Bispecific antibodies and uses thereof

Abstract

Provided are bispecific antibodies and uses thereof. The provided bispecific antibodies can specifically bind to TGF-β, GARP or GARP-TGF-β complex, either singly or simultaneously, and can also specifically bind to PD-L1. The provided bispecific antibodies exhibit high affinity and specificity for antigens and have tumor killing effects, and can therefore be used in tumor treatment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to a Chinese patent application filed on September 30, 2021, bearing application number 202111162303.X, which is incorporated herein in its entirety or in part. [Technical Field]

[0002] This application relates to the field of biopharmaceuticals, and in particular to bispecific antibodies and uses thereof. [Background technology]

[0003] Transforming growth factor-β (TGF-β), a member of the TGF-β superfamily, mediates the proliferation and differentiation of various immune cells, such as regulatory T cells (Tregs), DCs, and CD3 + TGF-β is expressed in T cells, M2 macrophages, and other cells, and regulates cell proliferation and differentiation. In the tumor microenvironment, immune cell activity is generally influenced by TGF-β in the environment, resulting in immunosuppression. Due to the potency and diversity of TGF-β, family members, including TGF-β1, β2, and β3, are produced as inactive latent forms and are unable to bind to their receptors without a strictly regulated extracellular activation step (Cold Spring Harb. Perspect. Biol. 2016;8(a022103), Cytokine Growth Factor Rev. 2013;24:355-372). TGF-β1 is the predominant isoform expressed by immune cells, such as Tregs. Treg cells produce active TGF-β and mediate immunosuppressive effects, thereby preventing the development of autoimmune diseases. However, malignant tumors exploit this mechanism to release large amounts of TGF-β, causing cancer cells to divide rapidly, while relying on Treg cells to inhibit the killing effects of other immune cells against cancer cells.

[0004] Glycoprotein A repeats predominant (GARP) is a type I transmembrane cell surface receptor that binds to latent transforming growth factor-β (TGF-β). It is primarily activated on the surface of Treg and B cells and platelets, and is highly expressed in human breast, lung, and colon tumors. GARP can form a complex with latent TGF-β (latency-associated peptide, LAP) (Proc Natl Acad Sci USA. 2009;106(32):13445-50), regulate the ability of membrane-bound latent TGF-β, and bind to αVβ8 or αVβ6 integrin, releasing TGF-β from the cell surface and activating it.

[0005] Given that high expression of GARP provides the activation and storage of TGF-β necessary for tumor growth, enhances the suppressive phenotype of Tregs, and maintains Treg-mediated peripheral immune tolerance, GARP inhibitors are gradually gaining popularity in tumor therapy. Antibodies targeting GARP are also being rapidly developed. For example, the first clinically used GARP inhibitor, ARGX-115, targets GARP and inhibits the release of active TGF-β and the immunosuppressive activity of Tregs, thereby reactivating the immune response of cancer cells and suppressing disease progression. DS-1055, a monoclonal antibody designed against GARP, developed by Daiichi Sankyo, is also undergoing clinical trials for relapsed / refractory late-stage or metastatic head and neck cancer, gastric cancer, and esophageal cancer.

[0006] Bispecific antibodies (BsAbs) are antibodies that can simultaneously bind specifically to two antigens or epitopes. Since the concept of BsAbs was first proposed by Nisonoff and coworkers in 1960, bispecific antibodies have been rapidly developed as recombinant antibody technology has gradually matured. Because BsAbs can target multiple antigens or epitopes, they offer many advantages over monoclonal antibodies and are gradually demonstrating superior efficacy compared to monoclonal antibody combination therapy. For example, they can redirect specific immune effector cells to nearby tumor cells to improve tumor killing. Furthermore, the interaction between two different cell surface antigens can enhance binding specificity, reducing development costs and clinical trials compared to the combination of single antibodies. Bispecific antibodies have become a research hotspot in the field of antibody engineering and are expected to be widely used in areas such as tumor therapy and autoimmune diseases. Several pharmaceutical companies in China and abroad are conducting research into bispecific antibodies targeting different targets.

[0007] However, bispecific antibodies are more complex than monoclonal antibodies because they involve the interaction of two or more entities. Further improvements are needed in the development of bispecific antibodies against different targets. Summary of the Invention

[0008] The present application provides bispecific antibodies and their use in disease treatment. The present application targets GARP, TGF-β, and / or the GARP-TGF-β complex to inhibit the release of active TGF-β and the immunosuppressive function of Tregs, thereby reactivating the immune response of cancer cells and suppressing disease progression. The provided bispecific antibodies can also specifically bind to PD-L1, thereby improving anti-tumor activity in cases of PD-1 / PD-L1 resistance.

[0009] A first aspect of the present application provides a bispecific antibody, the bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, the first antigen-binding portion comprising a heavy chain variable region, the heavy chain variable region comprising the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6. or comprising the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or comprising the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or comprising the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15. the first antigen-binding portion further comprises a light chain variable region comprising an LCDR sequence shown in SEQ ID NOs: 19, 20, and 21, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the ...22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 69, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 11 , 23 and 24, or a sequence having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24, or a LCDR sequence shown in SEQ ID NOs: 25, 20 and 26, or a sequence having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 25, 20 and 26, or a LCDR sequence shown in SEQ ID NOs: 27, 28 and 29, or a sequence having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29,comprising a sequence having a deletion or addition, or comprising the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or a sequence having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or comprising the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35, or a sequence having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35, The first antigen-binding moiety can bind to TGF-β, GARP, or a GARP-TGF-β complex, the second antigen-binding moiety can bind to PD-L1, and the HCDR sequence and the LCDR sequence are obtained according to the IMGT scheme.

[0010] The provided bispecific antibodies have a first antigen-binding moiety and a second antigen-binding moiety, where the first antigen-binding moiety specifically targets one, two, or three of GARP, TGF-β, or the GARP-TGF-β complex and can activate immune cell responses, and the second antigen-binding moiety specifically targets PD-L1 and can block the PD-1 / PD-L1 signaling pathway, thereby relieving immunosuppression and restoring the body's immune killing ability. The provided bispecific antibodies have been confirmed to exhibit excellent tumor therapeutic effects.

[0011] A second aspect of the present application provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises complementarity 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, and HCDR3 comprises the sequence shown in SEQ ID NO: 3, or 6, or 9, or 12, or 15, or 18. the first antigen-binding moiety is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex; the second antigen-binding moiety is capable of binding to PD-L1; and the HCDR1, HCDR2, and HCDR3 sequences and the LCDR1, LCDR2, and LCDR3 sequences are obtained based on the IMGT scheme.

[0012] A third aspect of the present application provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is capable of binding to GARP, TGFβ and / or a GARP-TGF-β complex; the second antigen-binding portion comprises CDR1, CDR2, and CDR3, wherein: (a) the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 60, 63 and 66, or a sequence having one or two amino acid substitutions, deletions or additions compared to SEQ ID NOs: 60, 63 and 66; (b) the CDR2 comprises a sequence selected from the following group: SEQ ID NOs: 61, 64, and 67, or a sequence having one or two amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 61, 64, and 67; (c) the CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 62, 65, 68 and 69, or a sequence having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NOs: 62, 65, 68 and 69; The CDR1, CDR2 and CDR3 sequences are obtained based on the IMGT scheme.

