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WO2024164807A1 - 多特异性抗体及其应用 - Google Patents

多特异性抗体及其应用 Download PDF

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Publication number
WO2024164807A1
WO2024164807A1 PCT/CN2024/072447 CN2024072447W WO2024164807A1 WO 2024164807 A1 WO2024164807 A1 WO 2024164807A1 CN 2024072447 W CN2024072447 W CN 2024072447W WO 2024164807 A1 WO2024164807 A1 WO 2024164807A1
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Prior art keywords
seq
antigen binding
binding portion
sequence shown
variable region
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PCT/CN2024/072447
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English (en)
French (fr)
Inventor
徐汶新
丁列明
Original Assignee
贝达药业股份有限公司
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Priority to CN202480001682.XA priority Critical patent/CN118829657A/zh
Publication of WO2024164807A1 publication Critical patent/WO2024164807A1/zh

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the present invention relates to the field of biomedicine, and in particular to a multispecific antibody and application thereof.
  • BsAb bispecific antibody
  • bispecific antibodies have now become a research hotspot in the field of antibody engineering, and have broad application prospects in the fields of tumor treatment and autoimmune diseases. Many domestic and foreign pharmaceutical companies have conducted research on bispecific antibodies for different targets.
  • Multispecific antibodies are antibodies that can specifically bind to two antigens or more antigenic epitopes at the same time, involving the interaction of two or more entities, which is more complex than monoclonal antibodies.
  • the development of multispecific antibodies targeting different targets still needs further improvement.
  • the present invention provides multispecific antibodies and their use in disease treatment.
  • the present invention inhibits the release of active TGF- ⁇ and the immunosuppressive function of Treg by targeting GARP, TGF- ⁇ and/or GARP-TGF- ⁇ complex, thereby reactivating the immune response of cancer cells and inhibiting disease progression.
  • the multispecific antibodies provided can also specifically bind to VEGF, block the formation of blood vessels, and thus inhibit tumor growth.
  • the multispecific antibodies provided can also be selectively designed to specifically bind to PD-L1 as needed, which can improve the anti-tumor activity of PD-1/PD-L1 resistance.
  • the first aspect of the present invention provides a multispecific antibody, comprising a first antigen binding portion and a third antigen binding portion; the first antigen binding portion is capable of binding to TGF- ⁇ , GARP or GARP-TGF- ⁇ complex, the first antigen binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region: comprises the HCDR sequence shown in SEQ ID NO: 1, 2 and 3, or comprises the HCDR sequence shown in SEQ ID NO: 4, 5 and 6, or comprises the HCDR sequence shown in SEQ ID NO: 7, 8 and 9, or comprises the HCDR sequence shown in SEQ ID NO: 10, 11 and 12, or comprises the HCDR sequence shown in SEQ ID NO: 13, 14 and 15, or comprises the HCDR sequence shown in SEQ ID NO: 16, 17 and 18.
  • the light chain variable region comprises the HCDR sequence shown in SEQ ID NO: 19, 20 and 21, or comprises the LCDR sequence shown in SEQ ID NO: 22, 23 and 24, or comprises the LCDR sequence shown in SEQ ID NO: 25, 20 and 26, or comprises the LCDR sequence shown in SEQ ID NO: 27, 28 and 29, or comprises the LCDR sequence shown in SEQ ID NO: 30, 31 and 32, or comprises the LCDR sequence shown in SEQ ID NO: 33, 34 and 35;
  • the third antigen binding portion is a VEGF receptor fusion protein, an anti-VEGF antibody or an antigen binding fragment or a VEGF binding molecule;
  • the HCDR sequence and the LCDR sequence are obtained based on the IMGT definition scheme.
  • the multispecific antibody has at least a first antigen binding portion and a third antigen binding portion, wherein the first antigen binding portion can specifically target GARP, TGF- ⁇ and/or GARP-TGF- ⁇ complex to activate the response of immune cells; the third antigen binding portion of the multispecific antibody can specifically bind to VEGF to block the formation of blood vessels, thereby inhibiting the growth of tumors.
  • the multispecific antibody comprising the first antigen binding portion and the third antigen binding portion can not only activate the response of immune cells, but also block the formation of blood vessels and inhibit the growth of tumors, showing an excellent tumor treatment effect.
  • the second aspect of the present invention provides a multispecific antibody, comprising at least a first antigen binding portion and a third antigen binding portion; the first antigen binding portion is capable of binding to TGF- ⁇ , GARP or GARP-TGF- ⁇ complex, the first antigen binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises complementary determining regions HCDR1, HCDR2 and HCDR3, the light chain variable region comprises complementary determining regions LCDR1, LCDR2 and LCDR3; wherein HCDR1 comprises the sequence shown in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16, HCDR2 comprises the sequence shown in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17, HCDR3 comprises the sequence shown in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16, ID NO:3 or 6 or 9 or 12 or 15 or 18, LCDR1 comprises the sequence shown in SEQ ID NO:19 or 22 or 25 or 27 or 30 or 33, LCDR2 comprises the sequence shown in SEQ ID
  • the third aspect of the present invention provides a multispecific antibody, including a first antigen binding portion, a second antigen binding portion and a third antigen binding portion; the first antigen binding portion can bind to TGF- ⁇ , GARP or GARP-TGF- ⁇ complex, the second antigen binding portion can bind to PD-L1, and the third antigen binding portion is a VEGF receptor fusion protein, an anti-VEGF antibody or antigen binding fragment or a VEGF binding molecule; the first antigen binding portion includes a heavy chain variable region and a light chain variable region, and the first antigen binding portion has: the HCDR sequence shown in SEQ ID NO: 1, 2 and 3 and the LCDR sequence shown in SEQ ID NO: 19, 20 and 21, or the sequence shown in SEQ ID NO: 4, 5 and 6 The HCDR sequence of SEQ ID NOs: 22, 23 and 24 and the LCDR sequence of SEQ ID NOs: 25, 20 and 26, or the HCDR sequence of SEQ ID NOs: 10, 11 and 12 and the LCDR sequence of SEQ ID
  • the second antigen binding portion has: the CDR1 sequence shown in SEQ ID NO:60, the CDR2 sequence shown in SEQ ID NO:61, and the CDR3 sequence shown in SEQ ID NO:62; or the CDR1 sequence shown in SEQ ID NO:63, the CDR2 sequence shown in SEQ ID NO:64, and the CDR3 sequence shown in SEQ ID NO:65; or the CDR1 sequence shown in SEQ ID NO:66, the CDR2 sequence shown in SEQ ID NO:67, and the CDR3 sequence shown in SEQ ID NO:68; or the CDR1 sequence shown in SEQ ID NO:60, the CDR2 sequence shown in SEQ ID NO:61, and the CDR3 sequence shown in SEQ ID NO:69;
  • the first antigen binding moiety forms an IgG type structure with the first constant domain CH1 of the heavy chain, the constant domain VL of the light chain and the Fc region, the second antigen binding moiety is connected to the constant domain VL of the light chain via a linker, and the third antigen binding moiety is connected to the carboxyl terminus of the Fc region;
  • HCDR sequence LCDR sequence
  • CDR1, CDR2, and CDR3 sequences are obtained based on the IMGT definition scheme.
  • the fourth aspect of the present invention provides a multispecific antibody comprising a first antigen binding portion, a second antigen binding portion and a third antigen binding portion;
  • the first antigen binding portion is capable of binding to TGF- ⁇ , GARP or GARP-TGF- ⁇ complex, and the first antigen binding portion comprises: the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO: 36 or 37 or 38 or 39 or 40 or 41 or 42 or 43 or 44 or 45 or 46 or 47, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO: 48 or 49 or 50 or 51 or 52 or 53 or 54 or 55 or 56 or 57 or 58 or 59;
  • the second antigen binding portion is capable of binding to PD-L1
  • the second antigen binding portion comprises:
  • the third antigen binding portion is a VEGF receptor fusion protein having an amino acid sequence shown in SEQ ID NO:92.
  • the fifth aspect of the present invention provides a bispecific antibody, comprising a first antigen binding portion that specifically binds to TGF- ⁇ , GARP and/or GARP-TGF- ⁇ complex, a second antigen binding portion that specifically binds to PD-L1 and a third antigen binding portion that specifically binds to VEGF; further comprising a first heavy chain constant domain CH1 and a light chain constant domain CL, the first antigen binding portion being covalently linked to the first heavy chain constant domain CH1 and the light chain constant domain CL, respectively; further comprising an Fc region, the Fc region being connected to the first heavy chain constant domain CH1 via a hinge region; the second antigen binding portion being connected to the Fc region and/or the light chain constant domain CL and/or the light chain variable region; the third antigen binding portion being connected to the Fc region and/or the light chain constant domain CL and/or the light chain variable region.
  • the sixth aspect of the present invention provides a polynucleotide encoding the multispecific antibody described in any one of the first to fifth aspects above.
  • the seventh aspect of the present invention provides a construct comprising the polynucleotide described in the sixth aspect.
  • the eighth aspect of the present invention provides a host cell comprising the polynucleotide described in the sixth aspect or the construct described in the seventh aspect.
  • the ninth aspect of the present invention provides a pharmaceutical composition, comprising: the multispecific antibody described in any one of the first to fifth aspects above; and a pharmaceutically acceptable carrier.
  • the tenth aspect of the present invention provides an antibody conjugate, comprising the multispecific antibody described in any one of the first to fifth aspects above; and a functional small molecule connected to the multispecific antibody.
  • the eleventh aspect of the present invention provides a kit, which comprises the multispecific antibody described in any one of the first to fifth aspects above.
  • the twelfth aspect of the present invention provides a method for producing the above-mentioned multispecific antibody, comprising: culturing the host cell described in the eighth aspect, and collecting the multispecific antibody from the culture.
  • the thirteenth aspect of the present invention provides a method for preventing and/or treating a disease, comprising: administering to a subject in need thereof an effective amount of the multispecific antibody described in any one of the first to fifth aspects, or the pharmaceutical composition described in the ninth aspect, or the antibody conjugate described in the tenth aspect.
  • a fourteenth aspect of the present invention provides a use of the multispecific antibody described in any one of the first to fifth aspects in preparing a drug or a kit or in preparing an antibody conjugate.
  • FIG1 is a schematic diagram of the structure of a multispecific antibody provided according to an embodiment of the present invention.
  • FIG2 is a schematic diagram of the structure of a multispecific antibody provided according to an embodiment of the present invention.
  • FIG3 is a schematic diagram of the structure of a multispecific antibody provided according to an embodiment of the present invention.
  • FIG. 4 is a graph showing the results of detecting the binding activity of different antibodies to VEGF using an ELISA method according to an embodiment of the present invention.
  • FIG. 5 is a graph showing the results of a reporter gene blocking experiment with different multispecific antibodies provided according to an embodiment of the present invention.
  • FIG. 6 is a graph showing the results of characterizing the binding activity of different multispecific antibodies to GARP/TGF- ⁇ through a reporter gene blocking experiment according to an embodiment of the present invention.
  • FIG. 7 shows the blocking effect of different multispecific antibodies on the PD-L1/PD-1 pathway according to an embodiment of the present invention.
  • multi-specific antibody includes antigen-binding portions that specifically bind to epitopes of at least two different biomolecules.
  • the multi-specific antibodies mentioned herein include bispecific antibodies, tri-specific antibodies, etc.
  • the “multi” mentioned herein includes two and more than two. In order to distinguish, the portions of the multi-specific antibody that can specifically target a specific antigen are referred to as “first antigen-binding portion”, “second antigen-binding portion”, “third antigen-binding portion”, etc. as needed. "First”, “second”, “third”, etc. are only used to distinguish, not to indicate the order of precedence, nor to indicate importance.
  • first antigen-binding portion The "first antigen-binding portion", "second antigen-binding portion” and “third antigen-binding portion” will also be explained and described below. Unless otherwise stated, the order of binding to antigens in the listed multi-specific antibodies is arbitrary. Of course, when a multi-specific antibody specifically binds to different biomolecules, it is possible to bind to more than one antigen epitope of the specific biomolecule.
  • antibody is used in the broadest sense to refer to a protein or polypeptide comprising an antigen binding site, covering natural antibodies and artificial antibodies of various structures, including but not limited to complete antibody forms or antigen-binding fragments of antibodies.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • the heavy chain constant region includes the first heavy chain constant domain (CH1), the second heavy chain constant domain (CH2) and the third heavy chain constant domain (CH3).
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region.
  • the light chain constant region is also the light chain constant domain (CL).
  • VH and VL regions can be further divided into complementary determining regions (also called hypervariable regions or high variable regions, abbreviated as CDR), with conserved framework regions (FR) inserted between them.
  • CDR complementary determining regions
  • FR conserved framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus) in the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the CDRs of the heavy chain variable region are called HCDR1, HCDR2 and HCDR3 respectively starting from the amino terminus of the heavy chain amino acid sequence
  • the CDRs of the light chain variable region are called LCDR1, LCDR2 and LCDR3 respectively starting from the amino terminus of the light chain amino acid sequence.
  • an "antigen-binding fragment”, or “antigen-binding portion” (such as a first antigen-binding portion, a second antigen-binding portion) is Refers to a part of a complete antibody, usually containing a variable domain or variable region that binds to an antigen.
  • the term "variable region” or “variable domain” refers to a domain that is involved in the binding of an antigen to an antibody heavy chain or light chain.
  • the variable regions of the heavy and light chains of a complete antibody usually have similar results, with each domain including four conserved framework regions and three hypervariable regions.
  • antigen-binding fragments or antigen-binding portions can be Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments, linear antibodies, single-chain antibodies, single-domain antibodies, etc.
  • the complete antibody is digested with papain to produce two identical antigen-binding fragments, called Fab fragments, each of which contains a heavy chain and light chain variable region and a light chain constant domain and a heavy chain first constant domain (CH1).
  • the Fab' fragment differs from the Fab fragment by adding a few residues at the carboxyl terminus of the first constant domain CH1 of the heavy chain, including one or more cysteines from the hinge region of the antibody.
  • the complete antibody is digested with pepsin to obtain a F(ab')2 fragment.
  • the F(ab')2 fragment has two antigen-binding F(ab) parts linked together by a disulfide bond, and the F(ab')2 fragment is a bivalent antibody.
  • Single-chain antibodies are fusion proteins formed by connecting the variable regions of the heavy chain and light chain of antibodies through a flexible short peptide consisting of about 10-25 amino acids.
  • Single-domain antibodies are antibody fragments composed of the variable regions of a single monomer. Since single-domain antibodies are usually derived from the variable regions of camelid antibodies or shark anti-heavy chains, they are often called nanobodies.
  • Nanobodies contain only the heavy chain CDR region and are the smallest antigen-binding fragment with complete functions. Nanobodies include three CDR regions (CDR1-CDR3) and four framework regions FR (FR1-FR4). Framework regions FR1, FR2, FR3, and FR4 are separated by complementary determining regions CDR1, CDR2, and CDR3, respectively.
  • 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 of the binding sites between an antigen and an antibody or an antigen-binding fragment.
  • Specific binding or “specifically binds to”, “binds to”, “specifically targets” a specific antigen or epitope, or “has specificity” or “can bind to” a specific antigen or epitope means to distinguish from non-specific interactions, and this specific binding can be measured by some methods commonly used in the art.
  • the ability of an antibody to bind to an antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, cells carrying an 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.
  • ELISA enzyme-linked immunosorbent assay
  • the antibodies provided have EC50 values of ⁇ 100nM, ⁇ 50nM, ⁇ 20nM, ⁇ 10nM, ⁇ 5nM, ⁇ 1nM, ⁇ 0.5nM, ⁇ 0.1nM, ⁇ 0.05nM, ⁇ 0.04nM, ⁇ 0.03nM, ⁇ 0.02nM, ⁇ 0.01nM.
  • the binding activity of antibodies and antigens can also be measured by surface plasmon resonance (SPR) or bio-film interferometry (BLI).
  • “Separable” means that it can be identified and separated and/or recovered from a cell or cell culture expressing a polypeptide or protein. Usually, the isolated polypeptide will be prepared by at least one purification step.
  • isolated antibody means that it is substantially free of different antigens.
  • “Recombinant” means that antibodies can be produced in exogenous host cells using genetic recombination technology.
  • Humanized antibodies generally refer to antibodies based on antigen-binding portions derived from non-human species and based on partial structures and sequences of human immunoglobulin molecules. For example, in humanized antibodies, the entire antibody except for CDR is encoded by a polynucleotide of human origin, which retains the antigen-binding activity while reducing immunogenicity.
  • the CDR sequences of the first antigen binding portion and the second antigen binding portion shown in this article are obtained by analysis according to existing definition schemes, for example, the CDR sequence of the first antigen binding portion and the CDR sequence of the second antigen binding portion are obtained according to the IMGT definition scheme. They can also be obtained by definition schemes such as Kabat (for example, see U.S. Dept. of Health and Human Servies, "Sequences of Proteins of Immunological Interest” (1983)), Chothia (for example, see J. Mol. Biol. 196: 901-917 (1987)). It is known to those skilled in the art that the difference in CDRs caused by the difference in definition methods are also included in the protection scope of the present invention.
  • the present invention provides a multispecific antibody, comprising at least a first antigen binding portion and a third antigen binding portion, wherein the first antigen binding portion and the third antigen binding portion target different target antigens, respectively.
  • the first antigen binding portion can specifically target one, two or three of TGF- ⁇ , GARP and the GARP-TGF- ⁇ complex; the third antigen binding portion specifically targets VEGF.
  • the first antigen binding portion specifically targets TGF- ⁇ , GARP, or the GARP-TGF- ⁇ complex, or Targeting any two or three of these three; all can directly or indirectly achieve the regulation of TGF- ⁇ , activate the immune response of cancer cells, and inhibit the progression of the disease.
