25或者27相比，具有一个、两个、三个或者四个保守氨基酸取代的序列；
    (4)FR4选自SEQ ID NO:19，或者与SEQ ID NO:19相比，具有一个或者两个保守氨基酸取代的序列。
  4. 根据权利要求1或2所述的抗PD-L1纳米抗体，其特征在于，还包括框架区FR，所述框架区FR分别包括：
    (1)FR1为SEQ ID NO:16所示的氨基酸序列、FR2为SEQ ID NO:17所示的氨基酸序列、FR3为SEQ ID NO:18所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列；或者
    (2)FR1为SEQ ID NO:20所示的氨基酸序列、FR2为SEQ ID NO:21所示的氨基酸序列、FR3为SEQ ID NO:22所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列；或者
    (3)FR1为SEQ ID NO:23所示的氨基酸序列、FR2为SEQ ID NO:24所示的氨基酸序列、FR3为SEQ ID NO:25所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列；或者
    (4)FR1为SEQ ID NO:26所示的氨基酸序列、FR2为SEQ ID NO:24所示的氨基酸序列、FR3为SEQ ID NO:25所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列； 或者
    (5)FR1为SEQ ID NO:16所示的氨基酸序列、FR2为SEQ ID NO:17所示的氨基酸序列、FR3为SEQ ID NO:27所示的氨基酸序列和FR4为SEQ ID NO:19所示的氨基酸序列。
  5. 根据权利要求1或2所述的抗PD-L1纳米抗体，其特征在于，所述抗PD-L1纳米抗体包括：
    (a)具有如SEQ ID NO:4、或SEQ ID NO:8、或SEQ ID NO:12、或SEQ ID NO:13或SEQ ID NO:15所示的氨基酸序列；
    (b)与(a)相比，同源性在85％以上、86％以上、87％以上、88％以上、89％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、或者99％以上的氨基酸序列。
  6. 根据权利要求1或2所述的抗PD-L1纳米抗体，其特征在于，所述抗PD-L1纳米抗体具有如SEQ ID NO:28、或SEQ ID NO:29、或SEQ ID NO:30、或SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列。
  7. 一种抗PD-L1纳米抗体，其特征在于，选自：
    i)具有如SEQ ID NO:4、或SEQ ID NO:8、或SEQ ID NO:12、或SEQ ID NO:13、或SEQ ID NO:15、或SEQ ID NO:28、或SEQ ID NO:29、或SEQ ID NO:30、或SEQ ID NO:31或SEQ ID NO:32所示的氨基酸序列；或者
    ii)与i)相比，同源性在85％以上、86％以上、87％以上、88％以上、89％以上、90％以上、91％以上、92％以上、93％以上、94％以上、95％以上、96％以上、97％以上、98％以上、或者99％以上的氨基酸序列。
  8. 一种融合蛋白，其特征在于，所述融合蛋白包括：
    第一结构域，所述第一结构域为权利要求1～7中任一项所述的抗PD-L1纳米抗体，以及
    第二结构域，所述第二结构域为不同于抗PD-L1纳米抗体的功能蛋白。
  9. 根据权利要求8所述的融合蛋白，其特征在于，所述第二结构域包括选自下列中的至少一种：
    1)免疫球蛋白Fc区，优选为人免疫球蛋白Fc区；
    2)细胞因子调控蛋白；
    3)不同于PD-L1抗原靶向蛋白。
  10. 一种抗体偶联物，其特征在于，包括：
    权利要求1～7中任一项所述的抗PD-L1纳米抗体或者权利要求8或9所述的融合蛋白；
    与所述抗PD-L1纳米抗体或者所述融合蛋白连接的功能小分子。
  11. 一种分离的多核苷酸，其特征在于，所述分离的多核苷酸编码权利要求1～7中任一项所述的抗PD-L1纳米抗体或者编码权利要求8或9所述的融合蛋白。
  12. 一种构建体，其特征在于，所述构建体含有权利要求11所述的分离的多核苷酸。
  13. 一种宿主细胞，其特征在于，所述宿主细胞含有权利要求11所述的分离的多核苷酸或者权利要求12所述的构建体。
  14. 一种生产纳米抗体或者融合蛋白的方法，其特征在于，包括：
    培养宿主细胞，所述宿主细胞为权利要求13所述的宿主细胞，以及
    从培养物中收集所述纳米抗体或者融合蛋白。
  15. 一种药物组合物，其特征在于，包括：
    权利要求1～7中任一项所述的抗PD-L1纳米抗体、或权利要求8或9所述的融合蛋白、或权利要求10所述的抗体偶联物；以及
    药学上可接受的载体。
  16. 一种试剂盒，其特征在于，包括：权利要求1～7中任一项所述的抗PD-L1纳米抗体、或权利要求8或9所述的融合蛋白、或权利要求10所述的抗体偶联物。
  17. 一种预防和/或治疗疾病的方法，其特征在于，包括：
    给予受试者预防和/或治疗有效量的权利要求1～7中任一项所述的抗PD-L1纳米抗体、或权利要求8或9所述的融合蛋白、或权利要求10所述的抗体偶联物。
  18. 权利要求1～7中任一项所述的抗PD-L1纳米抗体或权利要求8或9所述的融合蛋白或权利要求10所述的抗体偶联物在制备药物或试剂盒中的用途。
PCT/CN2022/110423 2021-08-06 2022-08-05 抗pd-l1纳米抗体及其应用 Ceased WO2023011614A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US18/294,516 US20240327518A1 (en) 2021-08-06 2022-08-05 Anti-pd-l1 nanobody and use thereof
CN202280007891.6A CN116547006B (zh) 2021-08-06 2022-08-05 抗pd-l1纳米抗体及其应用
EP22852325.4A EP4382166A4 (en) 2021-08-06 2022-08-05 ANTI-PD-L1 NANOBODY AND ITS USE