[0013] A fourth aspect of the present application provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, and the light chain variable region comprises the LCDR sequences shown in SEQ ID NOs: 19, 20 and 21. or comprising the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24, or comprising the LCDR sequences shown in SEQ ID NOs: 25, 20 and 26, or comprising the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29, or comprising the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or comprising the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35, and the second antigen-binding portion comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the sequence shown in SEQ ID NO: 60, 63 or 66, the CDR2 comprises the sequence shown in SEQ ID NO: 61, 64 or 67, and the CDR3 comprises the sequence shown in SEQ ID NO: 62, 65, 68 or 69.

[0014] A fifth aspect of the present application provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises the HCDR1, HCDR2, and HCDR3 sequences of a heavy chain variable region set forth 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 a light chain variable region set forth 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, wherein the first antigen-binding portion is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1.

[0015] A sixth aspect of the present application provides a bispecific antibody, the bispecific antibody comprising a first antigen-binding portion that specifically binds to TGFβ, GARP and / or the GARP-TGFβ complex, and a second antigen-binding portion that specifically binds to PD-L1, the bispecific antibody further comprising a heavy chain first constant domain, CH1, and a light chain constant domain, CL, wherein the first antigen-binding portion is covalently linked to the heavy chain first constant domain, CH1, and the light chain constant domain, CL, respectively; the bispecific antibody further comprising an Fc region, wherein the Fc region is connected to the heavy chain first constant domain, CH1, by a hinge region, and the second antigen-binding portion is covalently linked to the Fc region and / or the light chain constant domain, CL, by a linker.

[0016] A seventh aspect of the present application provides a monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region, wherein the heavy chain variable region comprises the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9. or comprises the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or comprises the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or comprises the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18.

[0017] An eighth aspect of the present application provides a monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the antibody or antigen-binding fragment has the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, and the LCDR sequences set forth in SEQ ID NOs: 19, 20, and 21, or the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, and the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 24, or the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, and the LCDR sequences set forth in SEQ ID NOs: 25, 20, and 26, or the HCDR sequences set forth in SEQ ID NOs: 10, 11, and 12, and the LCDR sequences set forth in SEQ ID NOs: 27, 28, and 29, or the HCDR sequences set forth in SEQ ID NOs: 13, 14, and 15, and the LCDR sequences set forth in SEQ ID NOs: 30, 31, and 32, or the HCDR sequences set forth in SEQ ID NOs: 16, 17, and 18, and the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35.

[0018] A ninth aspect of the present application provides a monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3; and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, or 16; HCDR2 comprises the sequence shown in SEQ ID NO: 2, 5, 8, 11, 14, or 17; and HCDR3 comprises the sequence shown in SEQ ID NO: 3, 6, or 9, or 12, 15, or 18; LCDR1 comprises the sequence shown in SEQ ID NO: 19, 22, 25, 27, 30, or 33; LCDR2 comprises the sequence shown in SEQ ID NO: 20, 23, 28, 31, or 34; and LCDR3 comprises the sequence shown in SEQ ID NO: 21, 24, 26, 29, 32, or 35.

[0019] A tenth aspect of the present application provides a monoclonal antibody or antigen-binding fragment comprising the HCDR1, HCDR2 and HCDR3 sequences of a heavy chain variable region 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 a light chain variable region 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.

[0020] An eleventh aspect of the present application provides a polynucleotide encoding the bispecific antibody according to any one of the above first to sixth aspects, or encoding the monoclonal antibody or antigen-binding fragment according to any one of the seventh to tenth aspects.

[0021] A twelfth aspect of the present application provides a construct comprising a polynucleotide according to the eleventh aspect above.

[0022] A thirteenth aspect of the present application provides a host cell containing a polynucleotide according to the eleventh aspect or a construct according to the twelfth aspect above.

[0023] A fourteenth aspect of the present application provides a pharmaceutical composition comprising the bispecific antibody according to any one of the above first to sixth aspects, or the monoclonal antibody or antigen-binding fragment according to any one of the above seventh to tenth aspects, and a pharmaceutically acceptable carrier.

[0024] A fifteenth aspect of the present application provides an antibody complex comprising the bispecific antibody according to any one of the above first to sixth aspects, or the monoclonal antibody or antigen-binding fragment according to any one of the above seventh to tenth aspects, and a functional small molecule linked to the bispecific antibody or the monoclonal antibody or antigen-binding fragment.

[0025] A sixteenth aspect of the present application provides a kit comprising a bispecific antibody described in any one of the examples of the first to sixth aspects above, or a monoclonal antibody or antigen-binding fragment described in any one of the seventh to tenth aspects.

[0026] A 17th aspect of the present application provides a method for producing a bispecific antibody or a monoclonal antibody or antigen-binding fragment thereof, comprising culturing a host cell according to the 13th aspect and recovering the bispecific antibody or monoclonal antibody or antigen-binding fragment thereof from the culture.

[0027] An 18th aspect of the present application provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof an effective amount of the bispecific antibody according to any one of the above-mentioned first to sixth aspects, or the monoclonal antibody or antigen-binding fragment according to any one of the above-mentioned seventh to tenth aspects, or the pharmaceutical composition according to the 14th aspect, or the antibody conjugate according to the 15th aspect.

[0028] A 19th aspect of the present application provides use of the bispecific antibody according to any one of the above-mentioned first to sixth aspects, or the monoclonal antibody or antigen-binding fragment according to any one of the above-mentioned seventh to tenth aspects, in the production of a drug or kit, or in the production of an antibody conjugate. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram of the structure of a bispecific antibody provided by the Examples of the present application. [Figure 2] FIG. 2 is a schematic diagram of the structure of a bispecific antibody provided by an example of the present application. [Figure 3] FIG. 3 is a schematic diagram of the structure of a bispecific antibody provided by an example of the present application. [Figure 4] FIG. 4 is a schematic diagram of the structure of a bispecific antibody provided by an example of the present application. [Figure 5] FIG. 5 shows the results of the binding activity of antibodies to the GARP-TGF-β complex detected using FACS, provided in the Examples of the present application. [Figure 6] FIG. 6 shows the results of the binding activity of antibodies provided by the examples of the present application against TGF-β. [Figure 7] FIG. 7 shows the results of the neutralizing activity of antibodies against TGF-β provided by the examples of the present application. [Figure 8] FIG. 8 shows the results of efficacy testing of antibodies provided by the examples of the present application in an animal model. DETAILED DESCRIPTION OF THE INVENTION

[0030] The technical solutions of the present application will be described in detail below with specific examples. In addition, some terms in the present application will be explained and explained to facilitate understanding by those skilled in the art. These explanations and explanations are for the purpose of facilitating understanding by those skilled in the art, and should not be considered as limitations on the protection scope of the present application.

[0031] The term "bispecific antibody" includes antigen-binding portions that specifically bind to epitopes of at least two different biomolecules, and for ease of distinction, these are referred to as the "first antigen-binding portion" and the "second antigen-binding portion," respectively. The terms "first antigen-binding portion" and "second antigen-binding portion" are also interpreted and explained below. Unless otherwise specified, there is no restriction on the order in which the listed bispecific antibodies bind to antigens. Of course, when specifically binding to different biomolecules, the bispecific antibody may bind to more than one epitope of the particular biomolecules.

[0032] As used herein, the term "antibody" is used in its broadest sense to refer to a protein or polypeptide that comprises an antigen-binding site, covering natural and artificial antibodies of various structures, including, but not limited to, forms of intact antibodies or antigen-binding fragments of antibodies.