  • first antigen-binding portion second antigen-binding portion
  • third antigen-binding portion respectively refer to portions that can bind to different target antigens, which can be complete antibodies or parts of complete antibodies, such as heavy chain variable regions, Fab, Fab', F(ab') 2 , single-chain antibodies (ScFv), or single-domain antibodies (sdAb), etc.
  • TGF- ⁇ Transforming growth factor- ⁇
  • TGF- ⁇ Transforming growth factor- ⁇
  • immune cells such as regulatory T cells (Tregs), DC cells, CD3+T cells, M2 macrophages, etc.
  • Tregs regulatory T cells
  • DC cells DC cells
  • CD3+T cells CD3+T cells
  • M2 macrophages etc.
  • TGF- ⁇ regulatory T cells
  • the activity of immune cells is usually affected by TGF- ⁇ in the environment, showing immunosuppression. It is precisely because of the potency and diversity of TGF- ⁇ that subtypes of the family members, such as TGF- ⁇ 1, ⁇ 2 and ⁇ 3, are produced in a latent, inactive form and require strictly regulated extracellular activation steps to acquire the ability to bind to their receptors (Cold Spring Harb. Perspect. Biol.
  • TGF- ⁇ 1 is the main subtype expressed by immune cells such as Tregs.
  • Treg cells mediate immunosuppression by producing active TGF- ⁇ , thereby preventing the occurrence of autoimmune diseases.
  • malignant tumors use this mechanism to release a large amount of TGF- ⁇ , helping cancer cells to divide rapidly, while using Treg cells to inhibit the killing effect of other immune cells on cancer cells.
  • Glycoprotein A repetitions predominant is a type I transmembrane cell surface receptor that binds latent transforming growth factor ⁇ (TGF- ⁇ ). It is mainly activated on the surface of Treg, B cells and platelets, and is highly expressed in human breast, lung and colon tumors. GARP can form a complex with latent TGF- ⁇ (latency associated peptide, LAP) (Proc Natl Acad Sci USA. 2009; 106(32): 13445-50), which can regulate the ability of membrane-bound latent TGF- ⁇ and bind to ⁇ V ⁇ 8 integrin or ⁇ V ⁇ 6 integrin to release TGF- ⁇ from the cell surface and activate it.
  • LAP latent TGF- ⁇
  • GARP inhibitors are gradually emerging in tumor treatment.
  • Monospecific antibodies targeting GARP are also advancing rapidly, such as ARGX-115, the first GARP inhibitor to enter the clinic, which targets GARP and inhibits the release of active TGF- ⁇ and the immunosuppressive activity of Treg, thereby reactivating the immune response of cancer cells and inhibiting disease progression.
  • ARGX-115 the first GARP inhibitor to enter the clinic
  • the first antigen-binding portion mentioned in this article can specifically target one, two or three of TGF- ⁇ , GARP and the GARP-TGF- ⁇ complex, so as to achieve the regulation of TGF- ⁇ , activate the immune response of cancer cells, and inhibit disease progression.
  • the third antigen binding portion can specifically bind to VEGF.
  • the third antigen binding portion is a VEGF receptor fusion protein, an anti-VEGF antibody or antigen binding fragment or a VEGF binding molecule.
  • the third antigen binding portion inhibits tumor growth by blocking the normal physiological activity of VEGF and preventing the formation of new blood vessels. By effectively blocking the formation of new blood vessels, tumor tissue cannot grow normally due to lack of vascular nutrition.
  • the multispecific antibodies provided can also be used for pathological blood vessel growth in other diseases besides tumors, such as diabetic retinopathy, psoriasis, and pathological corneal angiogenesis caused by transplantation, infection or trauma.
  • the present invention provides a multispecific antibody, comprising a first antigen binding portion that specifically binds to TGF- ⁇ , GARP and/or a GARP-TGF- ⁇ complex, and a third antigen binding portion that specifically binds to VEGF, and may also selectively comprise a second antigen binding portion that specifically binds to PD-L1.
  • the multispecific antibody further comprises a first heavy chain constant domain CH1 and a light chain constant domain CL, wherein the first antigen binding portion is covalently linked to the first heavy chain constant domain CH1 and the light chain constant domain CL, respectively; further comprising an Fc region, wherein the Fc region is connected to the first heavy chain constant domain CH1 through a hinge region; and the third antigen binding portion is directly or through a linker connected to the Fc region and/or to the light chain constant domain CL.
  • the multispecific antibody may also comprise a second antigen binding portion as required, wherein the second antigen binding portion is linked to the Fc region and/or the light chain constant domain CL and/or the light chain variable region.
  • the other sequences of the formed IgG-type structure may adopt natural sequences of mammalian origin (e.g., natural sequences of human origin).
  • the heavy chain constant domain sequence used is shown in SEQ ID NO:93
  • the light chain constant domain sequence used is shown in SEQ ID NO:94.
  • connection can be direct connection or indirect connection.
  • Direct connection refers to the connection of polypeptide or protein sequences directly through two amino acids to form a peptide bond.
  • Indirect connection refers to the connection of polypeptide or protein sequences through a linker.
  • the Fc region sequence is not limited and can be an Fc region of natural origin, such as an Fc region from a human. It may also be a modified Fc region.
  • the modified Fc region may be a mutated Fc region, for example, mutations may occur at certain sites in the Fc region to regulate the ADCC effector function of the antibody.
  • the multispecific antibodies provided have at least one of the following properties: (a) specifically bind to PD-L1; (b) specifically bind to the glycoprotein A repeat dominant sequence (GARP); (c) specifically bind to the GARP-TGF- ⁇ complex; (d) specifically bind to TGF- ⁇ ; (e) have active regulatory functions on immune cells, including but not limited to inhibitory activity on the immunosuppressive function of regulatory T cells, etc.; (f) have anti-tumor activity; (g) specifically bind to VEGF; (h) have activity in inhibiting pathological blood vessel growth.
  • GARP glycoprotein A repeat dominant sequence
  • the multispecific antibodies provided by the present invention simultaneously show the activity of specific binding to TGF- ⁇ , GARP and/or GARP-TGF- ⁇ complex, the activity of specific binding to PD-L1, and the activity of specific binding to VEGF.
  • detection methods such as TGF- ⁇ reporter gene experiment and Treg release TGF- ⁇ neutralization experiment, it is determined that the multispecific antibody can not only bind to the GARP-TGF- ⁇ 1 complex, but also capture the differentiated molecules of TGF- ⁇ 1 released into the microenvironment.
  • the multispecific antibodies provided can also specifically bind to VEGF, block the formation of new blood vessels, and thus inhibit tumor growth.
  • the multispecific antibodies provided can specifically bind to PD-L1, and by simultaneously blocking the PD-1/PD-L1 and TGF- ⁇ signaling pathways, immune suppression is relieved, and the possible complementary effects between the PD-L1 (PD-1) axis and the TGF- ⁇ axis pathways are avoided, and the body's immune capacity is restored exponentially, achieving the effect of synergistic killing.
  • the third antigen binding moiety is connected to the carboxyl end (C-terminus) of the Fc region via a linker, as shown in Figure 1. It should be noted that the disulfide bond is not specifically drawn in the schematic drawings of the multispecific antibodies provided herein. Figure 1 shows that a third antigen binding moiety is connected to the carboxyl end (C-terminus) of the Fc region.
  • the third antigen binding moiety is a fusion protein from VEGF trap, including the immunoglobulin domain of VEGFR1 and the immunoglobulin domain of VEGFR2.
  • the multispecific antibody provided may also include a second antigen binding moiety as needed.
  • the second antigen binding moiety is an anti-PD-L1 nanobody.
  • the second antigen binding moiety is connected to the carboxyl end (C-terminus) of the light chain constant domain.
  • the second antigen binding moiety can be directly connected to the carboxyl end of the light chain constant domain, or it can be connected to the carboxyl end of the light chain constant domain through a linker.
  • the second antigen binding portion is connected to the carboxyl terminus (C-terminus) of the light chain constant domain, it can be connected to one anti-PD-L1 nanobody respectively, or it can be connected to two or three anti-PD-L1 nanobodies respectively as needed.
  • the connected nanobodies may be the same or different.
  • the second antigen binding portion is connected to the amino terminus (N-terminus) of the light chain variable region.
  • the second antigen binding portion can be directly connected to the amino terminus of the light chain variable region, or it can be connected to the amino terminus of the light chain variable region through a linker.
  • the second antigen binding portion can be connected to the amino acid of the light chain variable region, it can be connected to one anti-PD-L1 nanobody respectively, or it can be connected to two or three anti-PD-L1 nanobodies respectively as needed.
  • the connected nanobodies may be the same or different. As shown in Figure 2.
  • connection relationship between the second antigen binding moiety and the third antigen binding moiety and the formed IgG structure can be replaced, that is, the second antigen binding moiety can be connected to the carboxyl terminus of the Fc region (directly or through a linker, the structure shown in Figure 3 is connected through a linker), and the third antigen binding moiety can be connected to the carboxyl terminus of the light chain constant domain CL (directly or through a linker, the structure shown in Figure 3 is connected through a linker).
  • the linkers mentioned can be the same or different.
  • the second antigen binding portion can be selected as a single-chain antibody, a single-domain antibody, or other forms of fusion proteins, etc. as needed.
  • the second antigen binding portion connected to the carboxyl terminus of the light chain constant domain may be the same or different, or the second antigen binding portion may be connected only to any one of the carboxyl termini of the light chain constant domain.
  • the carboxyl termini (C-termini) of the light chain constant domain are each connected to two anti-PD-L1 nanobodies.
  • the C-termini of the light chain constant domain may also be connected to an anti-PD-L1 nanobody or to two or more anti-PD-L1 nanobodies, etc.
  • the number of anti-PD-L1 nanobodies connected thereto may be the same or different.
  • the anti-PD-L1 nanobody provided may be a publicly available or commercially available nanobody, or may be obtained by screening an antibody library.
  • linkers are linkers commonly used in the art, and may be some oligopeptides or polypeptides. These oligopeptides or polypeptides may be any amino acid sequence that can provide flexibility.
  • Linkers include, but are not limited to, the following groups: GS, SG, GGS, GSG, SGG, GGG, GGGS, GGGGGSGS, GGGGSGS, GGGGSGGS, GGGGSGGGGSGGGGS, GGGGGGGSGGGGS, GGGGGSGGGGSGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGGGS, GGGGSGGGGSGGGGGGGSGGGGGGGS, GGGGSGGGGSGGGGGGGSGGGGGGGS, GGGGSGGGGSGGGGGGGSGGGGGGGS, GGGGSG
  • the present invention provides a multispecific antibody, comprising a first antigen-binding portion and a third antigen-binding portion;
  • the first antigen-binding portion comprises: the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region as shown in SEQ ID NO: 36 or 37 or 38 or 39 or 40 or 41 or 42 or 43 or 44 or 45 or 46 or 47, and the LCDR1, LCDR2 and LCDR3 sequences of the light chain variable region as shown in SEQ ID NO: 48 or 49 or 50 or 51 or 52 or 53 or 54 or 55 or 56 or 57 or 58 or 59;
  • the third antigen-binding portion is a VEGF receptor fusion protein, an anti-VEGF antibody or antigen-binding fragment or a VEGF binding molecule.
  • the HCDR1, HCDR2 and HCDR3 sequences of the heavy chain variable region and light chain variable region sequences and the LCDR1, LCDR2 and LCDR3 sequences shown are easy to analyze because of the antibody sequence.
  • the CDR sequences may be slightly different depending on the method, such as the classic Kabat CDR definition scheme, IMGT definition scheme, Chothia definition scheme, etc.
  • the different CDR sequences analyzed are all included in the protection scope of the present invention.
  • the present invention also provides a multispecific antibody, comprising a first antigen binding portion and a third antigen binding portion, the first antigen binding portion being capable of binding to TGF- ⁇ , GARP or a GARP-TGF- ⁇ complex, and the third antigen binding portion being capable of binding to VEGF; the first antigen binding portion comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the HCDR sequence shown in SEQ ID NO:1, 2 and 3, or a sequence having one or two amino acid substitutions, deletions or additions with the HCDR sequence shown in SEQ ID NO:1, 2 and 3; or comprising the HCDR sequence shown in SEQ ID NO:4, 5 and 6, or a sequence having one or two amino acid substitutions, deletions or additions with the HCDR sequence shown in SEQ ID NO:4, 5 and 6; or comprising the HCDR sequence shown in SEQ ID NO:7, 8 and 9, or or a sequence having one or two amino acid substitutions, deletions or additions with the HC
  • the first antigen binding portion may further comprise a light chain variable region, the light chain variable region comprising the LCDR sequence shown in SEQ ID NO: 19, 20 and 21, or a sequence having one or two amino acid substitutions, deletions or additions with the LCDR sequence shown in SEQ ID NO: 19, 20 and 21; or comprising the LCDR sequence shown in SEQ ID NO: 22, 23 and 24, or a sequence having one or two amino acid substitutions, deletions or additions with the LCDR sequence shown in SEQ ID NO: 22, 23 and 24; or comprising the LCDR sequence shown in SEQ ID NO: 25, 20 and 26, or a sequence having one or two amino acid substitutions, deletions or additions with the LCDR sequence shown in SEQ ID NO: 25, 20 and 26.
  • the sequences are shown below.
  • the first antigen-binding portion or the third antigen-binding portion of the multispecific antibody mentioned above can be selected as a complete antibody or only a part of the fragment thereof as needed, as long as it can target different target antigens.
  • the first antigen-binding portion or the second antigen-binding portion can be only a heavy chain variable region, Fab, Fab', F(ab') 2 , a single-chain antibody (ScFv), or a single-domain antibody (sdAb), etc.
  • the first antigen binding portion of the multispecific antibody provided can bind to GARP, TGF- ⁇ , or GARP-TGF- ⁇ complex, or can specifically bind to any one, two or three of the three.
  • the third antigen binding portion of the multispecific antibody can bind to VEGF, block the formation of blood vessels, and thus inhibit tumor growth.
  • the third antigen binding portion is a VEGF receptor fusion protein, an anti-VEGF receptor or antigen binding fragment or a VEGF binding molecule.
  • the multispecific antibodies provided have HCDR sequences shown in SEQ ID NOs: 1, 2, and 3 and LCDR sequences shown in SEQ ID NOs: 19, 20, and 21. In some embodiments, the multispecific antibodies provided have SEQ ID NOs: In some embodiments, the multispecific antibodies provided have the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6 and the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24. In some embodiments, the multispecific antibodies provided have the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9 and the LCDR sequences shown in SEQ ID NOs: 25, 20 and 26.
  • the multispecific antibodies provided have the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12 and the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29. In some specific embodiments, the multispecific antibodies provided have the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15 and the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32. In some specific embodiments, the multispecific antibodies provided have the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18 and the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35. The HCDR sequences and LCDR sequences shown as the first antigen binding portion of the multispecific antibodies specifically bind to the target antigen GARP, TGF- ⁇ or the GARP-TGF- ⁇ complex.
  • the multispecific antibody provided comprises at least one of the following: a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% affinity to the sequence shown in SEQ ID NO:36 or 37 or 38 or 39 or 40 or 41 or 42 or 43 or 44 or 45 or 46 or 47.
  • sequence identity to a heavy chain variable region, and a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence shown in SEQ ID NO: 48 or 49 or 50 or 51 or 52 or 53 or 54 or 55 or 56 or 57 or 58 or 59.
  • SEQ ID NO: 42, 43, 44, 45, 46 and 47 are humanized heavy chain variable region sequences, respectively.
  • SEQ ID NO: 54, 55, 56, 57, 58 and 59 are humanized light chain variable region sequences, respectively.
  • the heavy chain variable region and light chain variable region sequences shown are used as the first antigen binding portion of the multispecific antibody, specifically binding to the target antigen. After humanization, the sequences of the light chain variable region and the heavy chain variable region of the first antigen binding portion have lower immunogenicity and retain binding activity with the target antigen (such as GARP, TGF- ⁇ , and/or GARP-TGF- ⁇ complex).
  • the humanized antibody sequence can be obtained by methods commonly used in the art (such as complementary determining region transplantation). In some embodiments, the humanized antibody sequence provided retains the CDR region sequence and replaces the framework region sequence with a human framework region sequence.
  • the human framework region sequence can be obtained by published human sequences.
  • a human FR region template with the greatest homology is found, and a comprehensive consideration is given to determine whether the FR region needs to be reverse mutated and the key residues for restoring the mutation, to obtain a high-affinity humanized antibody.
  • sequences are usually recorded in commonly used databases, such as the PDB protein structure database, IMGT, Genebank and other databases.
  • Sequence identity refers to the degree of sequence identity by comparing amino acid or nucleotide sequences one by one. In order to determine the ratio of sequence identity, it can be achieved in a variety of ways known in the art. For example, publicly available software such as BLAST, ALIGN, BLAST-2 and other software can be used to obtain. In some embodiments, sequence identity is due to conservative amino acid substitution. "Conservative amino acid substitution” refers to the replacement of another amino acid residue with a different amino acid residue having a side chain with similar physiological and chemical properties.
  • conservative amino acid substitutions can occur between amino acid residues with hydrophobic side chains (such as Met, Ala, Val, Leu and Ile), conservative amino acid substitutions can occur between residues with neutral hydrophilic side chains (such as Cys, Ser, Thr, Asn and Gln), conservative amino acid substitutions can occur between residues with acidic side chains (such as Asp, Glu), conservative amino acid substitutions can occur between amino acids with basic side chains (such as His, Lys and Arg), or conservative amino acid substitutions can be performed between residues with aromatic side chains (such as Trp, Tyr and Phe).
  • conservative amino acid substitutions usually do not cause significant changes in protein conformational structure, and therefore can retain the biological activity of the protein.
  • the conservative amino acid substitutions mentioned can be 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, 9 conservative amino acid substitutions or 10 conservative amino acid substitutions, etc.