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202110903293 2021-08-06
CN202110903293.4 2021-08-06
CN202111162303.X 2021-09-30
CN202111162303 2021-09-30

Publications (1)

Publication Number Publication Date
WO2023011614A1 true WO2023011614A1 (zh) 2023-02-09

Family

ID=85155320

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/110423 Ceased WO2023011614A1 (zh) 2021-08-06 2022-08-05 抗pd-l1纳米抗体及其应用

Country Status (4)

Country Link
US (1) US20240327518A1 (zh)
EP (1) EP4382166A4 (zh)
CN (1) CN116547006B (zh)
WO (1) WO2023011614A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119912573B (zh) * 2025-01-23 2025-10-28 百吉生物医药(广州)股份有限公司 一种特异性结合pd-1的纳米抗体及其应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107686520A (zh) * 2016-08-04 2018-02-13 信达生物制药(苏州)有限公司 抗pd‑l1纳米抗体及其应用
CN109265548A (zh) * 2018-09-13 2019-01-25 东南大学 抗pd-l1纳米抗体及其编码序列、制备方法和应用
US20190177416A1 (en) * 2016-03-18 2019-06-13 Nanomab Technology Limited Anti-pd-l1 nanobody, coding sequence and use thereof
CN110003333A (zh) * 2019-04-12 2019-07-12 深圳普瑞金生物药业有限公司 多肽、pd-l1单域抗体、核苷酸序列及试剂盒
WO2019158113A1 (zh) * 2018-02-14 2019-08-22 上海洛启生物医药技术有限公司 阻断型pd-l1驼源单域抗体及其用途
CN111433224A (zh) * 2017-11-17 2020-07-17 南京传奇生物科技有限公司 抗pd-l1的单结构域抗体及其变体
CN112409483A (zh) * 2019-08-22 2021-02-26 浙江道尔生物科技有限公司 抗pd-l1纳米抗体