[0033] As used herein, the terms "intact antibody" and "complete antibody" are interchangeable and refer to a protein comprising at least two heavy chains (H) and two light chains (L) connected to each other by disulfide bonds. 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 comprises a first heavy chain constant domain (CH1), a second heavy chain constant domain (CH2), and a 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 the light chain constant domain (CL). The VH and VL regions are further divided into complementarity-determining regions (also called hypervariable regions or hypervariable regions, abbreviated as CDRs), separated by conserved framework regions (FR). Each of VH and VL contains three CDRs and four FRs arranged from the amino terminus (N-terminus) to the carboxy terminus (C-terminus) as the sequences FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDRs of the heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, respectively, from the amino terminus of the heavy chain amino acid sequence, and the CDRs of the light chain variable region are referred to as LCDR1, LCDR2, and LCDR3, respectively, from the amino terminus of the light chain amino acid sequence.

[0034] An "antigen-binding fragment" or "antigen-binding portion" (e.g., a first antigen-binding portion, a second antigen-binding portion) refers to a portion of a full-length antibody, typically comprising a variable domain or region that binds to an antigen. The term "variable region" or "variable domain" refers to the domain involved in antigen binding to the heavy or light chain of an antibody. As mentioned above, the variable regions of the heavy and light chains of an intact antibody generally have a similar organization, with each domain comprising four conserved framework regions and three hypervariable regions. Examples of antigen-binding fragments or portions include Fab, Fab', F(ab')2, bispecific Fab' and Fv fragments, linear antibodies, single-chain antibodies, single-domain antibodies, etc. Two identical antigen-binding fragments produced by papain digestion of an intact antibody are called Fab fragments, each containing heavy and light chain variable regions, a light chain constant domain, and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues, including one or more cysteines from the antibody hinge region, to the carboxy terminus of the first heavy chain constant domain CH1. F(ab')2 fragments are obtained by pepsin digestion of intact antibodies. F(ab')2 fragments contain two antigen-binding portions, F(ab), connected by disulfide bonds, making them bivalent antibodies. Single-chain antibodies are fusion proteins in which the heavy and light chain variable regions of an antibody are connected by a flexible short peptide chain consisting of approximately 10-25 amino acids. Single-domain antibodies are antibody fragments consisting of the variable region of a single monomer. Single-domain antibodies are commonly called nanobodies because they are typically derived from the variable region of the heavy chain of camelid or shark antibodies.

[0035] For example, a nanobody is the smallest fully functional antigen-binding fragment containing only the heavy chain CDR regions. Nanobodies contain three CDR regions (CDR1 to CDR3) and four framework regions FR (FR1 to FR4). Framework regions FR1, FR2, FR3, and FR4 are separated by complementarity-determining regions CDR1, CDR2, and CDR3, respectively.

[0036] The term "and / or," when used in conjunction with two or more options, is understood to mean any one of the options or two or more of the options.

[0037] The term "comprise" or "comprises" means including the elements or steps referred to, but not excluding other elements or steps. Naturally, unless otherwise specified, "comprise" or "comprises" also covers those consisting of the elements or steps referred to. For example, when a reference is made to including an antibody variable region of a particular sequence, it is intended to cover an antibody variable region consisting of that particular sequence.

[0038] "Affinity" or "binding affinity" as referred to herein is understood in the general sense in the art to reflect the strength and / or stability between an antigen and a binding site on an antibody or antigen-binding fragment.

[0039] The terms "specifically bind to," "specifically bind with," "bind to," "specifically target," or "have specificity for" or "can bind to" a particular antigen or epitope are distinct from nonspecific interactions, and such specific binding may be measured by several methods commonly used in the art. The ability of an antibody to bind to an antigen may be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art. For example, antigen-bearing cells may be detected by flow cytometry, and the competitive binding status of the test antibody to the labeled antibody may be detected by measuring the positive rate index of the cells. Because the spatial structure of antigens on the cell surface is closer to the form present in the body, this method can more accurately reflect actual conditions. According to specific embodiments of the present application, the provided antibodies have an EC of 100 nM or less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, or 0.05 nM or less. 50The binding activity of an antibody to an antigen may also be measured by surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0040] The monoclonal antibodies or antigen-binding fragments, bispecific antibodies, and polynucleotides provided herein are generally isolatable or recombinant. "Isolable" refers to the ability to be identified and isolated and / or recovered from a cell or cell culture that expresses the polypeptide or protein. Generally, an isolated polypeptide is produced by at least one purification step. An "isolated antibody" refers to one that is substantially free of a distinct antigen. "Recombinant" refers to the ability to produce the antibody in an exogenous host cell using genetic engineering techniques.

[0041] A "humanized" antibody generally refers to an antibody that consists of an antigen-binding portion derived from a non-human species and a partial structure and sequence of a human immunoglobulin molecule. For example, in a humanized antibody, the entire antibody except for the CDRs is encoded by a polynucleotide of human origin, retaining antigen-binding activity while reducing immunogenicity.

[0042] The CDR sequences of the first and second antigen-binding moieties described herein are obtained by analysis based on existing schemes. For example, the CDR sequences of the first and second antigen-binding moieties are obtained based on the IMGT scheme. They may also be obtained by schemes such as Kabat (see, e.g., US Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)) and Chothia (see, e.g., J. Mol. Biol. 196:901-917 (1987)). As those skilled in the art will recognize, CDR sequences that differ in their definition methods are also within the scope of protection of the present application.

[0043] "Bispecific antibody" The present application provides a bispecific antibody comprising a first antigen-binding moiety and a second antigen-binding moiety, each of which targets a different target antigen. Herein, the first antigen-binding moiety can specifically target one, two, or three of TGFβ, GARP, and the GARP-TGF-β complex, and the second antigen-binding moiety specifically targets PD-L1. Whether the first antigen-binding moiety specifically targets TGFβ, GARP, or the GARP-TGFβ complex, or two or three of these, it can directly or indirectly regulate TGF-β, activate the immune response of cancer cells, and suppress disease progression. The terms "first" and "second" used herein are for descriptive purposes only and should not be construed as indicating or implying relative importance or the number of technical features of interest, nor do they represent an order. The terms "first antigen-binding moiety" and "second antigen-binding moiety" refer to moieties capable of binding to different target antigens, and may be an intact antibody or a portion of an intact antibody, such as a heavy chain variable region, Fab, Fab', F(ab')2, a single-chain antibody (ScFv), or a single-domain antibody (sdAb).

[0044] The present application provides a bispecific antibody, which comprises a first antigen-binding moiety that specifically binds to TGF-β, GARP, and / or the GARP-TGF-β complex, and a second antigen-binding moiety that specifically binds to PD-L1. The bispecific antibody further comprises a heavy chain first constant domain CH1 and a light chain constant domain CL, wherein the first antigen-binding moiety is covalently linked to the heavy chain first constant domain CH1 and the light chain constant domain CL, respectively. The bispecific antibody further comprises an Fc region, wherein the Fc region is connected to the heavy chain first constant domain CH1 by a hinge region, and the second antigen-binding moiety is covalently linked to the Fc region and / or the light chain constant domain CL by a linker. In addition to the heavy chain and light chain variable region sequences mentioned above, other sequences may also be used for the IgG-type structure, including naturally occurring sequences derived from mammals (e.g., naturally occurring sequences derived from humans). In a specific embodiment, the sequence formed by the heavy chain first constant domain, heavy chain second constant domain, and heavy chain third constant domain is set forth in SEQ ID NO:92. ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)

[0045] In a specific embodiment, the sequence of the light chain constant domain is as set forth in SEQ ID NO:93. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93)

[0046] The Fc region sequence of the provided bispecific antibodies is not limited and may be naturally occurring or, for example, a human-derived Fc region or a modified Fc region. The modified Fc region may be a mutated Fc region, for example, by introducing mutations into several sites in the Fc region to control the ADCC effector function of the antibody.