  • the names of amino acids used herein are represented by standard single-letter or three-letter codes commonly used in the art.
  • the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:36 and a light chain variable region as set forth in SEQ ID NO:48. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:37 and a light chain variable region as set forth in SEQ ID NO:49. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:38 and a light chain variable region as set forth in SEQ ID NO:50. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:39 and a light chain variable region as set forth in SEQ ID NO:51.
  • the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:40 and a light chain variable region as set forth in SEQ ID NO:52. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:41 and a light chain variable region as set forth in SEQ ID NO:53. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:42 and a light chain variable region as set forth in SEQ ID NO:54. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:43 and a light chain variable region as set forth in SEQ ID NO:55.
  • the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:44 and a light chain variable region as set forth in SEQ ID NO:56. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:57. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:46 and a light chain variable region as set forth in SEQ ID NO:58. In some embodiments, the first antigen binding portion comprises a heavy chain variable region as set forth in SEQ ID NO:47 and a light chain variable region as set forth in SEQ ID NO:59.
  • the third antigen binding portion is capable of specifically binding to VEGF.
  • the third antigen binding portion is a VEGF receptor fusion protein, an anti-VEGF antibody or antigen binding fragment or a VEGF binding molecule.
  • the third antigen binding portion includes at least two different VEGFR immunoglobulin-like domains.
  • the third antigen binding portion is: (i) a VEGF receptor fusion protein comprising two polypeptides, having an immunoglobulin domain (Ig) of VEGFR1 and/or an immunoglobulin domain of VEGFR2 and/or an immunoglobulin domain of VEGFR3; (ii) a VEGF receptor fusion protein comprising two polypeptides, having an immunoglobulin-like domain 2 (domain 2) of VEGFR1 and an Ig domain 3 (domain 3) of VEGFR2; (iii) a VEGF receptor fusion protein comprising two polypeptides, having an immunoglobulin-like domain 2 (domain 2) of VEGFR1, an Ig domain 3 (domain 3) of VEGFR2, and an Ig domain 4 (domain 4) of VEGFR2.
  • a VEGF receptor fusion protein comprising two polypeptides, having an immunoglobulin domain (Ig) of VEGFR1 and/or an immunoglobulin domain of VEGFR2 and/or an immunoglobulin
  • the third antigen binding portion mentioned is derived from a fusion protein of VEGF trap.
  • VEGF trap is a fusion protein containing two different VEGFR extracellular domains, and is a soluble receptor formed by the fusion of the constant region of IgG (IgG-Fc1) and two different VEGFRs (domain 2 of VEGFR1, domain 3 of VEGFR2).
  • IgG-Fc1 constant region of IgG
  • VEGFRs domain 2 of VEGFR1, domain 3 of VEGFR2
  • VEGF trap blocks the normal physiological activity of VEGF, and cannot form new blood vessels, thereby inhibiting tumor growth.
  • VEGF trap can effectively block the formation of new blood vessels, so that tumor tissue cannot grow normally due to lack of vascular nutrition.
  • VEGF trap can also be used for pathological blood vessel growth in other diseases besides tumors, such as diabetic retinopathy, psoriasis, and pathological corneal angiogenesis caused by transplantation, infection or trauma.
  • the third antigen binding portion is derived from VEGF trap, which has two different VEGFRs (domain 2 of VEGFR1, domain 3 of VEGFR2). According to a specific embodiment, the third antigen binding portion has the sequence shown in SEQ ID NO:92.
  • the multispecific antibody provided may also include a second antigen binding portion as needed.
  • the second antigen binding portion can specifically target PD-L1.
  • the anti-tumor activity of PD-1/PD-L1 resistance can be improved.
  • its expression form is not limited.
  • it can be a complete antibody or a part of a complete antibody, including but not limited to a heavy chain variable region, Fab, Fab', F(ab') 2 , a single-chain antibody or a nanobody, etc.
  • the second antigen binding portion can be obtained by antibody library screening as needed, or it can be a commercially available or publicly disclosed antibody sequence.
  • the second antigen binding portion is a single domain antibody.
  • the structure of the bispecific antibody formed is a structure in which an IgG-type antibody and a second antigen binding portion are connected by a linker, and the IgG-type antibody comprises a first antigen binding portion.
  • the second antigen binding portion is connected to the IgG-type antibody to form a multispecific antibody structure.
  • the second antigen binding portion is connected to the carboxyl end of the light chain constant domain.
  • the second antigen binding portion is connected to the carboxyl end of the Fc region.
  • the second antigen binding portion provided comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises a sequence selected from the group consisting of SEQ ID NO: 60, 63 and 66, or a sequence having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO: 60, 63 and 66; CDR2 comprises a sequence selected from the group consisting of SEQ ID NO: 61, 64 and 67, or a sequence having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO: 61, 64 and 67; and CDR3 comprises a sequence selected from the group consisting of SEQ ID NO: 62, 65, 68 and 69, or a sequence having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NO: 62, 65, 68 and 69.
  • CDR1 comprises a sequence selected from the group consisting of SEQ ID NO: 60, 63 and 66, or a sequence having one or two amino acid substitutions,
  • the second antigen binding portion has a CDR1 sequence as shown in SEQ ID NO:60, a CDR2 sequence as shown in SEQ ID NO:61, and a CDR3 sequence as shown in SEQ ID NO:62. In some embodiments, the second antigen binding portion has a CDR1 sequence as shown in SEQ ID NO:63, a CDR2 sequence as shown in SEQ ID NO:64, and a CDR3 sequence as shown in SEQ ID NO:65. In some embodiments, the second antigen binding portion has a CDR1 sequence as shown in SEQ ID NO:66, a CDR2 sequence as shown in SEQ ID NO:67, and a CDR3 sequence as shown in SEQ ID NO:68.
  • the second antigen binding portion has a CDR1 sequence as shown in SEQ ID NO:66, a CDR2 sequence as shown in SEQ ID NO:67, and a CDR3 sequence as shown in SEQ ID NO:68.
  • the second antigen binding portion has a CDR1 sequence shown in SEQ ID NO: 60, a CDR2 sequence shown in SEQ ID NO: 61, and a CDR3 sequence shown in SEQ ID NO: 69.
  • the CDR1 sequence, CDR2 sequence, and CDR3 sequence shown can form a single domain antibody with the framework region FR.
  • the framework region FR sequence is not particularly limited and can be mouse-derived, human-derived, or partially mouse-derived and partially human-derived.
  • the second antigen binding portion further includes a framework region FR, the framework region includes FR1, FR2, FR3 and FR4, and the FR1, FR2, FR3 and FR4 respectively include the following amino acid sequences: (1) FR1 includes a sequence selected from SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, or a sequence having one, two or three conservative amino acid substitutions compared to SEQ ID NO:70, 74, 77 or 80; (2) FR2 includes a sequence selected from SEQ ID NO:71, SEQ ID NO:75, (3) FR3 comprises a sequence selected from SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 81, or a sequence having one, two, three or four conservative amino acid substitutions compared to SEQ ID NO: 72, 76, 79 or 81; (4) FR4 is selected from SEQ ID NO: 73, or a sequence
  • the second antigen binding portion further includes a framework region FR, which includes FR1, FR2, FR3 and FR4.
  • the framework region includes the FR1 sequence shown in SEQ ID NO: 70, the FR2 sequence shown in SEQ ID NO: 71, the FR3 sequence shown in SEQ ID NO: 72, and the FR4 sequence shown in SEQ ID NO: 73.
  • the framework region includes the FR1 sequence shown in SEQ ID NO: 74, the FR2 sequence shown in SEQ ID NO: 75, the FR3 sequence shown in SEQ ID NO: 76, and the FR4 sequence shown in SEQ ID NO: 73.
  • the framework region includes the FR1 sequence shown in SEQ ID NO: 77, the FR2 sequence shown in SEQ ID NO: 78, the FR3 sequence shown in SEQ ID NO: 79, and the FR4 sequence shown in SEQ ID NO: 73.
  • the framework region includes the FR1 sequence shown in SEQ ID NO: 80, the FR2 sequence shown in SEQ ID NO: 78, the FR3 sequence shown in SEQ ID NO: 79, and the FR4 sequence shown in SEQ ID NO: 73.
  • the framework region includes the FR1 sequence shown in SEQ ID NO: 70, the FR2 sequence shown in SEQ ID NO: 71, the FR3 sequence shown in SEQ ID NO: 81, and the FR4 sequence shown in SEQ ID NO: 73.
  • the second antigen binding portion is selected from a sequence as shown in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91.
  • the second antigen binding portion has a sequence identity of 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% compared to the amino acid sequence shown in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91.
  • the amino acid sequence shown in SEQ ID NO: 87, 88, 89, 90 or 91 provided is a humanized antibody sequence.
  • the provided humanized sequence has low immunogenicity and high affinity with the target protein.
  • the present invention also provides a multispecific antibody, comprising: a first antigen binding portion, a second antigen binding portion and a third antigen binding portion, wherein the first antigen binding portion can bind to TGF- ⁇ , GARP and/or GARP-TGF- ⁇ complex, the second antigen binding portion can bind to PD-L1, and the third antigen binding portion is a VEGF receptor fusion protein, an anti-VEGF antibody or antigen binding fragment or a VEGF binding molecule.
  • the first antigen binding moiety comprises the heavy chain variable region shown in SEQ ID NO: 36, and the light chain variable region shown in SEQ ID NO: 48;
  • the second antigen binding moiety comprises the sequence shown in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding moiety comprises the sequence shown in SEQ ID NO: 92.
  • the first antigen binding moiety comprises the heavy chain variable region shown in SEQ ID NO: 37, and the light chain variable region shown in SEQ ID NO: 49;
  • the second antigen binding moiety comprises the sequence shown in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding moiety comprises the sequence shown in SEQ ID NO: 92.
  • the first antigen binding portion comprises the heavy chain variable region of SEQ ID NO:38 and the light chain variable region of SEQ ID NO:50; the second antigen binding portion comprises the sequence of SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91; the third antigen binding portion comprises the sequence of SEQ ID NO:92.
  • the first antigen binding portion comprises SEQ ID NO:39
  • the second antigen binding portion comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91; the third antigen binding portion comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding portion comprises the heavy chain variable region shown in SEQ ID NO:40 and the light chain variable region shown in SEQ ID NO:52;
  • the second antigen binding portion comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding portion comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding moiety comprises the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:53;
  • the second antigen binding moiety comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding moiety comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding moiety comprises the heavy chain variable region shown in SEQ ID NO:42 and the light chain variable region shown in SEQ ID NO:54;
  • the second antigen binding moiety comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding moiety comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding portion comprises the heavy chain variable region shown in SEQ ID NO:43 and the light chain variable region shown in SEQ ID NO:55;
  • the second antigen binding portion comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding portion comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding portion comprises the heavy chain variable region shown in SEQ ID NO:44 and the light chain variable region shown in SEQ ID NO:56;
  • the second antigen binding portion comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding portion comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding moiety comprises the heavy chain variable region shown in SEQ ID NO:45 and the light chain variable region shown in SEQ ID NO:57;
  • the second antigen binding moiety comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding moiety comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding moiety comprises the heavy chain variable region shown in SEQ ID NO:46 and the light chain variable region shown in SEQ ID NO:58;
  • the second antigen binding moiety comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding moiety comprises the sequence shown in SEQ ID NO:92.
  • the first antigen binding portion comprises the heavy chain variable region shown in SEQ ID NO:47 and the light chain variable region shown in SEQ ID NO:59;
  • the second antigen binding portion comprises the sequence shown in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91;
  • the third antigen binding portion comprises the sequence shown in SEQ ID NO:92.
  • the present invention also provides a polynucleotide encoding the multispecific antibody.
  • the polynucleotide mentioned is separable, including but not limited to DNA, RNA or cDNA, etc.
  • the isolated polynucleotide sequence can be obtained by conventional methods in the art to encode the multispecific antibody or monoclonal antibody or antigen-binding fragment.
  • the polynucleotide sequence encoding the antibody can be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system.
  • the present invention also provides a construct comprising the polynucleotide described above.
  • a variety of methods commonly used in the art can be used to obtain the construct, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.
  • the polynucleotide can be inserted into the multiple cloning site of the expression vector to form a construct.
  • the construct can contain a variety of operating factors such as promoters, terminators, marker genes, etc.
  • the promoter is usually used to provide a signal to start transcription.
  • the promoter can select lactose promoter (Lac), Trp promoter, Tac promoter, PL and PR promoters of bacteriophage as needed; the terminator provides a signal for transcription termination during the transcription process, and the marker gene on the construct is often used for screening.
  • lactose promoter Lac
  • Trp promoter Trp promoter
  • Tac promoter Tac promoter
  • PL and PR promoters of bacteriophage as needed
  • the terminator provides a signal for transcription termination during the transcription process, and the marker gene on the construct is often used for screening.
  • enhancers as needed to enhance protein expression.
  • the expression vector is not particularly limited, and can be some commercially available expression vectors, or it can be an expression vector artificially modified as needed, such as a plasmid, a bacteriophage, a virus, etc.
  • the virus can be a plant cell virus, a mammalian cell virus, etc
  • the present invention also provides a host cell containing the above-mentioned polynucleotide or the above-mentioned construct.
  • Any cell suitable for expressing antibodies or proteins with polynucleotides or constructs can be used as a host cell.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a yeast cell, a mammalian cell, etc. Commonly used host cells can be yeast cells, CHO, HEK-293 cells, 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, etc.
  • the present invention also provides a pharmaceutical composition, comprising: the multispecific antibody described in any one of the first aspects above, and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier mentioned 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 disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sodium acetate, citric acid, sodium citrate, citrate, Tris; sugars, such as trehalose, polysorbate, sucrose, mannitol; surfactants such as polysorbate; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine or lysine, etc.
  • Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, such as by filtering with a sterile filter membrane. Those skilled in the art can select different pharmaceutically acceptable carriers for the pharmaceutical composition as needed to prepare different dosage forms, such as freeze-dried dosage forms, injections and other dosage forms.
  • the prepared different pharmaceutical dosage forms can be formulated for administration to a subject by any suitable route, including but not limited to intravenous, dermal, intramuscular, peritoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, etc.
  • the present invention also provides an antibody conjugate, the antibody conjugate includes the multi-specific antibody, and a functional small molecule or protease connected to the multi-specific antibody.
  • the functional small molecule mentioned can be a developed or undeveloped small molecule drug, which can enhance the therapeutic effect of anti-tumor drugs and reduce adverse reactions by chemically coupling with the multi-specific antibody.
  • Small molecules such as toxins, chemotherapeutic drugs, and photosensitizers can also be coupled to the antibodies mentioned through a connector.
  • the antibody Protac conjugate can also be obtained by coupling the antibody with a protac molecule, such as connecting the antibody and a protease targeting ligand (such as an E3 ligase ligand) through a connector, shortening the distance between the target protein and the E3 ubiquitin ligase in the cell, and utilizing the ubiquitin-proteasome pathway to specifically degrade the target protein.
  • a protac molecule such as connecting the antibody and a protease targeting ligand (such as an E3 ligase ligand) through a connector, shortening the distance between the target protein and the E3 ubiquitin ligase in the cell, and utilizing the ubiquitin-proteasome pathway to specifically degrade the target protein.
  • the present invention also provides a kit, which includes the above-mentioned multispecific antibody.
  • the kit may also include a container, a buffer reagent, and a control such as a positive control and a negative control as needed. Those skilled in the art can make corresponding selections as needed. Accordingly, the kit may also include instructions for use to facilitate operation and use by those skilled in the art.
  • the present invention further provides a method for producing the multispecific antibody, comprising: culturing the host cell, and collecting the multispecific antibody from the culture.
  • the multispecific antibodies collected from the culture can be purified to obtain a substantially pure product.
  • substantially pure means that the purity of the multispecific antibodies is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or even greater than 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.
  • the present invention provides a method for preventing and/or treating a disease, comprising: administering an effective amount of the above-mentioned multispecific antibody to a subject in need thereof.
  • the "therapeutically effective amount” mentioned can lead to a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic periods of the disease, or prevent a reduction in pain caused by the disease.
  • a “preventive effective amount” is usually lower than a therapeutic effective amount.
  • the inhibition rate of cells in the subject's body is 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or even 90% or more or 95% or more compared to a subject who has not been treated with the antibody.
  • the subject mentioned can be an animal or a human. For example, it can be a mammal, including cattle, sheep, mice, horses, etc.
  • the multispecific antibodies provided can be used to treat diseases including but not limited to cancer and autoimmune diseases.
  • the methods provided by the present invention can be used to treat TGF- ⁇ -related diseases, GARP-related diseases, VEGF-related diseases, and PD-1/PD-L1-related diseases.
  • TGF- ⁇ -related diseases include but are not limited to inflammatory diseases, chronic infections, cancer, fibrosis, cardiovascular diseases, cerebrovascular diseases, and neurodegenerative diseases.
  • the cancers mentioned include but are not limited to lung cancer, colon cancer, kidney cancer, urothelial cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, and blood cancers, etc.
  • pathological vascular growth of other diseases other than tumors can also be treated, such as diabetic retinopathy, psoriasis, and pathological corneal angiogenesis caused by transplantation, infection or trauma, and the corresponding diseases can be treated.
  • the present invention also provides the use of multispecific antibodies in preparing drugs or kits.
  • the drugs are used to treat cancer.
  • the provided multispecific antibodies can be used to prepare drugs for treating various diseases. They can also be used to prepare kits for use as immunodiagnostic reagents.
  • the sequences of the first antigen binding portion and the second antigen binding portion mentioned are recorded in the text of the international patent application with application number PCT/CN2022/122367.
  • the contents recorded may be cited in whole or in part herein as needed.