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
UY32917A (es) * 2009-10-02 2011-04-29 Boehringer Ingelheim Int Moléculas de unión a dll-4
CN109096396B (zh) * 2017-06-20 2022-01-04 华兰生物工程股份有限公司 一种抗pd-l1人源化纳米抗体及其应用
CN115947846A (zh) * 2019-09-12 2023-04-11 普米斯生物技术(珠海)有限公司 一种抗pd-l1纳米抗体及其衍生物和用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190177416A1 (en) * 2016-03-18 2019-06-13 Nanomab Technology Limited Anti-pd-l1 nanobody, coding sequence and use thereof
CN107686520A (zh) * 2016-08-04 2018-02-13 信达生物制药(苏州)有限公司 抗pd‑l1纳米抗体及其应用
CN111433224A (zh) * 2017-11-17 2020-07-17 南京传奇生物科技有限公司 抗pd-l1的单结构域抗体及其变体
WO2019158113A1 (zh) * 2018-02-14 2019-08-22 上海洛启生物医药技术有限公司 阻断型pd-l1驼源单域抗体及其用途
CN109265548A (zh) * 2018-09-13 2019-01-25 东南大学 抗pd-l1纳米抗体及其编码序列、制备方法和应用
CN110003333A (zh) * 2019-04-12 2019-07-12 深圳普瑞金生物药业有限公司 多肽、pd-l1单域抗体、核苷酸序列及试剂盒
CN112409483A (zh) * 2019-08-22 2021-02-26 浙江道尔生物科技有限公司 抗pd-l1纳米抗体

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Sequences of Proteins of Immunological Interest", 1983, U.S.DEPT. OF HEALTH AND HUMAN SERVIES
EHRENMANN F.KAAS QLEFRANC M.-P., NUCLEIC ACIDS RES., vol. 38, 2010, pages D301 - D307
EHRENMANN, F.LEFRANC, M.-P., COLD SPRING HARBOR PROTOC., vol. 6, 2011, pages 737 - 749
J. MOL. BIOL., vol. 196, 1987, pages 901 - 917
See also references of EP4382166A4

Also Published As

Publication number Publication date
CN116547006B (zh) 2024-09-20
CN116547006A (zh) 2023-08-04
EP4382166A4 (en) 2025-07-09
EP4382166A1 (en) 2024-06-12
US20240327518A1 (en) 2024-10-03

Similar Documents

Publication Publication Date Title
US20200399375A1 (en) Pd-l1 antibody, antigen-binding fragment thereof and medical application thereof
WO2020135201A1 (zh) 一种抗体及其用途
US12509520B2 (en) Anti-CD3 and anti-CD123 bispecific antibody and use thereof
KR20190134614A (ko) B7-h3 항체, 이의 항원-결합 단편 및 이의 의학적 용도
US20230357398A1 (en) Novel human antibodies binding to human cd3 epsilon
CN113227148B (zh) 抗gpc3抗体、其抗原结合片段及其医药用途
WO2023036215A1 (zh) 双特异性抗原结合分子及其应用
CN118696058A (zh) 抗bcma纳米抗体及其应用
CN110913894B (zh) 抗csf-1r抗体、其抗原结合片段及其医药用途
WO2024017326A1 (zh) 抗gprc5d纳米抗体及其应用
WO2023236991A1 (zh) 靶向her2，pd-l1和vegf的三特异性抗体
CN116547006B (zh) 抗pd-l1纳米抗体及其应用
CN116547300B (zh) 双特异性抗体及其应用
WO2022141378A1 (zh) 一种抗pd-1的单域抗体
CN115335402B (zh) 特异性抗原结合分子,其制备方法及医药用途
CN116813786B (zh) 抗cd73抗体及其应用
CN107250162A (zh) 新的人源化adam17抗体
WO2024164807A1 (zh) 多特异性抗体及其应用
HK40095576A (zh) 双特异性抗体及其应用
HK40095576B (zh) 双特异性抗体及其应用
WO2025025190A1 (zh) 抗cd73抗体及其应用
WO2026013159A1 (en) Antibodies binding to constant domains of gamma delta t-cell receptors
CN116284406A (zh) 一种pd-1结合蛋白及其应用
HK40048164B (zh) 一种抗体及其用途
HK40012331B (zh) 抗b7-h4抗体、其抗原结合片段及其医药用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22852325

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202280007891.6

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 18294516

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022852325

Country of ref document: EP

Effective date: 20240306