[0047] The provided bispecific antibodies have at least one of the following properties: (a) specific binding to PD-L1, (b) specific binding to glycoprotein A repeat dominant (GARP), (c) specific binding to the GARP-TGF-β complex, (d) specific binding to TGF-β, (e) a function to regulate the activity of immune cells and an inhibitory activity against the immunosuppressive function of regulatory T cells, including, but not limited to, (f) anti-tumor activity.

[0048] The bispecific antibodies provided herein simultaneously exhibit specific binding activity to TGF-β, GARP, and / or GARP-TGF-β complexes, and specific binding activity to PD-L1. Using detection methods such as TGF-β reporter gene assays and Treg-released TGF-β neutralization tests, it was determined that the bispecific antibodies can bind to GARP-TGF-β1 complexes and capture TGF-β1 differentiation molecules released into the microenvironment. Furthermore, the provided bispecific antibodies specifically bind to PD-L1, simultaneously blocking the PD-1 / PD-L1 and TGF-β signaling pathways, thereby relieving immunosuppression and avoiding the complementary effect that may exist between the PD-L1 (PD-1) axis and the TGF-β axis pathway, thereby restoring the body's immune capabilities by a factor of 2 and achieving a synergistic killing effect.

[0049] In at least some embodiments, as shown in Figures 1 and 2, the second antigen-binding moiety is attached to the carboxy terminus (C-terminus) of the Fc domain via a linker. Note that disulfide bonds are not specifically depicted in the drawings provided. Figure 1 shows that each second antigen-binding moiety is attached to the carboxy terminus (C-terminus) of the Fc domain. The second antigen-binding moiety may optionally be configured as a trap structure, nanobody, single-chain antibody, or other fusion protein capable of binding to PD-L1. According to specific embodiments, the second antigen-binding moiety may be an anti-PD-L1 nanobody. When nanobodies are attached to the C-terminus of the Fc domain, the same nanobody may be attached, or different nanobodies may be attached. Furthermore, depending on the circumstances, a nanobody may be attached to only one C-terminus of the Fc domain. Figure 2 shows two second antigen-binding moieties attached to the C-terminus of each Fc domain. According to specific embodiments, two PD-L1 nanobodies are attached to the C-terminus of each Fc domain. Similarly, the same Nanobody may be attached to each C-terminus of the Fc region, or different Nanobodies may be attached. Furthermore, the number of Nanobodies attached to each C-terminus of the Fc region may be the same (e.g., two or more) or different (e.g., two or more). Taking the carboxy terminus of any one Fc region as an example, the two or more attached Nanobodies may be the same or different. Anti-PD-L1 Nanobodies may be publicly available or commercially available Nanobodies, or may be obtained by screening an antibody library. For example, Chinese patent application No. 202110903293.4 discloses obtaining an anti-PD-L1 Nanobody library through alpaca immunization, followed by screening and identification to obtain Nanobodies with good affinity, specific binding, and tumor-killing effect. The contents of the international patent application No. PCT / CN2022 / 110423 (priority application No. 202110903293.4) ​​are incorporated herein in whole or in part, where appropriate. According to a specific embodiment, each Fc may be linked to a single chain antibody at its C-terminus.The PD-L1 single chain antibody may be any disclosed or commercially available single chain antibody. The single chain antibodies attached to the C-terminus of the Fc may be the same or different, or the single chain antibody may be attached to the C-terminus of only one of the Fc domains.

[0050] In at least some embodiments, the second antigen-binding moiety is connected to the carboxy terminus (C-terminus) of the light chain constant domain via a linker. In at least some specific embodiments, as shown in Figure 3, the second antigen-binding moieties are each connected to the carboxy terminus of the light chain constant domain via a linker. The second antigen-binding moieties may optionally be selected as single-chain antibodies, single-domain antibodies, or other forms of fusion proteins. The second antigen-binding moieties connected to the carboxy termini of the light chain constant domains may be the same or different, or the second antigen-binding moiety may be connected to the carboxy terminus of any one of the light chain constant domains. In some specific embodiments, two anti-PD-L1 nanobodies are connected to the carboxy termini (C-terminus) of each light chain constant domain. Similarly, depending on the circumstances, one anti-PD-L1 nanobody or two or more anti-PD-L1 nanobodies may also be connected to the C-terminus of each light chain constant domain. For example, the number of anti-PD-L1 nanobodies connected to the carboxy terminus of any one of the light chain constant domains may be the same or different.

[0051] In at least some embodiments, as shown in Figure 4, the second antigen-binding moiety is connected to the C-terminus of the Fc region by a first linker and to the C-terminus of the light chain constant domain by a second linker. Similarly, the second antigen-binding moiety connected to the C-terminus of the Fc region or the second antigen-binding moiety connected to the light chain constant domain may be the same or different. The second antigen-binding moiety may optionally be selected as a trap structure, a single-chain antibody, a single-domain antibody, or other form of fusion protein. The first linker and second linker may be the same or different.

[0052] The linkers usable herein are those commonly used in the art and may be any oligopeptide or polypeptide. These oligopeptides or polypeptides may be any amino acid sequence that can provide flexibility. Linkers include, but are not limited to, the following group: GS, SG, GGS, GSG, SGG, GGG, GGGS (SEQ ID NO: 94), SGGG (SEQ ID NO: 95), GGGGS (SEQ ID NO: 96), GGGGGSGS (SEQ ID NO: 97), GGGGSGS (SEQ ID NO: 98), GGGGSGGS (SEQ ID NO: 99), GGGGSGGGGSGGGGGS (SEQ ID NO: 100), GGGGSGGGGS (SEQ ID NO: 101), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 102), GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 103), GGGGSGGGGSGGGGSGGGGSGGGSGGGS (SEQ ID NO: 104), etc.

[0053] The provided bispecific antibodies bind to TGF-β, GARP, or the GARP-TGFβ complex, and to PD-L1, thereby activating the immune response of cancer cells and suppressing disease progression.

[0054] The present application provides bispecific antibodies comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion comprises the HCDR1, HCDR2, and HCDR3 sequences of a heavy chain variable region set forth in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, or 47, and the LCDR1, LCDR2, and LCDR3 sequences of a light chain variable region set forth in SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59, and the second antigen-binding portion is capable of binding to PD-L1. As mentioned above, the CDR sequences of the heavy and light chain variable region sequences shown may differ slightly due to differences in antibody sequence analysis methods, such as the classical Kabat CDR scheme, the IMGT scheme, and the Chothia scheme. All of the different CDR sequences obtained through analysis are within the scope of protection of the present application.

[0055] The present application also provides a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the second antigen-binding portion is capable of binding to PD-L1, and the first antigen-binding portion comprises a heavy chain variable region comprising the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, or sequences having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, or the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, or sequences having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, or the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, or sequences having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9. or comprising the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or comprising the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or comprising the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18.