  • the TGF- ⁇ , GARP or GARP-TGF- ⁇ targeting sequence derived from the first antigen binding portion, the sequence preparation and the method of obtaining the sequence are recorded in the text of the international patent application with application number PCT/CN2022/122367.
  • the PD-L1 binding portion derived from the second antigen binding portion, the sequence preparation and the method of obtaining the sequence are recorded in the text of the international patent application with application number PCT/CN2022/122367 and the text of the international application with application number PCT/CN2022/110423.
  • the contents recorded therein are partially cited below.
  • the sequence derived from the first antigen binding part is obtained by hybridoma.
  • Mice are immunized with human antigens (GARP, TGF- ⁇ and GARP-TGF- ⁇ complex) to produce specific antibodies in the mice.
  • Mouse myeloma cells and mouse spleen cells are fused and the fused cells are plated.
  • HAT selective medium is added for screening, and myeloma cells cannot survive, and spleen cells have a short survival time.
  • myeloma cells fused with lymphocytes survive, that is, hybridoma cells are screened.
  • monoclonal cells are screened by limiting dilution cloning cells and sequenced.
  • the obtained different antibodies are expressed and purified using a mammalian cell system to obtain different antibodies with a purity of at least 90%. They are named m212 antibody, m305 antibody, m107 antibody, m202 antibody, m301 antibody and m109 antibody respectively.
  • the m212 antibody contains the heavy chain variable region shown in SEQ ID NO:36 and the light chain variable region shown in SEQ ID NO:48.
  • the m305 antibody comprises a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:49.
  • the m107 antibody comprises a heavy chain variable region as shown in SEQ ID NO:38 and a light chain variable region as shown in SEQ ID NO:50.
  • the m202 antibody comprises a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:51.
  • the m301 antibody comprises a heavy chain variable region as shown in SEQ ID NO:40 and a light chain variable region as shown in SEQ ID NO:52.
  • the m109 antibody comprises a heavy chain variable region as shown in SEQ ID NO:41 and a light chain variable region as shown in SEQ ID NO:53.
  • Humanized antibodies were obtained by complementary determining region grafting. Accordingly, the humanized antibodies were numbered m212-hu, m305-hu, m107-hu, m202-hu, m301-hu and m109-hu.
  • the m212-hu antibody comprises a heavy chain variable region as shown in SEQ ID NO:42 and a light chain variable region as shown in SEQ ID NO:54.
  • the m305-hu antibody comprises a heavy chain variable region as shown in SEQ ID NO:43 and a light chain variable region as shown in SEQ ID NO:55.
  • the m107-hu antibody comprises a heavy chain variable region as shown in SEQ ID NO:44 and a light chain variable region as shown in SEQ ID NO:56.
  • the m202-hu antibody comprises a heavy chain variable region as shown in SEQ ID NO:45 and a light chain variable region as shown in SEQ ID NO:57.
  • the m301-hu antibody comprises a heavy chain variable region as shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:58.
  • the m109-hu antibody contains the heavy chain variable region shown in SEQ ID NO:47 and the light chain variable region shown in SEQ ID NO:59.
  • the binding activity of antibodies to 293-GARP/TGF- ⁇ stable cell lines overexpressing GARP-TGF- ⁇ complex was tested by FACS detection.
  • the results showed that m212 antibody, m305 antibody, m107 antibody, m202 antibody, m301 antibody and m109 antibody were able to bind to GARP-TGF- ⁇ complex, and the corresponding EC50 values were: ⁇ 2nM, 4.1nM, 3.2nM, 2.7nM, 2.5nM, >5nM;
  • the EC50 values of humanized antibodies m212-hu, m305-hu, m107-hu, m202-hu, m301-hu and m109-hu were: ⁇ 2nM, 2.1nM, 7.5nM, 2.5nM, 4.0nM, >5nM.
  • CHO-K1-GARP cell line that overexpresses GARP
  • FACS detection revealed that the m212 antibody, m305 antibody, and m107 antibody can all specifically bind to the GARP protein, with the corresponding EC50 values of 0.46nM, ⁇ 1nM, and >2nM, respectively; the corresponding EC50 values of the humanized antibodies m212-hu and m305-hu are 1.4nM and 0.45nM, respectively.
  • Human transforming growth factor ⁇ 1 was used to coat the cell culture plate, and the binding activity of different antibodies to TGF- ⁇ was detected by ELISA. The results showed that the m107 antibody, m202 antibody, m301 antibody, and m109 antibody could specifically bind to TGF- ⁇ .
  • the corresponding EC50 values were 0.021nM, ⁇ 1nM, ⁇ 1nM, and ⁇ 1nM, respectively; the EC50 values of humanized antibodies m107-hu, m202-hu, m301-hu, and m109-hu were 7.5nM, ⁇ 2nM, ⁇ 2nM, and 0.73nM, respectively.
  • TGF- ⁇ secretion neutralization experiment it was found that these antibodies could inhibit or neutralize TGF- ⁇ produced by Treg cells.
  • PD-L1 nanoantibodies were obtained by alpaca immunization to obtain anti-PD-L1 nanolibrary, which was screened and identified to obtain candidate nanoantibodies.
  • the fusion protein composed of the extracellular domain sequence of human PD-L1 protein and the Fc region sequence of human immunoglobulin was mixed with Freund's adjuvant, emulsified, and immunized into healthy alpacas to stimulate B cells to express antigen-specific nanoantibodies. 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 fragments were obtained by PCR amplification from the reverse transcribed cDNA.
  • the VHH gene fragment and the yeast display vector were co-electroporated into competent yeast, and the yeast homologous recombination enzyme connected the fragment to the vector to form a complete plasmid, and a yeast transformant library with high library insertion rate and excellent diversity was established, and the passage was stable in nutrient-deficient medium.
  • the yeast cells expressing the antibody were co-incubated with magnetic beads enriched with target protein antigens, and after multiple enrichment cultures, the magnetic bead enrichment products were identified by flow cytometry and sorted. After multiple rounds of screening, five highly expressed positive clones were obtained and named Antibody A to Antibody E.
  • the amino acid sequence is shown in SEQ ID NO: 82, the amino acid sequence of antibody B is shown in SEQ ID NO: 83, the amino acid sequence of antibody C is shown in SEQ ID NO: 84, the amino acid sequence of antibody D is shown in SEQ ID NO: 85, and the amino acid sequence of antibody E is shown in SEQ ID NO: 86.
  • the humanized antibodies are antibody Ahu, with an amino acid sequence shown in SEQ ID NO: 87; antibody Bhu, with an amino acid sequence shown in SEQ ID NO: 88; antibody Chu, with an amino acid sequence shown in SEQ ID NO: 89; antibody D1hu, with an amino acid sequence shown in SEQ ID NO: 90; and antibody D2hu, with an amino acid sequence shown in SEQ ID NO: 91.
  • the binding activity of antibodies and human PD-L1 is detected by FACS.
  • the experimental process is: the affinity of different antibodies to antigens is detected by flow cytometry. MDA-MB-231 cells endogenously expressing PD-L1 antigen are added to a 96-well plate at 1 ⁇ 10 5 cells/well. Then different concentrations of samples are added and incubated at 4 degrees Celsius for 30 minutes. Then a fluorescently labeled goat anti-human IgG secondary antibody (manufacturer: Abcam) is added to detect antibodies bound to the cell surface.
  • the antibody-antigen binding dose-response curve is generated using geometric values, and the original data of the four parameters are plotted using Graphpad Prism V6.0 software to determine the EC50 result of antibody binding to antigen.
  • the experimental results showed that the EC50 values of antibody A, antibody B, antibody C, antibody D and antibody E were 0.089nM, 0.031nM, 0.042nM, 0.039nM and 0.053nM, respectively, which were similar to the positive control (atezolizumab, 0.059nM), showing high binding activity.
  • the binding activity of humanized antibodies was determined by a similar method, and the EC50 values of antibody Ahu, antibody Bhu, antibody Chu, antibody D1hu and antibody D2hu were 0.051nM, 0.18nM, 0.025M, 0.18nM and 0.29nM, respectively.
  • Another example is to determine the blocking activity of antibodies for PD-1/PD-L1 by FACS.
  • the experimental process is: using a competitive flow cytometry-based method to detect the blocking effect of antibodies on PD-1 and its ligand PD-L1. Resuscitate the 293T-PD-1 cell line overexpressing PD-1 (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.), plate the cells at 1 ⁇ 10 5 /well in a 96-well plate, mix different concentrations of antibody dilutions (starting working concentration is 200nM, 4-fold dilution) and PD-L1-Biotin dilution (2ug/ml, 50 ⁇ l/well), and incubate at room temperature for 30 minutes.
  • the experimental results show that the IC50 values of antibody A, antibody B, antibody C, antibody D and antibody E are 1.024nM, 2.077nM, 1.678nM, 2.103nM, and 2.536nM, respectively, and the antibodies can block the binding of antigens and their ligands.
  • a similar method was used to determine the blocking activity of humanized antibodies, and the IC50 values of antibody Ahu, antibody Bhu, antibody Chu, antibody D1hu, and antibody D2hu were 4.0 nM, 3.4 nM, 1.0 nM, 2.6 nM, and 12 nM, respectively.
  • the blocking activity of the antibody for CD-80/PD-L1 was determined by FACS.
  • the experimental process was as follows: the blocking effect of the antibody on PD-L1 and CD-80 was detected by a competitive flow cytometry-based method.
  • the 293T-PD-L1 cell line overexpressing PD-L1 (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.) was revived, and the cells were plated in a 96-well plate at 1 ⁇ 10 5 per well.
  • Different concentrations of antibody dilutions starting working concentration was 200nM, 4-fold dilution) were added to the cells and incubated at 4°C for 60 minutes.
  • the experimental results show that the IC50 values of antibody A, antibody B, antibody C, antibody D and antibody E are 3.954nM, 3.355nM, 4.146nM, 4.757nM, and 2.979nM, respectively, and the antibodies can block the binding of antigen and its ligand.
  • a similar method was used to determine the blocking activity of humanized antibodies.
  • the IC50 values of antibody Ahu, antibody Bhu, antibody Chu and antibody D1hu were approximately 1.8nM, 1.7nM, 3.2nM and 1.5nM, respectively.
  • the positive control 1 (aflibercept) used is a fusion protein containing Fc, which can specifically target VEGFA and VEGFB.
  • the positive control 2 (bevacizumab) used is bevacizumab, which is a humanized IgG1 type vascular endothelial growth factor (VEGF) inhibitor.
  • VEGF vascular endothelial growth factor
  • Example 1 mainly describes how to construct a bispecific antibody expressing anti-GARP, anti-TGF- ⁇ , GARP-TGF- ⁇ complex and VEGF, and construct a trispecific antibody expressing anti-GARP, anti-TGF- ⁇ , GARP-TGF- ⁇ complex, anti-VEGF and anti-PD-L1.
  • Bispecific antibodies are named with b at the beginning, to distinguish monoclonal antibodies starting with and m, and trispecific antibodies are named with t at the beginning.
  • the two antigen-binding parts are connected with "-", and the two antigen-binding parts still retain the previous numbering.
  • the antibody number "b301-hu-Vtrap” represents a bispecific antibody, which is completed by connecting the antibody sequence of m301-hu and the antibody sequence from Vtrap, and the bispecific antibody structure formed is shown in Figure 1.
  • the antibody number "t301-hu-VTrap-D2hu” represents a trispecific antibody, which is completed by connecting the antibody sequence of m301-hu and the antibody sequence from Vtrap and the antibody sequence of D2hu, and the trispecific antibody structure formed is shown in Figure 2.
  • N or G 4 S is connected to the antibody number through "-" it means that the linkers used are different.
  • the second antigen-binding portion and the third antigen-binding portion are connected to the formed IgG type antibody structure through (G 4 S) 4.
  • N is connected to the antibody number through "-", for example, "b301-hu-Vtrap-N"
  • G 4 S is connected to the antibody number through "-"
  • b301-hu-Vtrap-G 4 S it means that the second antigen-binding portion and the third antigen-binding portion are connected to the formed IgG type antibody structure through one G 4 S.
  • the sequence of Vtrap in the following antibody numbering is the sequence shown in SEQ ID NO:92.
  • Example 2 The binding activity of the prepared bispecific antibody and trispecific antibody to VEGF was characterized by ELISA.
  • the experimental contents are as follows:
  • VEGF plays a certain role between tumor cells and lymphatic epithelial cells, allowing the two to regulate each other, which can promote the generation of new lymphatic vessels. The increase in lymphatic vessels makes it easier for tumors to metastasize.
  • a VEGF response element was inserted into 293 cells. Under VEGF stimulation, the VEGF signaling pathway was activated, inducing the expression of luciferase, and releasing fluorescent signals under the action of fluorescent substrates. After adding antibodies to block the binding of VEGF to its receptor, the VEGF signaling pathway was inhibited, thereby inhibiting the fluorescent signal.
  • the binding activity of the prepared bispecific and trispecific antibodies to VEGF was characterized by a reporter gene blocking experiment. The experimental content is as follows:
  • Culture 293-VEGF-Res cells (purchased from Nearshore Bio), then plate the cells for later use. Add antibody solutions of different concentrations to the cell culture plate. Prepare VEGF protein dilutions of different concentrations. Mix the antibody solution and protein dilution, and incubate at 37°C for 30 minutes. Then add the diluted 293-VEGF-res cells to the VEGF/antibody mixture and incubate at 37°C for 6 hours. Then add One-Glo reagent (One Glo luciferase assay kit, purchased from Progema), incubate for 5-10 minutes, and read the fluorescence value with an enzyme reader. The experimental results are shown in Table 2 and Figure 5.
  • the experimental results show that the VEGF reporter gene blocking activity of the prepared bispecific antibody or trispecific antibody is similar to that of the control aflibercept.
  • Example 4 characterized the binding activity of the prepared bispecific and trispecific antibodies to GARP/TGF- ⁇ through a reporter gene blocking experiment.
  • GARP, LAP, and ⁇ v ⁇ 6 genes were inserted into the 293F cell line. During the growth process, the cell line will shear the LAP protein through the tension of the GARP-LAP- ⁇ v ⁇ 6 complex, thereby releasing TGF- ⁇ .
  • the TGF- ⁇ After the released TGF- ⁇ enters the 293-SBE-res (1E9) cells inserted with the TGF- ⁇ signal response element, it will activate the TGF ⁇ signaling pathway and induce the release of luciferase, which will then release fluorescence under the action of the fluorescent substrate. When the GARP/TGF ⁇ antibody is added, the TGF ⁇ signaling pathway is blocked, thereby reducing the fluorescence signal.
  • the experimental steps are as follows:
  • Culture 293F-GARP/TGF- ⁇ -av ⁇ 6-4D11 cells add cells (cell density 0.6x10 6 /mL) to the cell culture plate at 50ul/well; continue to add 293-SBE-res cells to the cell culture plate. Then add different concentrations of the antibody to be tested to the cell culture plate, mix well and culture in an incubator. Add luciferase substrate One-Glo TM reagent to each well, incubate in dark for 5-10min, and read the luminescent signal value using an enzyme reader. Use data processing software Graphpad Prism6.0 to calculate the IC50 value of the sample to be tested. As shown in Table 3 and Figure 6 below.
  • Example 5 The blocking effect of the antibody on the PD-L1/PD-1 pathway was studied by reporter gene experiments. The experiment was completed with the help of two cell lines.
  • Jurkat-PD1-CD3zeta-NFAT-Luc2 cell line (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.) stably expresses a fusion protein composed of PD-1ECD and CD3zeta in Jurkat cells, and inserts a luciferase reporter gene driven by NF-AT; 293T-hPD-L1 cells (manufacturer: Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd.), that is, 293T cells expressing human PD-L1 When the two cells are co-cultured, the PD-1/PD-L1 interaction serves as the first signal, and the intracellular CD3zeta chain serves as the second signal to transmit the activation signal inward, and the NFAT-driven luciferase reporter
  • 293T-hPD-L1 cells were plated in a 96-well plate at 2 ⁇ 10 4 cells/well, and Jurkat-PD1-CD3zeta-NFAT-Luc2 effector cells were added to the 96-well plate at 2 ⁇ 10 4 /well; then different concentrations of the antibody to be tested (starting concentration was 60ug/ml, 4-fold gradient dilution) were added and incubated at 37°C for 18-24h. 100 ⁇ l of luciferase substrate ONE-Glo TM Luciferase Assay system detection reagent was added to each well and incubated in the dark for 5 minutes; the microplate reader read the fluorescence signal in the 96-well plate.
  • Example 6 evaluates the effect of bispecific antibodies or trispecific antibodies on HUVEC cell migration.
  • HUVEC cells express VEGFRI and VEGFRII receptors on their surface. When HUVEC cells are stimulated by VEGF, they activate downstream signaling pathways by binding to their receptors, thereby mediating the growth and migration of HUVEC cells. When antibodies are added to block the binding of VEGF to its receptors, VEGF growth signals are inhibited and cell proliferation is inhibited.
  • the experimental contents are as follows:
  • HUVEC cells were plated in a 96-well culture plate at 100uL/well. Different concentrations of antibodies were added to a new cell culture plate, and different concentrations of diluted VEGF were added and incubated at 37°C for 1 hour. Then the antigen-antibody mixture was added to the HUVEC cell culture plate; the test was performed after incubation at 37°C, 5% CO 2 incubator for 72 hours.
  • the experimental results show that the provided bispecific antibody or multispecific antibody can inhibit the proliferation of HUVEC cells.
  • the description with reference to the terms “embodiment”, “implementation method”, “specific implementation method”, etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention.
  • the schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.