[0056] In some embodiments, the first antigen-binding portion may further comprise a light chain variable region, wherein the light chain variable region comprises an LCDR sequence set forth in SEQ ID NOs: 19, 20, and 21, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence set forth in SEQ ID NOs: 19, 20, and 21, or an LCDR sequence set forth in SEQ ID NOs: 22, 23, and 24, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence set forth in SEQ ID NOs: 22, 23, and 24, or an LCDR sequence set forth in SEQ ID NOs: 25, 20, and 26, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence set forth in SEQ ID NOs: 25, 20, and 26. or comprises the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29, or comprises the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or comprises the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35.

[0057] The sequences are as follows: The first and second antigen-binding portions of the bispecific antibodies mentioned may optionally be selected from intact antibodies or fragments thereof, as long as they are capable of targeting different target antigens. The first and second antigen-binding portions may be heavy chain variable regions, Fab, Fab', F(ab')2, single-chain antibodies (ScFv), single-domain antibodies (sdAbs), or the like. JPEG0007734856000001.jpg33156JPEG0007734856000002.jpg34156

[0058] The first antigen-binding portion of the provided bispecific antibody can bind to GARP, TGFβ, or the GARP-TGFβ complex, or specifically bind to two or three of these, and can bind to PD-L1, blocking the PD-1 / PD-L1 pathway and enhancing the killing effect of immune cells against tumor cells.

[0059] In some specific embodiments, bispecific antibodies are provided that have HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, and have LCDR sequences set forth in SEQ ID NOs: 19, 20, and 21. In some specific embodiments, bispecific antibodies are provided that have HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, and have LCDR sequences set forth in SEQ ID NOs: 22, 23, and 24. In some embodiments, bispecific antibodies are provided that have HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, and have LCDR sequences set forth in SEQ ID NOs: 25, 20, and 26. In some embodiments, bispecific antibodies are provided that have HCDR sequences set forth in SEQ ID NOs: 10, 11, and 12, and have LCDR sequences set forth in SEQ ID NOs: 27, 28, and 29. In some specific embodiments, bispecific antibodies are provided that have HCDR sequences set forth in SEQ ID NOs: 13, 14, and 15, and have LCDR sequences set forth in SEQ ID NOs: 30, 31, and 32. In some specific embodiments, the bispecific antibodies provided have HCDR sequences set forth in SEQ ID NOs: 16, 17, and 18, and LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35. The HCDR and LCDR sequences set forth specifically bind to the target antigen as the first antigen-binding portion of the bispecific antibody.

[0060] In some embodiments, the bispecific antibodies provided have a heavy chain that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth 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 at least one 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%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59, where SEQ ID NOs: 42, 43, 44, 45, 46, and 47 are humanized heavy chain variable region sequences, respectively. SEQ ID NOs: 54, 55, 56, 57, 58, and 59 are humanized light chain variable region sequences, respectively. The heavy and light chain variable region sequences shown specifically bind to a target antigen as a first antigen-binding portion of a bispecific antibody. The sequences of the light chain variable region and heavy chain variable region of the first antigen-binding moiety are humanized to reduce immunogenicity while retaining binding activity to the target antigen (e.g., GARP, TGFβ, and / or GARP-TGFβ complex). Humanized antibody sequences can be obtained by methods commonly used in the art (e.g., complementarity-determining region grafting). In some embodiments, the CDR region sequences are retained in the provided humanized antibody sequences, and the framework region sequences are replaced with human-derived framework region sequences. The human-derived framework region sequences are obtained from published human sequences. For example, database comparison and computer homology modeling are used to find the most homologous human FR region template, and a comprehensive review is performed to determine whether or not restoration mutations in the FR region are necessary and the critical residues for restoration mutations, thereby obtaining a high-affinity humanized antibody. These sequences are generally found in commonly used databases, such as the PDB protein structure database, IMGT, and Genebank.

[0061] "Sequence identity" as referred to herein refers to the degree of identity between amino acid or nucleotide sequences when compared one by one. Determining the sequence identity ratio can be achieved in various ways known in the art. For example, it may be obtained using publicly available software such as BLAST, ALIGN, BLAST-2, etc. In some specific embodiments, the sequence identity is based on conservative amino acid substitutions. "Conservative amino acid substitution" refers to replacing one amino acid residue with another amino acid residue having a side chain with similar physiological and chemical properties. For example, conservative amino acid substitutions may be made between residues having hydrophobic side chains (e.g., Met, Ala, Val, Leu, Ile), between residues having neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, Gln), between residues having acidic side chains (e.g., Asp, Glu), between residues having basic side chains (e.g., His, Lys, Arg), or between residues having aromatic side chains (e.g., Trp, Tyr, Phe). As is known in the art, conservative amino acid substitutions generally do not significantly alter the conformation of a protein, thereby allowing the biological activity of the protein to be retained. The conservative amino acid substitution referred to may be one conservative amino acid substitution, two conservative amino acid substitutions, three conservative amino acid substitutions, four conservative amino acid substitutions, five conservative amino acid substitutions, six conservative amino acid substitutions, seven conservative amino acid substitutions, eight conservative amino acid substitutions, nine conservative amino acid substitutions, or ten conservative amino acid substitutions, etc. The names of amino acids used herein are represented by the standard one-letter or three-letter code used in the art.

[0062] The sequences shown are as follows: JPEG0007734856000003.jpg235169

[0063] In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 36 and a light chain variable region set forth in SEQ ID NO: 48. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 49. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 38 and a light chain variable region set forth in SEQ ID NO: 50. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 39 and a light chain variable region set forth in SEQ ID NO: 51. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 40 and a light chain variable region set forth in SEQ ID NO: 52. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 41 and a light chain variable region set forth in SEQ ID NO: 53. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 42 and a light chain variable region set forth in SEQ ID NO: 54. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 43 and a light chain variable region set forth in SEQ ID NO: 55. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 44 and a light chain variable region set forth in SEQ ID NO: 56. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 45 and a light chain variable region set forth in SEQ ID NO: 57. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 46 and a light chain variable region set forth in SEQ ID NO: 58. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 47 and a light chain variable region set forth in SEQ ID NO: 59.

[0064] The second antigen-binding moiety can specifically target PD-L1. As mentioned above, the second antigen-binding moiety may take any form, as long as it can specifically target PD-L1. For example, it may be an intact antibody or a portion of an intact antibody, including, but not limited to, a heavy chain variable region, Fab, Fab', F(ab')2, a single-chain antibody, or a nanobody. The second antigen-binding moiety may optionally be obtained by screening an antibody library or may be a commercially available or disclosed antibody sequence. In some specific embodiments, the second antigen-binding moiety is a single-domain antibody. In some preferred embodiments, the bispecific antibody structure is formed by connecting an IgG antibody and the second antigen-binding moiety via a linker, and the IgG antibody comprises the first antigen-binding moiety. The bispecific antibody structure is formed by connecting the first antigen-binding moiety with the Fc of the IgG antibody and the second antigen-binding moiety to the IgG antibody via a linker.

[0065] In some specific embodiments, the second antigen-binding portion provided comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the following group: SEQ ID NOs: 60, 63, and 66, or a sequence having one or two amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 60, 63, and 66; CDR2 comprises a sequence selected from the following group: SEQ ID NOs: 61, 64, and 67, or a sequence having one or two amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 61, 64, and 67; and CDR3 comprises a sequence selected from the following group: SEQ ID NOs: 62, 65, 68, and 69, or a sequence having one, two, or three amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 62, 65, 68, and 69.

[0066] In some specific embodiments, the second antigen-binding portion has the CDR1 sequence set forth in SEQ ID NO: 60, the CDR2 sequence set forth in SEQ ID NO: 61, and the CDR3 sequence set forth in SEQ ID NO: 62. In some specific embodiments, the second antigen-binding portion has the CDR1 sequence set forth in SEQ ID NO: 63, the CDR2 sequence set forth in SEQ ID NO: 64, and the CDR3 sequence set forth in SEQ ID NO: 65. In some specific embodiments, the second antigen-binding portion has the CDR1 sequence set forth in SEQ ID NO: 66, the CDR2 sequence set forth in SEQ ID NO: 67, and the CDR3 sequence set forth in SEQ ID NO: 68. In some specific embodiments, the second antigen-binding portion has the CDR1 sequence set forth in SEQ ID NO: 60, the CDR2 sequence set forth in SEQ ID NO: 61, and the CDR3 sequence set forth in SEQ ID NO: 69. The CDR1 sequence, CDR2 sequence, and CDR3 sequence set forth above can form a single domain antibody together with the framework region FR. The framework region FR sequence is not particularly limited and may be derived from a murine or human, or may be derived partially from a murine or human.

[0067] The present application also provides bispecific antibodies, which comprise a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is capable of specifically binding to GARP, TGF-β, and / or a GARP-TGF-β complex, and the second antigen-binding portion is capable of specifically binding to PD-L1. In some specific embodiments, the second antigen-binding portion is an anti-PD-L1 nanobody.

[0068] In some embodiments, the second antigen-binding portion comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises a sequence selected from the following group: SEQ ID NOs: 60, 63, and 66, or a sequence having one or two amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 60, 63, and 66; CDR2 comprises a sequence selected from the following group: SEQ ID NOs: 61, 64, and 67, or a sequence having one or two amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 61, 64, and 67; and CDR3 comprises a sequence selected from the following group: SEQ ID NOs: 62, 65, 68, and 69, or a sequence having one, two, or three amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 62, 65, 68, and 69. According to specific embodiments of the present application, the amino acid substitutions, deletions, or additions referred to may be due to conservative amino acid substitutions or may be due to differences in the representation of CDR regions according to different schemes. It should be known to those skilled in the art that when different schemes are used to display CDR regions, there will be differences in the displayed CDR region sequences, and these differences should also be included in the scope of protection of the present application.

[0069] In some specific embodiments, the second antigen-binding portion has: (1) CDR1 is the amino acid sequence set forth in SEQ ID NO: 60, CDR2 is the amino acid sequence set forth in SEQ ID NO: 61, and CDR3 is the amino acid sequence set forth in SEQ ID NO: 62, or (2) CDR1 is the amino acid sequence set forth in SEQ ID NO: 63, CDR2 is the amino acid sequence set forth in SEQ ID NO: 64, and CDR3 is the amino acid sequence set forth in SEQ ID NO: 65, or (3) CDR1 is the amino acid sequence set forth in SEQ ID NO: 66, CDR2 is the amino acid sequence set forth in SEQ ID NO: 67, and CDR3 is the amino acid sequence set forth in SEQ ID NO: 68, or (4) CDR1 is the amino acid sequence set forth in SEQ ID NO: 60, CDR2 is the amino acid sequence set forth in SEQ ID NO: 61, and CDR3 is the amino acid sequence set forth in SEQ ID NO: 69.

[0070] In at least some embodiments, the second antigen-binding portion further comprises a framework region FR, the framework region comprising FR1, FR2, FR3, and FR4, wherein FR1, FR2, FR3, and FR4 each comprise the amino acid sequence shown below: (1) FR1 is selected from sequences having one, two, or three conservative amino acid substitutions compared to SEQ ID NO:70, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, or SEQ ID NO:70, 74, 77, or 80; (2) FR2 is selected from sequences having one, two, or three conservative amino acid substitutions compared to SEQ ID NO:71, SEQ ID NO:75, SEQ ID NO:78, or SEQ ID NO:71, 75, or 78; (3) FR3 is selected from sequences having one, two, three, or four conservative amino acid substitutions compared to SEQ ID NO:72, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:81, or SEQ ID NO:72, 76, 79, or 81; and (4) FR4 is selected from SEQ ID NO:73, or sequences having one or two conservative amino acid substitutions compared to SEQ ID NO:73.

[0071] In some specific embodiments, FR1 included in the framework regions has the amino acid sequence set forth in SEQ ID NO: 70, FR2 has the amino acid sequence set forth in SEQ ID NO: 71, FR3 has the amino acid sequence set forth in SEQ ID NO: 72, and FR4 has the amino acid sequence set forth in SEQ ID NO: 73. In some specific embodiments, FR1 included in the framework regions has the amino acid sequence set forth in SEQ ID NO: 74, FR2 has the amino acid sequence set forth in SEQ ID NO: 75, FR3 has the amino acid sequence set forth in SEQ ID NO: 76, and FR4 has the amino acid sequence set forth in SEQ ID NO: 73. In some specific embodiments, FR1 included in the framework regions has the amino acid sequence set forth in SEQ ID NO: 77, FR2 has the amino acid sequence set forth in SEQ ID NO: 78, FR3 has the amino acid sequence set forth in SEQ ID NO: 79, and FR4 has the amino acid sequence set forth in SEQ ID NO: 73. In some specific embodiments, FR1 included in the framework regions has the amino acid sequence set forth in SEQ ID NO: 80, FR2 has the amino acid sequence set forth in SEQ ID NO: 78, FR3 has the amino acid sequence set forth in SEQ ID NO: 79, and FR4 has the amino acid sequence set forth in SEQ ID NO: 73. In some specific embodiments, FR1 included in the framework region is the amino acid sequence shown in SEQ ID NO: 70, FR2 is the amino acid sequence shown in SEQ ID NO: 71, FR3 is the amino acid sequence shown in SEQ ID NO: 81, and FR4 is the amino acid sequence shown in SEQ ID NO: 73.

[0072] In some embodiments, the second antigen-binding portion is selected from the sequence set forth in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some embodiments, the second antigen-binding portion has sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence set forth 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 set forth in SEQ ID NO: 88, 89, 90, 91, or 92 provided is the antibody sequence after humanization. The provided humanized sequence has low immunogenicity and high affinity for the target protein. JPEG0007734856000004.jpg5877 JPEG0007734856000005.jpg66127 JPEG0007734856000006.jpg126141

[0073] The present application also provides a bispecific antibody, the bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is capable of binding to TGF-β, GARP and / or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1.

[0074] In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, or the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, or the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, or the HCDR sequences set forth in SEQ ID NOs: 10, 11, and 12, or the HCDR sequences set forth in SEQ ID NOs: 13, 14, and 15, or the HCDR sequences set forth in SEQ ID NOs: 16, 17, and 18; and the light chain variable region comprises the LCDR sequences set forth in SEQ ID NOs: 19, 20, and 21, or the LCDR sequences set forth in SEQ ID NOs: 22, 23, and 24, or the LCDR sequences set forth in SEQ ID NOs: 25, 20, and 26, or the LCDR sequences set forth in SEQ ID NOs: 27, 28, and 29, or the LCDR sequences set forth in SEQ ID NOs: 30, 31, and 32, or the LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35. The second antigen-binding portion comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the sequence shown in SEQ ID NO: 60, 63 or 66, the CDR2 comprises the sequence shown in SEQ ID NO: 61, 64 or 67, and the CDR3 comprises the sequence shown in SEQ ID NO: 62, 65, 68 or 69.