  • those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

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Abstract

提供了多特异性抗体及其应用。所提供的抗体能够特异性单一地或者同时结合TGF-β、GARP或者GARP-TGF-β复合物，同时还能够特异性结合VEGF，还可以选择性地特异性结合PD-L1。所提供的多特异性抗体表现出与抗原的高亲和力、特异性，具有肿瘤杀伤的效果，能够应用于治疗肿瘤。

Description

多特异性抗体及其应用 技术领域
本发明涉及生物医药领域，具体涉及一种多特异性抗体及其应用。
背景技术
近年来肿瘤免疫疗法取得了巨大的进步，在免疫检查点抑制、CAR-T细胞等多个方向上发展迅速。然而单特异性抗体的局限性日渐突出，多特异性抗体(multispecific antibody)尤其是双特异性抗体(bispecific antibody，BsAb)得到越来越多的关注。自1960年Nisonoff及其合作者首次提出BsAb的概念以来，双特异性抗体随着基因工程抗体技术的逐渐成熟，得到了快速发展。由于BsAb可以靶向多个抗原或者抗原表位，相较于单克隆抗体表现出更多的优势，而且也逐渐展现出比单克隆抗体联用疗法更高的疗效。例如，可以将特异性免疫效应细胞重定向到邻近的肿瘤细胞，以增强肿瘤杀伤；而且可以通过两个不同的细胞表面抗原的相互作用增加结合的特异性；同时相较于单一抗体联用来说，可以降低开发成本、临床试验。双特异性抗体现已成为抗体工程领域的研究热点，在肿瘤治疗及自身免疫疾病等领域中具有广泛的应用前景。国内国外多个制药公司针对不同的靶点展开了双特异性抗体的研究。
多特异性抗体能够同时特异性结合两个抗原或者两个以上抗原表位的抗体，涉及两个或者两个以上实体的相互作用，相较于单克隆抗体来说更加复杂。然而针对不同靶点的多特异性抗体的开发还需要进一步改进。
发明内容
本发明提供了多特异性抗体及其在疾病治疗中的用途。本发明通过靶向GARP、TGF-β和/或GARP-TGF-β复合体来抑制活性TGF-β的释放以及Treg的免疫抑制功能，从而重新激活癌细胞的免疫应答，抑制疾病进展。所提供的多特异性抗体还能够特异性结合VEGF，阻断血管的形成，从而抑制肿瘤的生长。而且所提供的多特异性抗体还可以根据需要选择性地设计成能够特异性结合PD-L1，能够提高PD-1/PD-L1耐药的抗肿瘤活性。
本发明的第一方面提供了一种多特异性抗体，包括第一抗原结合部分和第三抗原结合部分；所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区：包含SEQ ID NO:1、2和3所示的HCDR序列，或者包含SEQ ID NO:4、5和6所示的HCDR序列，或者包含SEQ ID NO:7、8和9所示的HCDR序列，或者包含SEQ ID NO:10、11和12所示的HCDR序列，或者包含SEQ ID NO:13、14和15所示的HCDR序列，或者包含SEQ ID NO:16、17和18所示的HCDR序列；所述轻链可变区：包含SEQ ID NO:19、20和21所示的LCDR序列，或者包含SEQ ID NO:22、23和24所示的LCDR序列，或者包含SEQ ID NO:25、20和26所示的LCDR序列，或者包含SEQ ID NO:27、28和29所示的LCDR序列，或者包含SEQ ID NO:30、31和32所示的LCDR序列，或者包含SEQ ID NO:33、34和35所示的LCDR序列；所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子；所述HCDR序列和所述LCDR序列是基于IMGT定义方案获得的。
所提供的多特异性抗体至少具有第一抗原结合部分和第三抗原结合部分，第一抗原结合部分能够特异性靶向GARP、TGF-β和/或GARP-TGF-β复合物，激活免疫细胞的应答；多特异性抗体的第三抗原结合部分能够特异性结合VEGF，阻断血管的形成，从而抑制肿瘤的生长。包含第一抗原结合部分和第三抗原结合部分的多特异性抗体，不仅能够激活免疫细胞的应答，同时可以阻断血管的形成，抑制肿瘤的生长，表现出优异的肿瘤治疗效果。
本发明的第二方面提供了一种多特异性抗体，至少包含第一抗原结合部分和第三抗原结合部分；所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含互补决定区LCDR1、LCDR2和LCDR3；其中HCDR1包含SEQ ID NO:1或4或7或10或13或16所示的序列，HCDR2包含SEQ ID NO:2或5或8或11或14或17所示的序列，HCDR3包含SEQ  ID NO:3或6或9或12或15或18所示的序列，LCDR1包含SEQ ID NO:19或22或25或27或30或33所示的序列，LCDR2包含SEQ ID NO:20或23或28或31或34所示的序列，LCDR3包含SEQ ID NO:21或24或26或29或32或35所示的序列；所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子；所述HCDR1、HCDR2、HCDR3序列和所述LCDR1、LCDR2、LCDR3序列是基于IMGT定义方案获得的。
本发明的第三方面提供了一种多特异性抗体，包括第一抗原结合部分、第二抗原结合部分和第三抗原结合部分；所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第二抗原结合部分能够结合PD-L1，所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子；所述第一抗原结合部分包括重链可变区和轻链可变区，所述第一抗原结合部分具有：SEQ ID NO:1、2和3所示的HCDR序列和SEQ ID NO:19、20和21所示的LCDR序列，或者SEQ ID NO:4、5和6所示的HCDR序列和SEQ ID NO:22、23和24所示的LCDR序列，或者SEQ ID NO:7、8和9所示的HCDR序列和SEQ ID NO:25、20和26所示的LCDR序列，或者SEQ ID NO:10、11和12所示的HCDR序列和SEQ ID NO:27、28和29所示的LCDR序列，或者SEQ ID NO:13、14和15所示的HCDR序列和SEQ ID NO:30、31和32所示的LCDR序列，或者SEQ ID NO:16、17和18所示的HCDR序列和SEQ ID NO:33、34和35所示的LCDR序列；
所述第二抗原结合部分具有：SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:62所示的CDR3序列；或者SEQ ID NO:63所示的CDR1序列、SEQ ID NO:64所示的CDR2序列和SEQ ID NO:65所示的CDR3序列；或者SEQ ID NO:66所示的CDR1序列、SEQ ID NO:67所示的CDR2序列和SEQ ID NO:68所示的CDR3序列；或者SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:69所示的CDR3序列；
所述第一抗原结合部分与重链第一恒定结构域CH1、轻链恒定结构域VL以及Fc区形成IgG型结构，所述第二抗原结合部分通过连接子与轻链恒定结构域VL连接，所述第三抗原结合部分与Fc区的羧基端连接；
所述HCDR序列、LCDR序列以及CDR1、CDR2和CDR3序列是基于IMGT定义方案获得的。
本发明的第四方面提供了一种多特异性抗体，包含第一抗原结合部分、第二抗原结合部分和第三抗原结合部分；所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第一抗原结合部分包括：如SEQ ID NO:36或37或38或39或40或41或42或43或44或45或46或47所示重链可变区的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:48或49或50或51或52或53或54或55或56或57或58或59所示轻链可变区的LCDR1、LCDR2和LCDR3序列；所述第二抗原结合部分能够结合PD-L1，所述第二抗原结合部分包含：
如SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示氨基酸的CDR1、CDR2和CDR3序列；
所述第三抗原结合部分为VEGF受体融合蛋白，具有SEQ ID NO:92所示的氨基酸序列。
本发明的第五方面提供了一种双特异性抗体，包含与TGF-β、GARP和/或GARP-TGF-β复合物特异性结合的第一抗原结合部分、与PD-L1特异性结合的第二抗原结合部分和与VEGF特异性结合的第三抗原结合部分；进一步包括重链第一恒定结构域CH1和轻链恒定结构域CL，所述第一抗原结合部分分别和重链第一恒定结构域CH1、轻链恒定结构域CL共价连接；进一步包括Fc区，所述Fc区通过铰链区和重链第一恒定结构域CH1连接；所述第二抗原结合部分与Fc区和/或所述轻链恒定结构域CL和/或轻链可变区连接；所述第三抗原结合部分与Fc区和/或所述轻链恒定结构域CL和/或轻链可变区连接。
本发明的第六方面提供了一种多核苷酸，所述多核苷酸编码上述第一方面至第五方面任一所述的多特异性抗体。
本发明的第七方面提供了一种构建体，包含上述第六方面所述的多核苷酸。
本发明的第八方面提供了一种宿主细胞，含有上述第六方面所述的多核苷酸或者第七方面所述的构建体。
本发明的第九方面提供了一种药物组合物，包括：上述第一方面至第五方面任一所述的多特异性抗体；以及药学上可接受的载体。
本发明的第十方面提供了一种抗体偶联物，包括上述第一方面至第五方面任一所述的多特异性抗体；以及与多特异性抗体连接的功能小分子。
本发明的第十一方面提供了一种试剂盒，所述试剂盒包括上述第一方面至第五方面任一所述的多特异性抗体。
本发明的第十二方面提供了一种生产上述多特异性抗体的方法，包括：培养第八方面所述的宿主细胞，以及从培养物中收集所述的多特异性抗体。
本发明的第十三方面提供了一种预防和/或治疗疾病的方法，包括：给予有需要的受试者有效量的上述第一方面至第五方面任一所述的多特异性抗体，或者第九方面所述的药物组合物，或者第十方面所述的抗体偶联物。
本发明的第十四方面提供了一种上述第一方面至第五方面任一所述的多特异性抗体在制备药物或者试剂盒或者在制备抗体偶联物中的用途。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明的实施例提供的一种多特异性抗体的结构示意图。
图2是根据本发明的实施例提供的一种多特异性抗体的结构示意图。
图3是根据本发明的实施例提供的一种多特异性抗体的结构示意图。
图4是根据本发明的实施例提供的利用ELISA的方法检测不同抗体与VEGF的结合活性结果图。
图5是根据本发明的实施例提供的不同多特异性抗体的报告基因阻断实验结果图。
图6是根据本发明的实施例提供的通过报告基因阻断实验对于不同多特异性抗体与GARP/TGF-β的结合活性进行表征的结果图。
图7是根据本发明的实施例提供的不同多特异性抗体对于PD-L1/PD-1通路的阻断作用结果。
具体实施方式
下面结合具体示例对于本发明的技术方案进行详细说明。同时，为了方便本领域技术人员理解，对于本发明的一些术语进行解释和说明，需要说明的是，这些解释和说明仅用来方便本领域技术人员的理解，而不应看作是本发明保护范围的限制。
术语“多特异性抗体”包含与至少两种不同生物分子的表位特异性结合的抗原结合部分。本文所提到的多特异性抗体包括双特异性抗体、三特异性抗体等。本文中提到的“多”包含两个以及两个以上。本文中为了区分，根据需要将多特异性抗体所包含的能够特异性靶向特定抗原的部分分别称为“第一抗原结合部分”、“第二抗原结合部分”、“第三抗原结合部分”等。“第一”、“第二”、“第三”等仅用于区分，不用于表示先后顺序，也不表示重要性。下文对于“第一抗原结合部分”、“第二抗原结合部分”和“第三抗原结合部分”也会有解释和说明。除非另外说明，否则所列出的多特异性抗体中与抗原的结合顺序是任意的。当然，多特异性抗体在与不同生物分子特异性结合时，有可能和该特定生物分子的不止一个抗原表位结合。
本文中，术语“抗体”作最广泛意义使用，指包含抗原结合位点的蛋白质或者多肽，涵盖各种结构的天然抗体和人工抗体，包括但不限于完整抗体形式或者抗体的抗原结合片段。
“完整抗体”或“完全抗体”等均用来表示相同的含义，指示包含二硫键相互连接的至少两个重链(H)和两条轻链(L)的蛋白。每条重链由重链可变区(缩写为VH)和重链恒定区(缩写为CH)组成。重链恒定区包括重链第一恒定结构域(CH1)、重链第二恒定结构域(CH2)和重链第三恒定结构域(CH3)。每条轻链由轻链可变区(缩写为VL)和轻链恒定区组成。轻链恒定区也就是轻链恒定结构域(CL)。VH和VL区可以进一步划分为互补决定区(也称为超变区或者高变区，缩写为CDR)，其间插有保守的框架区(FR)。每个VH和VL包含三个CDR和四个FR，从氨基端(N端)到羧基端(C端)按照如下序列排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链可变区的CDR从重链氨基酸序列的氨基末端开始分别被称为HCDR1、HCDR2和HCDR3，轻链可变区的CDR从轻链氨基酸序列的氨基末端开始分别称为LCDR1、LCDR2和LCDR3。
相应地，“抗原结合片段”，或者“抗原结合部分”(如第一抗原结合部分、第二抗原结合部分)是 指包含完全抗体的一部分，通常包含与抗原结合的可变域或者可变区。术语“可变区”或“可变域”是指涉及抗原与抗体重链或轻链结合的域。如上文提到的，完整抗体的重链和轻链的可变区通常具有相似的结果，每个域包括四个保守的框架区和三个高变区。抗原结合片段或者抗原结合部分的实例可以为Fab、Fab’、F(ab’)2、双特异性Fab’和Fv片段、线性抗体、单链抗体、单域抗体等。完整抗体经木瓜蛋白酶消化产生两个完全相同的抗原结合片段，称为Fab片段，其各自含有重链和轻链可变区以及轻链恒定结构域和重链第一恒定结构域(CH1)。Fab’片段在重链第一恒定结构域CH1的羧基末端增加了少数残基而与Fab片段有所不同，包括来自抗体铰链区的一个或多个半胱氨酸。完整抗体经胃蛋白酶消化获得F(ab’)2片段。F(ab’)2片段具有通过二硫键连接在一起的两个抗原结合F(ab)部分，F(ab’)2片段为双价抗体。单链抗体由抗体重链可变区和轻链可变区通过一个约10-25个氨基酸组成的柔性短肽连接而成的融合蛋白。单域抗体是由单个单体的可变区组成的抗体片段。由于单域抗体通常来源于骆驼科动物抗体或鲨鱼科动物抗重链的可变区，因此也常称为纳米抗体。纳米抗体仅仅含有重链CDR区，是具有完整功能的最小的抗原结合片段。纳米抗体包括三个CDR区(CDR1-CDR3)和四个框架区FR(FR1-FR4)。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。
术语“和/或”用于连接两个或者多个可选项时，应理解为意指可选项中的任一项或者可选项中的任意两项或者多项。
术语“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的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
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”、“结合”、“特异性靶向”特定抗原或表位，或对特定抗原或表位“具有特异性”或者“能够结合”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。抗体与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情况。结合本发明的具体实施方式，所提供的抗体具有≤100nM,≤50nM,≤20nM,≤10nM,≤5nM,≤1nM,≤0.5nM,≤0.1nM,≤0.05nM，≤0.04nM，≤0.03nM，≤0.02nM，≤0.01nM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。
本文中所提供的无论是多特异性抗体，或者是多核苷酸，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，分离的多肽将通过至少一个纯化步骤来制备。“分离的抗体”是指基本上不含具有不同抗原。“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。
“人源化”抗体通常是指基于来源于非人物种的抗原结合部分，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。例如，人源化抗体中，除CDR之外的整个抗体由人类来源的多核苷酸编码，其保留了抗原的结合活性，同时降低了免疫原性。
本文中所示出的第一抗原结合部分和第二抗原结合部分的CDR序列是根据已有的定义方案分析获得的，例如第一抗原结合部分的CDR序列，以及第二抗原结合部分的CDR序列是根据IMGT定义方案获得的。还可以通过Kabat(例如可以参见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))等定义方案获得。本领域技术人员可知的是，由于定义方法的差异所带来的差异CDR，也都包含在本发明的保护范围之内。
多特异性抗体
本发明提供了一种多特异性抗体，至少包括第一抗原结合部分和第三抗原结合部分，第一抗原结合部分和第三抗原结合部分分别靶向不同的靶抗原。在本文中，第一抗原结合部分能够特异性靶向TGF-β、GARP以及GARP-TGF-β复合物中的一个，两个或者三个；第三抗原结合部分特异性靶向VEGF。第一抗原结合部分无论是特异性靶向TGF-β，还是靶向GARP，或者是靶向GARP-TGF-β复合物，亦或者是 靶向这三者的任意两个或者三个；均能够直接或者间接实现对于TGF-β的调节，激活癌细胞的免疫应答，抑制疾病进展。需要说明的是，如文中所示，术语“第一”、“第二”、“第三”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量，也不用来表示先后顺序。术语“第一抗原结合部分”、“第二抗原结合部分”、“第三抗原结合部分”分别意指能够与不同靶抗原结合的部分，其可以是完整抗体，也可以是完整抗体的一部分，例如重链可变区、Fab、Fab’、F(ab’)2、单链抗体(ScFv)、或者单域抗体(sdAb)等等。
转化生长因子-β(Transforming growth factor-β，TGF-β)作为TGF-β超家族，在多种免疫细胞，例如调节性T细胞(regulatory T cells，Tregs)、DC细胞、CD3+T细胞、M2巨噬细胞等中表达，并调节细胞的生长和分化。在肿瘤微环境中，免疫细胞的活性通常会受到环境中TGF-β的影响，表现出免疫抑制。正是由于TGF-β所发挥的效力和多样性，家族成员中诸如TGF-β1、β2和β3等亚型以潜伏的、无活性的形式产生，而且需要严格调控的细胞外激活步骤才能获得与其受体相结合的能力(Cold Spring Harb.Perspect.Biol.2016；8(a022103),Cytokine Growth Factor Rev.2013；24:355-372)。TGF-β1是被免疫细胞例如Tregs表达的主要亚型。Treg细胞正是通过产生活性TGF-β来介导免疫抑制效应，从而防止自身免疫等疾病的产生。然而，恶性肿瘤会利用该机制释放出大量TGF-β，帮助癌细胞快速分裂，同时借助于Treg细胞抑制其他免疫细胞对癌细胞的杀伤功效。
糖蛋白A为主的重复序列(glycoprotein A repetitions predominant,GARP)是一种结合潜伏转化生长因子β(TGF-β)的I型跨膜细胞表面受体，主要活化在Treg、B细胞表面和血小板中，并在人类乳腺、肺和结肠肿瘤中高表达。GARP能够与潜伏的TGF-β(latency associated peptide，LAP)形成复合物(Proc Natl Acad Sci USA.2009；106(32):13445-50)，可以调节膜结合潜伏的TGF-β的能力，并与αVβ8整合素或者αVβ6整合素相结合，从细胞表面释放TGF-β并使其活化。
鉴于GARP的高表达为肿瘤提供了生长所需TGF-β的活化和储存，也增强了Treg的抑制性表型和维持Treg介导的外周耐受性，GARP抑制剂逐渐在肿瘤治疗中崭露头角。靶向GARP的单特异性抗体也在快速推进，例如首个进入临床的GARP抑制剂ARGX-115，其以GARP为靶点，抑制活性TGF-β的释放以及Treg的免疫抑制活性，从而重新激活癌细胞的免疫应答，抑制疾病进展。针对GARP或者GARP-TGF-β的多特异性抗体研究还较少。本文中所提到的第一抗原结合部分能够特异性靶向TGF-β、GARP以及GARP-TGF-β复合物中的一个，两个或者三个，从而可以实现对于TGF-β的调节，激活癌细胞的免疫应答，抑制疾病进展。