[0075] In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 36 and a light chain variable region set forth in SEQ ID NO: 48, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 49, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 38 and a light chain variable region set forth in SEQ ID NO: 50, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:39 and a light chain variable region set forth in SEQ ID NO:51, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:40 and a light chain variable region set forth in SEQ ID NO:52, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:41 and a light chain variable region set forth in SEQ ID NO:53, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 42 and a light chain variable region set forth in SEQ ID NO: 54, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91.In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:43 and a light chain variable region set forth in SEQ ID NO:55, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:44 and a light chain variable region set forth in SEQ ID NO:56, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO:45 and a light chain variable region set forth in SEQ ID NO:57, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO:82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 46 and a light chain variable region set forth in SEQ ID NO: 58, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91. In some specific embodiments, the first antigen-binding portion comprises a heavy chain variable region set forth in SEQ ID NO: 47 and a light chain variable region set forth in SEQ ID NO: 59, and the second antigen-binding portion comprises the sequence set forth in SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91.

[0076] "Monoclonal antibody or antigen-binding fragment" The present application provides a monoclonal antibody or antigen-binding fragment comprising the HCDR1, HCDR2 and HCDR3 sequences of a heavy chain variable region 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 a light chain variable region 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.

[0077] The present application also provides a monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region, wherein the heavy chain variable region comprises an HCDR sequence set forth in SEQ ID NOs: 1, 2, and 3, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, or an HCDR sequence set forth in SEQ ID NOs: 4, 5, and 6, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, or an HCDR sequence set forth in SEQ ID NOs: 7, 8, and 9, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9. or comprising the HCDR sequences set forth in SEQ ID NOs: 10, 11 and 12, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences set forth in SEQ ID NOs: 10, 11 and 12, or comprising the HCDR sequences set forth in SEQ ID NOs: 13, 14 and 15, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences set forth in SEQ ID NOs: 13, 14 and 15, or comprising the HCDR sequences set forth in SEQ ID NOs: 16, 17 and 18, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences set forth in SEQ ID NOs: 16, 17 and 18. In some embodiments, the provided sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences may be due to differences when calculating CDR sequences in different databases.

[0078] In some embodiments, the provided monoclonal antibody or antigen-binding fragment further comprises a light chain variable region, wherein the light chain variable region comprises an LCDR sequence set forth in SEQ ID NOs: 19, 20, and 21, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence set forth in SEQ ID NOs: 19, 20, and 21, or an LCDR sequence set forth in SEQ ID NOs: 22, 23, and 24, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence set forth in SEQ ID NOs: 22, 23, and 24, or an LCDR sequence set forth in SEQ ID NOs: 25, 20, and 26, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence set forth in SEQ ID NOs: 25, 20, and 26. or comprises the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29, or comprises the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or comprises the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35.

[0079] The present application also provides a monoclonal antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence set forth in SEQ ID NO: 1, 4, 7, 10, 13, or 16, HCDR2 comprises the sequence set forth in SEQ ID NO: 2, 5, 8, 11, 14, or 17, and HCDR3 comprises the sequence set forth in SEQ ID NO: 3, 6, 9, 12, 15, or 18, wherein LCDR1 comprises the sequence set forth in SEQ ID NO: 19, 22, 25, 27, 30, or 33, wherein LCDR2 comprises the sequence set forth in SEQ ID NO: 20, 23, 28, 31, or 34, and wherein LCDR3 comprises the sequence set forth in SEQ ID NO: 21, 24, 26, 29, 32, or 35.

[0080] In some specific embodiments, the monoclonal antibodies provided comprise HCDR sequences set forth in SEQ ID NOs: 1, 2, and 3, and comprise LCDR sequences set forth in SEQ ID NOs: 19, 20, and 21. In some specific embodiments, the monoclonal antibodies provided comprise HCDR sequences set forth in SEQ ID NOs: 4, 5, and 6, and comprise LCDR sequences set forth in SEQ ID NOs: 22, 23, and 24. In some specific embodiments, the monoclonal antibodies provided comprise HCDR sequences set forth in SEQ ID NOs: 7, 8, and 9, and comprise LCDR sequences set forth in SEQ ID NOs: 25, 20, and 26. In some specific embodiments, the monoclonal antibodies provided comprise HCDR sequences set forth in SEQ ID NOs: 10, 11, and 12, and comprise LCDR sequences set forth in SEQ ID NOs: 27, 28, and 29. In some specific embodiments, the monoclonal antibodies provided comprise HCDR sequences set forth in SEQ ID NOs: 13, 14, and 15, and comprise LCDR sequences set forth in SEQ ID NOs: 30, 31, and 32. In some specific embodiments, the monoclonal antibodies provided comprise HCDR sequences set forth in SEQ ID NOs: 16, 17, and 18, and LCDR sequences set forth in SEQ ID NOs: 33, 34, and 35.

[0081] In some specific embodiments, the monoclonal antibodies or antigen-binding fragments provided comprise a heavy chain variable region having at least 80%, 85%, or 90% sequence identity to the sequence set forth in SEQ ID NO: 36 or 37 or 38 or 39 or 40 or 41, and a light chain variable region having at least 80%, 85%, or 90% sequence identity to the sequence set forth in SEQ ID NO: 42 or 43 or 44 or 45 or 46 or 47.

[0082] In some specific embodiments, the monoclonal antibody or antigen-binding fragment provided is the m212 antibody, which comprises a heavy chain variable region set forth in SEQ ID NO:36 and a light chain variable region set forth in SEQ ID NO:48. In some embodiments, the monoclonal antibody or antigen-binding fragment provided is the m305 antibody, which comprises a heavy chain variable region set forth in SEQ ID NO:37 and a light chain variable region set forth in SEQ ID NO:49. In some embodiments, the monoclonal antibody or antigen-binding fragment provided is the m107 antibody, which comprises a heavy chain variable region set forth in SEQ ID NO:38 and a light chain variable region set forth in SEQ ID NO:50. In some embodiments, the monoclonal antibody or antigen-binding fragment provided is the m202 antibody, which comprises a heavy chain variable region set forth in SEQ ID NO:39 and a light chain variable region set forth in SEQ ID NO:51. In some embodiments, the monoclonal antibody or antigen-binding fragment provided is the m301 antibody, which comprises a heavy chain variable region set forth in SEQ ID NO:40 and a light chain variable region set forth in SEQ ID NO:52. In some embodiments, the provided monoclonal antibody or antigen-binding fragment is an m109 antibody comprising a heavy chain variable region set forth in SEQ ID NO: 41 and a light chain variable region set forth in SEQ ID NO: 53. The provided antibody specifically binds to a GARP / TGFβ complex, for example, exhibits strong specific binding activity to a mammalian human GARP / TGFβ complex or a cynomolgus monkey GARP / TGFβ complex. The provided antibody specifically binds to GARP or TGFβ, for example, exhibits strong specific binding to GARP or TGFβ from humans, cynomolgus monkeys, etc.