第三抗原结合部分能够特异性结合VEGF。第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子。第三抗原结合部分通过阻断VEGF的正常生理活性，不能形成新的血管从而抑制肿瘤的生长。通过有效地阻断新生血管的形成，从而使肿瘤组织由于缺乏血管的营养补给而不成正常生长。除此之外，因为能够特异性靶向VEGF，所以所提供的多特异性抗体还可用于除肿瘤外的其他疾病的病理性血管生长，比如糖尿病性视网膜病、银屑病和由移植、感染或创伤引起的病理性角膜血管形成。
本发明提供了一种多特异性抗体，包含与TGF-β、GARP和/或GARP-TGF-β复合物特异性结合的第一抗原结合部分、和与VEGF特异性结合的第三抗原结合部分，还可以选择性地包含与PD-L1特异性结合的第二抗原结合部分。所提供的多特异性抗体进一步包括重链第一恒定结构域CH1和轻链恒定结构域CL，所述第一抗原结合部分分别和重链第一恒定结构域CH1、轻链恒定结构域CL共价连接；进一步包括Fc区，所述Fc区通过铰链区和重链第一恒定结构域CH1连接；第三抗原结合部分与Fc区和/或与轻链恒定结构域CL直接或者通过连接子连接。多特异性抗体还可以根据需要包括第二抗原结合部分，所述第二抗原结合部分与Fc区和/或所述轻链恒定结构域CL和/或轻链可变区连接。所形成的IgG型结构除了提到的重链可变区以及轻链可变区序列之外，其他序列可以采用哺乳动物来源的天然序列(例如可以是人源的天然序列)。根据具体实施方式，在形成IgG型结构时，所用到的重链恒定结构域序列如SEQ ID NO:93所示，所用到的轻连恒定结构域序列如SEQ ID NO:94所示。
本文中所提到的“连接”、“相连”可以是直接连接，也可以间接连接。直接连接是指多肽或者蛋白序列直接通过两个氨基酸形成肽键进行连接。间接连接多肽或者蛋白序列可以通过连接子进行连接。
所提供的多特异性抗体中，Fc区序列不受限制，可以是天然来源的Fc区，例如来自于人的Fc区， 也可以是经过改造的Fc区。经过改造的Fc区可以为发生突变的Fc区，例如可以在Fc区的某些位点发生突变，调节抗体的ADCC效应功能。
所提供的多特异性抗体至少具有下列性质之一：(a)与PD-L1特异性结合；(b)与糖蛋白A重复主导序列(GARP)特异性结合；(c)与GARP-TGF-β复合物特异性结合；(d)与TGF-β特异性结合；(e)对免疫细胞具有活性调节功能包括但不限于对调节性T细胞的免疫抑制功能具有抑制活性等；(f)具有抗肿瘤活性；(g)与VEGF特异性结合；(h)具有抑制病理性血管生长的活性。
本发明所提供的多特异性抗体同时显示出与TGF-β、GARP和/或GARP-TGF-β复合物特异性结合的活性，与PD-L1特异性结合的活性，以及与VEGF特异性结合的活性。通过TGF-β报告基因实验和Treg释放TGF-β中和实验等检测方法，确定了多特异性抗体既能结合GARP-TGF-β1复合物、又能捕获已释放到微环境中TGF-β1的差异化分子。所提供的多特异性抗体还能够特异性结合VEGF，阻断新生血管的形成，从而抑制肿瘤的生长。而且所提供的多特异性抗体能够与PD-L1特异性结合，通过同时阻断PD-1/PD-L1和TGF-β信号通路，解除免疫抑制，避免PD-L1(PD-1)轴和TGF-β轴途径之间可能存在的互补作用，成倍恢复机体免疫能力，达到增效杀伤的效果。
在至少一些实施方式中，所述第三抗原结合部分通过连接子与Fc区的羧酸端(C端)连接，如图1所示。需要说明的是，本文中所提供的多特异性抗体的示意性附图中没有专门绘出二硫键。图1示出了分别有一个第三抗原结合部分与Fc区的羧基端(C端)连接。根据具体实施方式，第三抗原结合部分为来自于VEGF trap的融合蛋白，包括VEGFR1的免疫球蛋白结构域和VEGFR2的免疫球蛋白结构域。根据具体实施方式，所提供的多特异性抗体还可以根据需要包括第二抗原结合部分。根据具体实施方式，第二抗原结合部分为抗PD-L1纳米抗体。如图2所示，第二抗原结合部分与轻链恒定结构域的羧基端(C端)连接。第二抗原结合部分可以直接与轻链恒定结构域的羧基端连接，也可以通过连接子与轻链恒定结构域的羧基端连接。第二抗原结合部分在与轻链恒定结构域的羧基端(C端)连接时，可以分别连接一个抗PD-L1纳米抗体，也可以根据需要分别连接两个或者三个抗PD-L1纳米抗体。所连接的纳米抗体可以相同，也可以不同。根据具体实施方式，第二抗原结合部分与轻链可变区的氨基端(N端)连接。第二抗原结合部分可以直接与轻链可变区的氨基端连接，也可以通过连接子与轻链可变区的氨基端连接。第二抗原结合部分在与轻链可变区的氨基酸连接时，可以分别连接一个抗PD-L1纳米抗体，也可以根据需要分别连接两个或者三个抗PD-L1纳米抗体。所连接的纳米抗体可以相同，也可以不同。如图2所示。
在至少一些实施方式中，第二抗原结合部分和第三抗原结合部分与所形成的IgG结构的连接关系可以替换，即第二抗原结合部分可以与Fc区的羧基端连接(直接或者通过连接子连接，图3示出的结构为通过连接子连接)，第三抗原结合部分可以与轻链恒定结构域CL的羧基端连接(直接或者通过连接子连接，图3示出的结构为通过连接子连接)。如图3所示。在进行连接时，所提到的连接子可以相同，也可以不同。
第二抗原结合部分可以根据需要，选择单链抗体、单域抗体，或者其他形式的融合蛋白等等。轻链恒定结构域的羧基端所连接的第二抗原结合部分可以相同，也可以不同，或者仅在轻链恒定结构域的任意其中一个羧基端连接第二抗原结合部分。在一些具体实施方式中，轻链恒定结构域的羧基端(C端)各自连接两个抗PD-L1纳米抗体。同样地，根据需要，轻链恒定结构域的C端也可以分别连接一个抗PD-L1纳米抗体或者连接两个以上的抗PD-L1纳米抗体等等。以轻链恒定结构域的任意其中一个羧基端为例，其所连接的抗PD-L1纳米抗体的个数可以相同，也可以不同。根据具体实施方式，所提供抗PD-L1纳米抗体可以是已公开或者市售的纳米抗体，也可以通过抗体库筛选获得。
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。
本发明提供了一种多特异性抗体，包括第一抗原结合部分和第三抗原结合部分；第一抗原结合部分包括：如SEQ ID NO:36或37或38或39或40或41或42或43或44或45或46或47所示重链可变区的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:48或49或50或51或52或53或54或55或56或57或58或59所示轻链可变区的LCDR1、LCDR2和LCDR3序列；所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子。所示出的重链可变区和轻链可变区序列的HCDR1、HCDR2和HCDR3序列以及LCDR1、LCDR2和LCDR3序列，因为抗体序列的分析方 法的不同，CDR序列也会稍有差异，如上文提到的经典的Kabat CDR定义方案、IMGT定义方案、Chothia定义方案等。所分析出的不同的CDR序列，均包含在本发明的保护范围之内。
本发明还提供了一种多特异性抗体，包括第一抗原结合部分和第三抗原结合部分，第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，第三抗原结合部分能够结合VEGF；第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:1、2和3所示的HCDR序列，或者与SEQ ID NO:1、2和3所示的HCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:4、5和6所示的HCDR序列，或者与SEQ ID NO:4、5和6所示的HCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:7、8和9所示的HCDR序列，或者与SEQ ID NO:7、8和9所示的HCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:10、11和12所示的HCDR序列，或者与SEQ ID NO:10、11和12所示的HCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:13、14和15所示的HCDR序列，或者与SEQ ID NO:13、14和15所示的HCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:16、17和18所示的HCDR序列，或者与SEQ ID NO:16、17和18所示的HCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列。
在一些实施方式中，所述第一抗原结合部分还可以进一步包含轻链可变区，所述轻链可变区包含SEQ ID NO:19、20和21所示的LCDR序列，或者与SEQ ID NO:19、20和21所示的LCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:22、23和24所示的LCDR序列，或者与SEQ ID NO:22、23和24所示的LCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:25、20和26所示的LCDR序列，或者与SEQ ID NO:25、20和26所示的LCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:27、28和29所示的LCDR序列，或者与SEQ ID NO:27、28和29所示的LCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:30、31和32所示的LCDR序列，或者与SEQ ID NO:30、31和32所示的LCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列；或者包含SEQ ID NO:33、34和35所示的LCDR序列，或者与SEQ ID NO:33、34和35所示的LCDR序列具有一个或者两个氨基酸取代、缺失或者增加的序列。
各序列如下所示。所提到的多特异性抗体的第一抗原结合部分或者第三抗原结合部分可以根据需要选择完整抗体或者仅仅其中的一部分片段，只要能够靶向不同的靶抗原即可。第一抗原结合部分或者第二抗原结合部分可以仅仅是重链可变区、Fab、Fab’、F(ab’)2、单链抗体(ScFv)、或者单域抗体(sdAb)等。

所提供的多特异性抗体的第一抗原结合部分能够结合GARP，TGF-β，或者GARP-TGF-β复合物，或者能够特异性结合这三者的任意一种、两种或者三种。多特异性抗体的第三抗原结合部分能够结合VEGF，阻断血管的形成，从而抑制肿瘤的生长。根据具体实施方式，第三抗原结合部分为VEGF受体融合蛋白、抗VEGF受体或抗原结合片段或VEGF结合分子。
在一些具体实施方式中，所提供的多特异性抗体具有SEQ ID NO:1、2和3所示的HCDR序列和SEQ ID NO:19、20和21所示的LCDR序列。在一些具体实施方式中，所提供的多特异性抗体具有SEQ ID NO: 4、5和6所示的HCDR序列和SEQ ID NO:22、23和24所示的LCDR序列。在一些实施方式中，所提供的多特异性抗体具有SEQ ID NO:7、8和9所示的HCDR序列和SEQ ID NO:25、20和26所示的LCDR序列。在一些实施方式中，所提供的多特异性抗体具有SEQ ID NO:10、11和12所示的HCDR序列和SEQ ID NO:27、28和29所示的LCDR序列。在一些具体实施方式中，所提供的多特异性抗体具有SEQ ID NO:13、14和15所示的HCDR序列和SEQ ID NO:30、31和32所示的LCDR序列。在一些具体实施方式中，所提供的多特异性抗体具有包含SEQ ID NO:16、17和18所示的HCDR序列和SEQ ID NO:33、34和35所示的LCDR序列。所示出的HCDR序列和LCDR序列作为多特异性抗体的第一抗原结合部分，与靶抗原GARP、TGF-β或者GARP-TGF-β复合物特异性结合。
在一些实施方式中，所提供的多特异性抗体包括下列中的至少一种：与SEQ ID NO:36或37或38或39或40或41或42或43或44或45或46或47所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链可变区，和与SEQ ID NO:48或49或50或51或52或53或54或55或56或57或58或59所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链可变区。其中SEQ ID NO:42、43、44、45、46和47分别为人源化的重链可变区序列。SEQ ID NO:54、55、56、57、58和59分别为人源化的轻链可变区序列。所示出的重链可变区和轻链可变区序列作为多特异性抗体的第一抗原结合部分，与靶抗原特异性结合。第一抗原结合部分的轻链可变区和重链可变区的序列，经过人源化之后，免疫原性更低，而且保留了与靶抗原(如GARP、TGF-β，和/或GARP-TGF-β复合物)的结合活性。可以通过本领域常用的方法(例如互补决定区移植法)获得人源化的抗体序列。在一些实施方式中，所提供的人源化的抗体序列保留了CDR区序列，将框架区序列替换为人源的框架区序列。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区需要是否需要进行回复突变以及恢复突变的关键残基，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。
本文中所提到的“序列同一性”是指逐个氨基酸或者核苷酸序列比较，序列相同的程度。为了确定序列同一性的比值，可以按照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。在一些具体实施方式中，序列同一性是由于保守氨基酸取代带来的。“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。所提到的保守氨基酸取代可以是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个保守氨基酸取代等等。本文中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。
所示出的各序列分别如下：

在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:36所示的重链可变区和SEQ ID NO:48所示的轻链可变区。在一些具体实施方式中，所述第一抗原结合部分包含SEQ ID NO:37所示的重链可变区和SEQ ID NO:49所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:38所示的重链可变区和SEQ ID NO:50所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:39所示的重链可变区和SEQ ID NO:51所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:40所示的重链可变区和SEQ ID NO:52所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:41所示的重链可变区和SEQ ID NO:53所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:42所示的重链可变区和SEQ ID NO:54所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:43所示的重链可变区和SEQ ID NO:55所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:44所示的重链可变区和SEQ ID NO:56所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:45所示的重链可变区和SEQ ID NO:57所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:46所示的重链可变区和SEQ ID NO:58所示的轻链可变区。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:47所示的重链可变区和SEQ ID NO:59所示的轻链可变区。
第三抗原结合部分能够特异性结合VEGF。根据具体实施方式，所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子。根据具体实施方式，第三抗原结合部分包括至少两种不同的VEGFR免疫球蛋白样结构域。根据具体实施方式，第三抗原结合部分为：(i)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白结构域(Ig)和/或VEGFR2的免疫球蛋白结构域和/或VEGFR3的免疫球蛋白结构域；(ii)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白样结构域2(domain 2)和VEGFR2的Ig结构域3(domain 3)；(iii)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白样结构域2(domain 2)、VEGFR2的Ig结构域3(domain 3)和VEGFR2的Ig结构域4(domain 4)。根据具体实施方式，所提到的第三抗原结合部分来自于VEGF trap的融合蛋白。VEGF trap是一种包含两种不同的VEGFR胞外结构域的融合蛋白，是一种可溶性受体，由IgG的恒定区(IgG-Fc1)和两种不同的VEGFR(VEGFR1的domain 2、VEGFR2的domain 3)融合而成。VEGF trap阻断了VEGF的正常生理活性，不能形成新的血管从而抑制肿瘤的生长。VEGF trap可以有效地阻断新生血管的形成，从而使肿瘤组织由于缺乏血管的营养补给而不成正常生长。除此之外，VEGF Trap还可用于除肿瘤外的其他疾病的病理性血管生长，比如糖尿病性视网膜病、银屑病和由移植、感染或创伤引起的病理性角膜血管形成。第三抗原结合部分来自于VEGF trap，具有两种不同的VEGFR(VEGFR1的domain 2、VEGFR2的domain 3)。根据具体实施方式，所述第三抗原结合部分具有SEQ ID NO:92所示的序列。
根据具体实施方式，所提供的多特异性抗体还可以根据需要包括第二抗原结合部分。第二抗原结合部分能够特异性靶向PD-L1。通过特异性结合PD-L1，能够提高PD-1/PD-L1耐药的抗肿瘤活性。如上文中所提到的，第二抗原结合部分只要能够特异性靶向PD-L1即可，其表现形式不受限制。例如可以是完整抗体，也可以是完整抗体的一部分，包括但不限于重链可变区、Fab、Fab’、F(ab’)2、单链抗体或者纳米抗体等等。第二抗原结合部分可以根据需要通过抗体库筛选获得，也可以是市售的或者已经公开的抗体序列。在一些具体实施方式中，所述第二抗原结合部分为单域抗体。在一些优选实施方式中，所形成的双特异性抗体的结构为通过连接子连接IgG型抗体与第二抗原结合部分的结构，IgG型抗体包含第一抗原结合部分。第二抗原结合部分与IgG型抗体连接，形成多特异性抗体结构。根据具体实施方式，第二抗原结合部分与轻链恒定结构域的羧基端连接。根据具体实施方式，第二抗原结合部分与Fc区的羧基端连接。
在一些具体实施方式中，所提供的第二抗原结合部分包含CDR1、CDR2和CDR3，所述CDR1包含选自下组的序列：SEQ ID NO:60、63和66，或者与SEQ ID NO:60、63和66相比，具有一个或者两个氨基酸取代、缺失或者增加的序列；CDR2包含选自下组的序列：SEQ ID NO:61、64和67，或者与SEQ ID NO:61、64和67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CDR3包含选自下组的序列：SEQ ID NO:62、65、68和69，或者与SEQ ID NO:62、65、68和69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。
在一些具体实施方式中，所述第二抗原结合部分具有SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:62所示的CDR3序列。在一些具体实施方式中，所述第二抗原结合部分具有SEQ ID NO:63所示的CDR1序列、SEQ ID NO:64所示的CDR2序列和SEQ ID NO:65所示的CDR3序列。在一些具体实施方式中，所述第二抗原结合部分具有SEQ ID NO:66所示的CDR1序列、SEQ ID NO:67所示的CDR2序列和SEQ ID NO:68所示的CDR3序列。在一些具体实施方式中，第 二抗原结合部分具有SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:69所示的CDR3序列。所示出的CDR1序列、CDR2序列和CDR3序列可以和框架区FR形成单域抗体。框架区FR序列不做特殊限制，可以是鼠源的，也可以是人源的，或者是部分鼠源、部分人源的。
在至少一些实施方式中，所述第二抗原结合部分进一步包括框架区FR，框架区包括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:70、SEQ ID NO:74、SEQ ID NO:77、SEQ ID NO:80，或者与SEQ ID NO:70、74、77或者80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:71、SEQ ID NO:75、SEQ ID NO:78，或者与SEQ ID NO:71、75或者78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:72、SEQ ID NO:76、SEQ ID NO:79、SEQ ID NO:81，或者与SEQ ID NO:72、76、79或者81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:73，或者与SEQ ID NO:73相比，具有一个或者两个保守氨基酸取代的序列。
第二抗原结合部分进一步包括框架区FR，所述框架区包括FR1、FR2、FR3和FR4。在一些具体实施方式中，所述框架区包括SEQ ID NO:70所示的FR1序列、SEQ ID NO:71所示的FR2序列、SEQ ID NO:72所示的FR3序列和SEQ ID NO:73所示的FR4序列。在一些具体实施方式中，所述框架区包括SEQ ID NO:74所示的FR1序列、SEQ ID NO:75所示的FR2序列、SEQ ID NO:76所示的FR3序列和SEQ ID NO:73所示的FR4序列。在一些具体实施方式中，所述框架区包括SEQ ID NO:77所示的FR1序列、SEQ ID NO:78所示的FR2序列、SEQ ID NO:79所示的FR3序列和SEQ ID NO:73所示的FR4序列。在一些具体实施方式中，所述框架区包括SEQ ID NO:80所示的FR1序列、SEQ ID NO:78所示的FR2序列、SEQ ID NO:79所示的FR3序列和SEQ ID NO:73所示的FR4序列。在一些具体实施方式中，所述框架区包括SEQ ID NO:70所示的FR1序列、SEQ ID NO:71所示的FR2序列、SEQ ID NO:81所示的FR3序列和SEQ ID NO:73所示的FR4序列。
在一些实施方式中，所述第二抗原结合部分选自具有如SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列。在一些实施方式中，所述第二抗原结合部分与SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的氨基酸序列相比，序列同一性在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。所提供的SEQ ID NO:87、88、89、90或91所示的氨基酸序列为人源化后的抗体序列。所提供的人源化的序列具有低的免疫原性，而且具有和靶蛋白高的亲和力。


本发明还提供了一种多特异性抗体，包括：第一抗原结合部分、第二抗原结合部分和第三抗原结合部分，所述第一抗原结合部分能够结合TGF-β、GARP和/或GARP-TGF-β复合物，所述第二抗原结合部分能够结合PD-L1，所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子。
在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:36所示的重链可变区，和SEQ ID NO:48所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:37所示的重链可变区，和SEQ ID NO:49所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:38所示的重链可变区和SEQ ID NO:50所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:39 所示的重链可变区和SEQ ID NO:51所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:40所示的重链可变区和SEQ ID NO:52所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:41所示的重链可变区和SEQ ID NO:53所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:42所示的重链可变区和SEQ ID NO:54所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:43所示的重链可变区和SEQ ID NO:55所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:44所示的重链可变区和SEQ ID NO:56所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:45所示的重链可变区和SEQ ID NO:57所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:46所示的重链可变区和SEQ ID NO:58所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。在一些具体实施方式中，第一抗原结合部分包含SEQ ID NO:47所示的重链可变区和SEQ ID NO:59所示的轻链可变区；所述第二抗原结合部分包含SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的序列；所述第三抗原结合部分包含SEQ ID NO:92所示的序列。
多核苷酸
本发明还提供了一种多核苷酸，其编码所述的多特异性抗体。所提到的多核苷酸是可分离的，包括但不限于DNA、RNA或者cDNA等等。可以采用本领域的常规方法获得分离的多核苷酸序列，编码多特异性抗体或者单克隆抗体或抗原结合片段。
构建体
为了制备本发明所提到的抗体，可以将编码抗体的多核苷酸序列插入到可复制的表达载体中，并在宿主细胞或者无细胞表达系统中表达。本发明还提供了一种构建体，包含上述所述的多核苷酸。本领域常用的多种方法都可以用来获得构建体，包括体外重组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，含有上述多核苷酸或者上述构建体。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细胞、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。
药物组合物
本发明还提供了一种药物组合物，包括：上述第一方面任一所述的多特异性抗体，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，例如磷酸氢二钠、磷酸二氢钠，氯化钠、醋酸钠、枸橼酸、枸橼酸钠、柠檬酸盐、Tris；糖类，如海藻糖、聚山梨醇、蔗糖、甘露醇；表面活性剂如聚山梨酯；防腐剂如氯己双铵、苯扎氯铵、苄索氯铵；氨基酸如组氨酸、盐酸组氨酸、甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸等。可以通过本领域常用的方法获得无菌的药物制剂，例如通过无菌滤膜过滤的方法获得。本领域技术人员可以根据需要将药物组合物选择不同的药学上可接受的载体，制备成不同的剂型，例如冻干剂型、注射剂等多种剂型。所制备的不同的药物剂型可以被配制成任何合适的施用途径施用于受试者，包括但不限于静脉、真皮、肌肉、腹膜、皮下、经鼻、口服、经直肠、局部、吸入、透皮等等。
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多特异性抗体，以及与所述多特异性抗体连接的功能小分子或蛋白酶。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，小分子药物通过与多特异性抗体进行化学偶联，增强抗肿瘤药物的治疗效果，并可以减少不良反应。还可以通过连接子将毒素、化疗药物、光敏剂等小分子偶联到所提到的抗体上。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。
试剂盒
本发明还提供了一种试剂盒，所述试剂盒包括上述多特异性抗体。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。
生产抗体的方法
本发明又提供了一种生产上述多特异性抗体的方法，包括：培养上述宿主细胞，以及从培养物中收集所述的多特异性抗体。
从培养物中收集的多特异性抗体经过纯化可以获得基本上纯的产物。“基本上纯的”是指多特异性抗体的纯度达到95％以上，96％以上，97％以上，98％以上，99％以上，甚至是99.5％、99.6％、99.7％、99.8％、99.9％以上。
预防和/或治疗疾病的方法
另外，本发明又提供了一种预防和/或治疗疾病的方法，包括：给予有需要的受试者有效量的上述多特异性抗体。
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。经过多特异性抗体的治疗后，相较于未经过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。
所提供的多特异性抗体，可以用于治疗的疾病包括但不限于癌症以及自身免疫性疾病。在一些实施方式中，本发明所提供的方法可以用于治疗TGF-β相关的疾病，GARP相关的疾病，也可以用于治疗VEGF相关的疾病，以及PD-1/PD-L1相关的疾病。TGF-β相关的疾病包括但不限于炎性疾病，慢性感染，癌症，纤维化，心血管疾病，脑血管疾病和神经退行性疾病等。在一些实施方式中，所提到癌症包括但不限于肺癌、结肠癌、肾癌、泌尿道上皮癌、前列腺癌、多形性成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑素瘤、肝癌、膀胱癌、胃癌、食道癌和血液癌症等等。除此之外，还可以治疗除了肿瘤外的其他疾病的病理性血管生长，比如糖尿病性视网膜病、银屑病和由移植、感染或创伤引起的病理性角膜血管形成，并治疗相应的疾病。
本发明还提供了多特异性抗体在制备药物或者试剂盒中的用途。所述药物用于治疗癌症。应用所提供的多特异性抗体，可以制备药物，用于治疗多种疾病。还可以用来制备试剂盒，作为免疫诊断试剂使用。
所提到的第一抗原结合部分和第二抗原结合部分的序列记载在申请号为PCT/CN2022/122367的国际专利申请文本中。可以根据需要将所记载的内容全部或者部分援引在本文中。来源于第一抗原结合部分的TGF-β、GARP或者GARP-TGF-β靶向序列，序列制备以及获得方式在申请号为PCT/CN2022/122367的国际专利申请文本中有记载。来源于第二抗原结合部分的PD-L1结合部分，序列制备以及获得方式在申请号为PCT/CN2022/122367的国际专利申请文本以及申请号为PCT/CN2022/110423的国际申请申请文本中均有记载。下面对其中所记载的内容进行部分援引。
来源于第一抗原结合部分的序列通过杂交瘤的方式获得。利用人抗原(GARP、TGF-β和GARP-TGF-β复合物)免疫小鼠，使小鼠体内产生特异性抗体。将小鼠骨髓瘤细胞和小鼠的脾脏细胞融合，融合后的细胞铺板。然后加入HAT选择性培养基筛选，骨髓瘤细胞无法存活，而脾脏细胞存活时间也很短。最终使得只有融合了淋巴细胞的骨髓瘤细胞存活，即筛选获得杂交瘤细胞。然后通过有限稀释克隆细胞的方法筛选获得了单克隆细胞，测序。然后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％以上的不同的抗体。分别命名为m212抗体、m305抗体、m107抗体、m202抗体、m301抗体和m109抗体。m212抗体包含SEQ ID NO:36所示的重链可变区和SEQ ID NO:48所示的轻链可变区。m305抗体包含SEQ ID NO:37所示的重链可变区和SEQ ID NO:49所示的轻链可变区。m107抗体包含SEQ ID NO:38所示的重链可变区和SEQ ID NO:50所示的轻链可变区。m202抗体包含SEQ ID NO:39所示的重链可变区和SEQ ID NO:51所示的轻链可变区。m301抗体包含SEQ ID NO:40所示的重链可变区和SEQ ID NO:52所示的轻链可变区。m109抗体包含SEQ ID NO:41所示的重链可变区和SEQ ID NO:53所示的轻链可变区。
通过互补决定区移植法获得人源化抗体，相应地，人源化的抗体编号分别为m212-hu、m305-hu、m107-hu、m202-hu、m301-hu和m109-hu。m212-hu抗体包含SEQ ID NO:42所示的重链可变区和SEQ ID NO:54所示的轻链可变区。m305-hu抗体包含SEQ ID NO:43所示的重链可变区和SEQ ID NO:55所示的轻链可变区。m107-hu抗体包含SEQ ID NO:44所示的重链可变区和SEQ ID NO:56所示的轻链可变区。m202-hu抗体包含SEQ ID NO:45所示的重链可变区和SEQ ID NO:57所示的轻链可变区。m301-hu抗体包含SEQ ID NO:46所示的重链可变区和SEQ ID NO:58所示的轻链可变区。m109-hu抗体包含SEQ ID NO:47所示的重链可变区和SEQ ID NO:59所示的轻链可变区。
利用过表达GARP-TGF-β复合物的293-GARP/TGF-β稳转细胞系，通过FACS检测对于抗体与其的结合活性进行了测试。结果表明：m212抗体、m305抗体、m107抗体、m202抗体、m301抗体以及m109抗体均能够结合GARP-TGF-β复合物，相应的EC50值分别为：<2nM、4.1nM、3.2nM、2.7nM、2.5nM、>5nM；人源化的抗体m212-hu、m305-hu、m107-hu、m202-hu、m301-hu和m109-hu的EC50值分别为：<2nM、2.1nM、7.5nM、2.5nM、4.0nM、>5nM。利用过表达GARP的CHO-K1-GARP细胞系，通过FACS检测发现：m212抗体、m305抗体、m107抗体均能够特异性结合GARP蛋白，相应的EC50值分别为0.46nM、<1nM、>2nM；人源化的抗体m212-hu、m305-hu相应的EC50值分别为1.4nM、0.45nM。
使用人转化生长因子β1包被细胞培养板，通过ELISA方法检测不同抗体与TGF-β的结合活性。结果表明：m107抗体、m202抗体、m301抗体、m109抗体均能够与TGF-β发生特异性结合。相应的EC50值分别为0.021nM、<1nM、<1nM、<1nM；人源化抗体m107-hu、m202-hu、m301-hu、m109-hu的EC50值分别为7.5nM、<2nM、<2nM、0.73nM。而且通过TGF-β分泌中和实验发现，这些抗体均可以抑制或中和Treg细胞产生的TGF-β。
PD-L1纳米抗体通过羊驼免疫的方法获得抗PD-L1纳米文库，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A～抗体E。抗体A的 氨基酸序列如SEQ ID NO:82所示，抗体B的氨基酸序列如SEQ ID NO:83所示，抗体C的氨基酸序列如SEQ ID NO:84所示，抗体D的氨基酸序列如SEQ ID NO:85所示，抗体E的氨基酸序列如SEQ ID NO:86所示。经过人源化的抗体为抗体Ahu，氨基酸序列如SEQ ID NO:87所示；抗体Bhu，氨基酸序列如SEQ ID NO:88所示；抗体Chu，氨基酸序列如SEQ ID NO:89所示；抗体D1hu，氨基酸序列如SEQ ID NO:90所示；抗体D2hu，氨基酸序列如SEQ ID NO:91所示。
这些抗体以及人源化后的抗体经FACS和ELISA实验证实和hPD-L1、食蟹猴PD-L1具有高的结合活性。而且表现出对于PD-1/PD-L1的阻断活性以及对于CD80/PD-L1的阻断活性。
例如，通过FACS检测抗体和人源的PD-L1的结合活性。实验过程为：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细胞按1×105个/孔加入到96孔板中。然后加入不同浓度的样品，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结果。实验结果表明，抗体A、抗体B、抗体C、抗体D和抗体E的EC50值分别为0.089nM、0.031nM、0.042nM、0.039nM、0.053nM，和阳性对照(阿替利珠单抗，0.059nM)相似，表现出高的结合活性。采用类似的方法测定人源化的抗体的结合活性，抗体Ahu、抗体Bhu、抗体Chu、抗体D1hu、抗体D2hu的EC50值分别为0.051nM、0.18nM、0.025M、0.18nM和0.29nM。
再例如通过FACS测定抗体对于PD-1/PD-L1的阻断活性。实验过程为：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1×105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，50μl/孔)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FACS缓冲液(含2％FBS的PBS)洗涤，在1200rpm下离心4分钟，弃掉上清。按照100ul/孔加入PE标记的链霉亲和素，避光孵育30分钟，孵育结束后用FACS缓冲液洗涤。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。实验结果表明，抗体A、抗体B、抗体C、抗体D和抗体E的IC50值分别为1.024nM、2.077nM、1.678nM、2.103nM、2.536nM，抗体均能阻断抗原和其配体的结合。采用类似的方法测定人源化的抗体的阻断活性，抗体Ahu、抗体Bhu、抗体Chu、抗体D1hu、抗体D2hu的IC50值分别为4.0nM、3.4nM、1.0nM、2.6nM和12nM。
通过FACS测定抗体对于CD-80/PD-L1的阻断活性。实验过程为：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1×105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FACS缓冲液洗涤，在1200rpm条件下离心4分钟，弃掉上清。然后按照100μl/孔加入human-CD80-mFc(2ug/ml)，4℃避光孵育30分钟。孵育结束后用FACS缓冲液洗涤。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FACS缓冲液洗涤，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。实验结果表明，抗体A、抗体B、抗体C、抗体D和抗体E的IC50值分别为3.954nM、3.355nM、4.146nM、4.757nM、2.979nM，抗体均能阻断抗原和其配体的结合。类似的方法测定人源化的抗体的阻断活性，抗体Ahu、抗体Bhu、抗体Chu和抗体D1hu的IC50值分别约为1.8nM、1.7nM、3.2nM和1.5nM。
下面参考实施例对本发明的技术方案进行详细说明。需要说明的是，这些实施例仅用于方便本领域技术人员的理解，不应看作是对本发明保护范围的限制。下面详细描述本发明的实施例，所描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
其中所用到的阳性对照1(aflibercept)为含有Fc的融合蛋白，能够特异性靶向VEGFA和VEGFB。所用到的阳性对照2(bevacizumab)为贝伐珠单抗，是一种人源化的IgG1型的血管生长因子(VEGF)抑制剂。
实施例1
实施例1主要描述了如何构建表达抗GARP、抗TGF-β、GARP-TGF-β复合物和VEGF的双特异性抗体，以及构建表达抗GARP、抗TGF-β、GARP-TGF-β复合物、抗VEGF和抗PD-L1的三特异性抗体。双特异性抗体在命名时以b开头，以和m开头的单抗区分，三特异性抗体在命名时以t开头。在命名时两个抗原结合部分中间用“-”连接，两个抗原结合部分仍然保留之前的编号。例如，抗体编号“b301-hu-Vtrap”表示双特异性抗体，其通过利用m301-hu的抗体序列和来自于Vtrap的抗体序列连接完成，所形成的双特异性抗体结构如图1所示。抗体编号“t301-hu-VTrap-D2hu”表示三特异性抗体，其是通过m301-hu的抗体序列和来自于Vtrap的抗体序列以及D2hu的抗体序列连接完成，所形成的三特异性抗体结构如图2所示。相应地抗体编号后若通过“-”连接有N或G4S时，代表所用到的连接子不同。在默认情况下(即没有连接有N或者G4S时)，第二抗原结合部分和第三抗原结合部分通过(G4S)4与所形成IgG型抗体结构连接。若抗体编号后通过“-”连接有N，例如“b301-hu-Vtrap-N”代表第二抗原结合部分和第三抗原结合部分直接与所形成的IgG型抗体结构连接。若抗体编号后通过“-”连接有G4S，例如“b301-hu-Vtrap-G4S”代表第二抗原结合部分和第三抗原结合部分通过一个G4S与所形成的IgG型抗体结构连接。其中，下述抗体编号中Vtrap的序列为SEQ ID NO:92所示的序列。
实施例2
实施例2通过ELISA的方法表征了所制备得到的双特异性抗体和三特异性抗体与VEGF的结合活性。实验内容如下：
用VEGF包被细胞培养板，4摄氏度包被过夜。使用缓冲液洗涤，每孔加入封闭缓冲液(2％BSA-PBS)，室温封闭1小时。使用缓冲液洗涤，每孔加入不同的抗体稀释液，室温孵育1到2小时。用缓冲液洗涤，每孔加入HRP标价的检测抗体稀释液到相应孔中，室温孵育1小时。用缓冲液洗涤，加入显色液，室温显色5-15分钟后终止显色，在波长为450nm的吸光度下读取相应数值。不同抗体的EC50结果如下表1和图4所示：
表1
实施例3
VEGF在肿瘤细胞和淋巴管上皮细胞之间起到了一定的作用，使两者进行相互调节，可促使新的淋巴管生成，淋巴管的增加使得肿瘤更容易进行转移。实施例3中，在293细胞中插入了VEGF响应元件，在VEGF刺激下，激活VEGF信号通路激活，诱导luciferase的表达，在荧光底物的作用下，释放荧光信号。加入抗体阻断VEGF与其受体的结合后，VEGF信号通路受到抑制，进而抑制荧光信号。通过报告基因阻断实验对所制备得到的双特异性抗体和三特异性抗体与VEGF的结合活性进行了表征。实验内容如下：
培养293-VEGF-Res细胞(购自近岸生物)，然后将细胞铺板，备用。将不同浓度的抗体溶液加入到细胞培养板中。准备不同浓度的VEGF蛋白稀释液。将抗体溶液和蛋白稀释液混匀，于37℃孵育30min。然后将稀释好的293-VEGF-res细胞加入VEGF/抗体混合液中，于37℃孵育6h。然后加入One-Glo试剂(One Gloluciferase assay kit，购自于Progema)，孵育5-10min，酶标仪读荧光值。实验结果如表2和图5所示。
表2
实验结果表明：所制备的双特异性抗体或者三特异性抗体的VEGF报告基因阻断活性与对照aflibercept类似。
实施例4
为了评价Anti-GARP/TGF-β抗体对下游信号通路的激活的抑制作用，实施例4通过报告基因阻断实验对所制备得到的双特异性抗体和三特异性抗体与GARP/TGF-β的结合活性进行了表征。在293F细胞系中插入GARP,LAP，以及αvβ6基因，该细胞系在生长过程中会通过GARP-LAP-αvβ6复合体张力作用剪切LAP蛋白，从而释放TGF-β，释放的TGF-β进入到插入了TGF-β信号响应元件的293-SBE-res(1E9)细胞后，会激活TGFβ信号通路，诱导荧光素酶的释放，进而在荧光底物的作用下释放荧光，当加入GARP/TGFβ抗体后，TGFβ信号通路被阻断，从而荧光信号降低。实验步骤如下：
培养293F-GARP/TGF-β-avβ6-4D11细胞，将细胞(细胞密度0.6x106个/mL)按照50ul/孔加入细胞培养板中；将；293-SBE-res细胞继续加入细胞培养板中。然后将不同浓度的待测抗体加入到细胞培养板中，混匀后至于培养箱中培养。每孔加入荧光素酶底物One-GloTM试剂，避光孵育5-10min，使用酶标仪读发光信号值。使用数据处理软件Graphpad Prism6.0计算得到待测样品的IC50值。如下表3和图6所示。
表3
实施例5
实施例5通过报告基因实验研究了抗体对于PD-L1/PD-1通路的阻断作用。该实验借助于两种细胞系完成。Jurkat-PD1-CD3zeta-NFAT-Luc2细胞株(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及CD3zeta组成的融合蛋白，同时插入了NF-AT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2×104个/孔铺于96孔板中，将Jurkat-PD1-CD3zeta-NFAT-Luc2效应细胞按照2×104/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)，于37℃下孵育18-24h。每孔加入100μl的荧光素酶底物ONE-GloTM Luciferase Assay system检测试剂，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 6.0软件分析该曲线并得出双抗的IC50值，如下表4和图7所示。
表4
实施例6
实施例6评估了双特异性抗体或者三特异性抗体对HUVEC细胞迁移的影响。HUVEC细胞表面表达VEGFRI及VEGFRII受体，当HUVEC细胞在VEGF的刺激下，通过与其受体的结合激活下游的信号通路，进而介导HUVEC细胞的增长与迁移。当加入抗体阻断VEGF与其受体的结合后，VEGF生长信号被抑制，细胞增殖受到抑制。实验内容如下：
将HUVEC细胞按100uL/孔铺于96孔培养板中。将不同浓度的抗体加入新的细胞培养板中，加入不同浓度的稀释的VEGF，37℃下孵育1h。然后将抗原抗体混合液加入HUVEC细胞培养板中；于37℃、5％CO2培养箱孵育72h后进行检测。
实验结果表明：所提供的双特异性抗体或多特异性抗体能够抑制HUVEC细胞的增殖。
在本说明书的描述中，参考术语“实施例”、“实施方式”、“具体实施方式”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 (23)

  1. 一种多特异性抗体，其特征在于，至少包括第一抗原结合部分和第三抗原结合部分；
    所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区：
    包含SEQ ID NO:1、2和3所示的HCDR序列；
    或者包含SEQ ID NO:4、5和6所示的HCDR序列，
    或者包含SEQ ID NO:7、8和9所示的HCDR序列，
    或者包含SEQ ID NO:10、11和12所示的HCDR序列，
    或者包含SEQ ID NO:13、14和15所示的HCDR序列，
    或者包含SEQ ID NO:16、17和18所示的HCDR序列；
    所述轻链可变区：
    包含SEQ ID NO:19、20和21所示的LCDR序列，
    或者包含SEQ ID NO:22、23和24所示的LCDR序列，
    或者包含SEQ ID NO:25、20和26所示的LCDR序列，
    或者包含SEQ ID NO:27、28和29所示的LCDR序列，
    或者包含SEQ ID NO:30、31和32所示的LCDR序列，
    或者包含SEQ ID NO:33、34和35所示的LCDR序列；
    所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子；
    所述HCDR序列和所述LCDR序列是基于IMGT定义方案获得的。
  2. 根据权利要求1所述的多特异性抗体，其特征在于，所述第一抗原结合部分具有：
    (1)SEQ ID NO:1、2和3所示的HCDR序列和SEQ ID NO:19、20和21所示的LCDR序列；或者
    (2)SEQ ID NO:4、5和6所示的HCDR序列和SEQ ID NO:22、23和24所示的LCDR序列；或者
    (3)SEQ ID NO:7、8和9所示的HCDR序列和SEQ ID NO:25、20和26所示的LCDR序列；或者
    (4)SEQ ID NO:10、11和12所示的HCDR序列和SEQ ID NO:27、28和29所示的LCDR序列；或者
    (5)SEQ ID NO:13、14和15所示的HCDR序列和SEQ ID NO:30、31和32所示的LCDR序列；或者
    (6)SEQ ID NO:16、17和18所示的HCDR序列和SEQ ID NO:33、34和35所示的LCDR序列；
    任选地，所述第三抗原结合部分包括至少两种不同的VEGFR免疫球蛋白样结构域；
    任选地，所述VEGF受体融合蛋白或抗VEGF抗体或抗原结合片段或VEGF结合分子为：
    (i)包含两个多肽的VEGF受体融合蛋白，具有SEQ ID NO:92所示的VEGFR1组分和VEGFR2组分；
    (ii)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白样(Ig)结构域2和VEGFR2的Ig结构域3；
    (iii)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白样(Ig)结构域2、VEGFR2的Ig结构域3和VEGFR2的Ig结构域4。
  3. 一种多特异性抗体，其特征在于，至少包含第一抗原结合部分和第三抗原结合部分；
    所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第一抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含互补决定区HCDR1、HCDR2和HCDR3，所述轻链可变区包含互补决定区LCDR1、LCDR2和LCDR3；其中：
    HCDR1包含SEQ ID NO:1或4或7或10或13或16所示的序列，
    HCDR2包含SEQ ID NO:2或5或8或11或14或17所示的序列，
    HCDR3包含SEQ ID NO:3或6或9或12或15或18所示的序列，
    LCDR1包含SEQ ID NO:19或22或25或27或30或33所示的序列，
    LCDR2包含SEQ ID NO:20或23或28或31或34所示的序列，
    LCDR3包含SEQ ID NO:21或24或26或29或32或35所示的序列；
    所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子；
    所述HCDR1、HCDR2、HCDR3序列和所述LCDR1、LCDR2、LCDR3序列是基于IMGT定义方案 获得的。
  4. 根据权利要求1～3中任一项所述的多特异性抗体，其特征在于，所述第一抗原结合部分包含：
    与SEQ ID NO:36或37或38或39或40或41或42或43或44或45或46或47所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链可变区，和
    与SEQ ID NO:48或49或50或51或52或53或54或55或56或57或58或59所示的序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、1％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链可变区。
  5. 根据权利要求1～4中任一项所述的多特异性抗体，其特征在于，还包括第二抗原结合部分，所述第二抗原结合部分能够结合PD-L1，所述第二抗原结合部分为单域抗体；
    任选地，所述第二抗原结合部分选自：
    (1)具有SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:62所示的CDR3序列；或者
    (2)具有SEQ ID NO:63所示的CDR1序列、SEQ ID NO:64所示的CDR2序列和SEQ ID NO:65所示的CDR3序列；或者
    (3)具有SEQ ID NO:66所示的CDR1序列、SEQ ID NO:67所示的CDR2序列和SEQ ID NO:68所示的CDR3序列；或者
    (4)具有SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:69所示的CDR3序列；
    所述CDR1、CDR2和CDR3是基于IMGT定义方案获得的。
  6. 根据权利要求5所述的多特异性抗体，其特征在于，所述第二抗原结合部分选自：
    (a)具有如SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的氨基酸序列；
    (b)与(a)相比，序列同一性在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。
  7. 根据权利要求3所述的多特异性抗体，其特征在于，所述第三抗原结合部分包括至少两种不同的VEGFR胞外结构域；
    任选地，所述VEGF受体融合蛋白或抗VEGF抗体或抗原结合片段或VEGF结合分子为：
    (i)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白结构域(Ig)和/或VEGFR2的免疫球蛋白结构域和/或VEGFR3的免疫球蛋白结构域；
    (ii)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白样结构域2和VEGFR2的Ig结构域3；
    (iii)包含两个多肽的VEGF受体融合蛋白，具有VEGFR1的免疫球蛋白样(Ig)结构域2、VEGFR2的Ig结构域3和VEGFR2的Ig结构域4。
  8. 一种多特异性抗体，其特征在于，包括第一抗原结合部分、第二抗原结合部分和第三抗原结合部分，所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第二抗原结合部分能够结合PD-L1，所述第三抗原结合部分为VEGF受体融合蛋白、抗VEGF抗体或抗原结合片段或VEGF结合分子；
    所述第一抗原结合部分包括重链可变区和轻链可变区，所述第一抗原结合部分具有：
    SEQ ID NO:1、2和3所示的HCDR序列和SEQ ID NO:19、20和21所示的LCDR序列，
    或者SEQ ID NO:4、5和6所示的HCDR序列和SEQ ID NO:22、23和24所示的LCDR序列，
    或者SEQ ID NO:7、8和9所示的HCDR序列和SEQ ID NO:25、20和26所示的LCDR序列，
    或者SEQ ID NO:10、11和12所示的HCDR序列和SEQ ID NO:27、28和29所示的LCDR序列，
    或者SEQ ID NO:13、14和15所示的HCDR序列和SEQ ID NO:30、31和32所示的LCDR序列，
    或者SEQ ID NO:16、17和18所示的HCDR序列和SEQ ID NO:33、34和35所示的LCDR序列；
    所述第二抗原结合部分具有；
    SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:62所示的CDR3序列；
    或者SEQ ID NO:63所示的CDR1序列、SEQ ID NO:64所示的CDR2序列和SEQ ID NO:65所示的CDR3序列；
    或者SEQ ID NO:66所示的CDR1序列、SEQ ID NO:67所示的CDR2序列和SEQ ID NO:68所示的 CDR3序列；
    或者SEQ ID NO:60所示的CDR1序列、SEQ ID NO:61所示的CDR2序列和SEQ ID NO:69所示的CDR3序列；
    所述第一抗原结合部分与重链第一恒定结构域CH1、轻链恒定结构域VL以及Fc区形成IgG型结构，所述第二抗原结合部分与轻链恒定结构域VL连接，所述第三抗原结合部分与Fc区的羧基端连接；
    所述HCDR序列、LCDR序列以及CDR1、CDR2和CDR3序列是基于IMGT定义方案获得的。
  9. 根据权利要求8所述的多特异性抗体，其特征在于，所述第一抗原结合部分包括：
    (1)SEQ ID NO:36或37或38或39或40或41所示的重链可变区，和SEQ ID NO:48或49或50或51或52或53所示的轻链可变区；或者
    (2)SEQ ID NO:42或43或44或45或46或47所示的重链可变区，和SEQ ID NO:54或55或56或57或58或59所示的轻链可变区；
    所述第二抗原结合部分包括：
    具有SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示的氨基酸序列；
    所述第三抗原结合部分具有SEQ ID NO:92所示的序列。
  10. 一种多特异性抗体，其特征在于，包含第一抗原结合部分、第二抗原结合部分和第三抗原结合部分；
    所述第一抗原结合部分能够结合TGF-β、GARP或者GARP-TGF-β复合物，所述第一抗原结合部分包括：
    如SEQ ID NO:36或37或38或39或40或41或42或43或44或45或46或47所示重链可变区的HCDR1、HCDR2和HCDR3序列，以及如SEQ ID NO:48或49或50或51或52或53或54或55或56或57或58或59所示轻链可变区的LCDR1、LCDR2和LCDR3序列；
    所述第二抗原结合部分能够结合PD-L1，所述第二抗原结合部分包含：
    如SEQ ID NO:82或83或84或85或86或87或88或89或90或91所示氨基酸的CDR1、CDR2和CDR3序列；
    所述第三抗原结合部分为VEGF受体融合蛋白，具有SEQ ID NO:92所示的序列。
  11. 一种多特异性抗体，其特征在于，包含：
    与TGF-β、GARP和/或GARP-TGF-β复合物特异性结合的第一抗原结合部分、与PD-L1特异性结合的第二抗原结合部分和与VEGF特异性结合的第三抗原结合部分；
    进一步包括重链第一恒定结构域CH1和轻链恒定结构域CL，所述第一抗原结合部分分别和重链第一恒定结构域CH1、轻链恒定结构域CL共价连接；
    进一步包括Fc区，所述Fc区通过铰链区和重链第一恒定结构域CH1连接；
    所述第二抗原结合部分与Fc区和/或所述轻链恒定结构域CL和/或轻链可变区连接；
    所述第三抗原结合部分与Fc区和/或与轻链恒定结构域CL和/或轻链可变区连接。
  12. 根据权利要求11所述的多特异性抗体，其特征在于，选自下列中的至少一种：
    (i)所述第二抗原结合部分与轻链恒定结构域CL连接，所述第三抗原结合部分与所述Fc区的羧基端(C端)连接；
    (ii)所述第二抗原结合部分与轻链可变区的氨基端(N端)连接，所述第三抗原结合部分与所述Fc区的羧基端(C端)连接；
    (iii)所述第二抗原结合部分与Fc区的羧基端(C端)连接，所述第三抗原结合部分与轻链恒定结构域CL连接。
  13. 根据权利要求11所述的多特异性抗体，其特征在于，至少具有下列性质之一：
    (a)与PD-L1特异性结合；
    (b)与糖蛋白A重复主导序列(GARP)特异性结合；
    (c)与GARP-TGF-β复合物特异性结合；
    (d)与TGF-β特异性结合；
    (e)对调节性T细胞的免疫抑制功能具有抑制活性；
    (f)具有抗肿瘤活性；
    (g)与VEGF特异性结合；
    (h)具有抑制病理性血管生长的活性。
  14. 一种多核苷酸，其特征在于，所述多核苷酸编码权利要求1～13中任一项所述的多特异性抗体。
  15. 一种构建体，其特征在于，包含权利要求14所述的多核苷酸。
  16. 一种宿主细胞，其特征在于，含有权利要求14所述的多核苷酸或者权利要求15所述的构建体。
  17. 一种药物组合物，其特征在于，包括：
    权利要求1～13中任一项所述的多特异性抗体；以及
    药学上可接受的载体。
  18. 一种抗体偶联物，其特征在于，包括：
    权利要求1～13中任一项所述的多特异性抗体；以及
    与所述多特异性抗体连接的功能小分子。
  19. 一种试剂盒，其特征在于，包括权利要求1～13中任一项所述的多特异性抗体。
  20. 一种生产权利要求1～13中任一项所述的多特异性抗体的方法，其特征在于，包括：
    培养权利要求16所述的宿主细胞，以及
    从培养物中收集所述的多特异性抗体。
  21. 一种预防和/或治疗疾病的方法，其特征在于，包括：
    给予有需要的受试者有效量的权利要求1～13中任一项所述的多特异性抗体，或者权利要求17所述的药物组合物，或者权利要求18所述的抗体偶联物。
  22. 根据权利要求21所述的方法，其特征在于，所述疾病选自癌症或者自身免疫性疾病。
  23. 权利要求1～13中任一项所述的多特异性抗体在制备药物或者试剂盒或抗体偶联物中的用途。
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