[0083] The provided monoclonal antibodies or antigen-binding fragments may have undergone humanization. Humanization reduces the immunogenicity of the antibodies or antigen-binding fragments while retaining their binding activity to the target antigen. In some embodiments, the provided monoclonal antibodies or antigen-binding fragments are m212-hu antibodies comprising a heavy chain variable region set forth in SEQ ID NO: 42 and a light chain variable region set forth in SEQ ID NO: 54. In some embodiments, the provided monoclonal antibodies or antigen-binding fragments are m305-hu antibodies comprising a heavy chain variable region set forth in SEQ ID NO: 43 and a light chain variable region set forth in SEQ ID NO: 55. In some embodiments, the provided monoclonal antibodies or antigen-binding fragments are m107-hu antibodies comprising a heavy chain variable region set forth in SEQ ID NO: 44 and a light chain variable region set forth in SEQ ID NO: 56. In some embodiments, the provided monoclonal antibodies or antigen-binding fragments are m202-hu antibodies comprising a heavy chain variable region set forth in SEQ ID NO: 45 and a light chain variable region set forth in SEQ ID NO: 57. In some embodiments, the monoclonal antibody or antigen-binding fragment provided is the m301-hu antibody, which comprises a heavy chain variable region set forth in SEQ ID NO: 46 and a light chain variable region set forth in SEQ ID NO: 58. In some embodiments, the monoclonal antibody or antigen-binding fragment provided is the m109-hu antibody, which comprises a heavy chain variable region set forth in SEQ ID NO: 47 and a light chain variable region set forth in SEQ ID NO: 59.

[0084] "Polynucleotide" The present application also provides a polynucleotide encoding the bispecific antibody or monoclonal antibody or antigen-binding fragment. The polynucleotide referred to is isolatable and includes, but is not limited to, DNA, RNA, or cDNA. An isolated polynucleotide sequence encoding the bispecific antibody or monoclonal antibody or antigen-binding fragment may be obtained using conventional methods in the art.

[0085] "Construction" To produce the antibodies described herein, a polynucleotide sequence encoding the antibody may be inserted into a replicable expression vector and expressed in a host cell or a cell-free expression system. The present application also provides constructs containing the above-described polynucleotides. To obtain the constructs, various methods commonly used in the art, including in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology, can be used. For example, a polynucleotide may be inserted into a multiple cloning site of an expression vector to form a construct. The construct may optionally contain various operational elements, such as a promoter, terminator, or marker gene, which are operably linked to the polynucleotide. The promoter generally provides a signal to initiate transcription. Examples of the promoter include the lactose operon (Lac), Trp promoter, Tac promoter, and phage PL and PR promoters. A signal to terminate transcription is provided during the transcription of the terminator. The marker gene on the construct is often used for screening. Of course, an enhancer may also be present to improve protein expression. The expression vector is not particularly limited and may be any of several commercially available expression vectors, or may be an artificially modified expression vector, such as a plasmid, phage, or virus. The virus may be a plant cell virus, a mammalian cell virus, or the like. The construct may express an antibody or protein in vitro, or may be introduced into a cell to express an antibody or protein.

[0086] "Host cell" The present application also provides a host cell containing the above-described polynucleotide or construct. Any cell suitable for expressing an antibody or protein from a polynucleotide or construct may be used as the host cell. The host cell may be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a yeast cell or a mammalian cell. Commonly used host cells include yeast cells, CHO cells, HEK-293 cells, COS cells, and insect cells such as Drosophila S2 or Sf9. Host cells containing the polynucleotide or construct can be obtained using methods commonly used in the art, such as microinjection, electroporation, chemical transfection, or viral-mediated transformation.

[0087] Pharmaceutical Composition The present application also provides a pharmaceutical composition comprising the bispecific antibody of any of the first aspects above, or the monoclonal antibody or antigen-binding fragment as described above, and a pharmaceutically acceptable carrier.

[0088] The pharmaceutically acceptable carriers mentioned are tolerated by subjects at the dosage 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 salts, Tris; carbohydrates such as trehalose, polysorbitol, sucrose, mannitol; surfactants such as polysorbates; preservatives such as hexamethonium chloride, benzalkonium chloride, benzethonium chloride; amino acids such as histidine, histidine hydrochloride, glycine, glutamine, asparagine, arginine, or lysine. Sterile pharmaceutical preparations can be obtained by methods commonly used in the art, for example, by sterile membrane filtration. Those skilled in the art may select different pharmaceutically acceptable carriers for pharmaceutical compositions in various dosage forms, such as lyophilized dosage forms and injections. The different drug dosage forms manufactured may be formulated to be administered to a subject by any suitable route of administration, including, but not limited to, intravenous, intradermal, intramuscular, intraperitoneal, subcutaneous, nasal, oral, rectal, topical, inhalation, transdermal, etc.

[0089] "Antibody complex" The present application also provides an antibody conjugate, which comprises the bispecific antibody, monoclonal antibody, or antigen-binding fragment thereof and a functional small molecule or protease linked to the bispecific antibody, monoclonal antibody, or antigen-binding fragment. The functional small molecule may be a small molecule drug that has already been developed or is yet to be developed. By chemically coupling the small molecule drug to the bispecific antibody, monoclonal antibody, or antigen-binding fragment, the therapeutic effect of the antitumor drug can be improved and adverse events can be reduced. Small molecules such as toxins, chemotherapeutic drugs, and photosensitizers may also be coupled to the antibody via a linker. Of course, an antibody-Protac conjugate may also be obtained by coupling the antibody to a protac molecule, for example, by connecting the antibody to a ligand targeting a protease (e.g., an E3 ligase ligand) via a linker, bringing the target protein close to an intracellular E3 ubiquitin ligase for specific degradation via the ubiquitin-proteasome pathway.

[0090] "kit" The present application also provides a kit, which includes the bispecific antibody or the monoclonal antibody or antigen-binding fragment. The kit may optionally include a container, a buffer reagent, and controls, such as a positive control and a negative control. Those skilled in the art may select these appropriately, depending on the situation. Accordingly, the kit may include instructions for use to enable those skilled in the art to operate and use the kit.

[0091] "Method for producing antibodies" The present application also provides a method for producing the bispecific antibody, comprising culturing the host cell and recovering the bispecific antibody from the culture.

[0092] The present application also provides a method for producing a monoclonal antibody or antigen-binding fragment, comprising culturing the host cell described above and recovering the monoclonal antibody or antigen-binding fragment from the culture.

[0093] The bispecific or monoclonal antibody or antigen-binding fragment recovered from the culture can be purified to obtain a substantially pure product. "Substantially pure" refers to a bispecific or monoclonal antibody or antigen-binding fragment that is 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or even 99.5%, 99.6%, 99.7%, 99.8% or more pure.

[0094] "Method for preventing and / or treating diseases" The present application also provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof an effective amount of the above-described bispecific antibody, or monoclonal antibody or antigen-binding fragment.

[0095] The "therapeutically effective amount" referred to herein can reduce the severity of disease symptoms, increase the frequency and duration of symptom-free periods, and reduce or prevent pain caused by the disease. A "prophylactically effective amount" is generally less than the therapeutically effective amount. After treatment with a bispecific antibody or monoclonal antibody or antigen-binding fragment, the subject's body cell suppression rate reaches 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 90% or more, or 95% or more compared to subjects not receiving antibody treatment. The subject referred to herein may be an animal or a human. For example, the subject may be a mammal, including cows, goats, mice, horses, etc.

[0096] Diseases that can be treated with the provided bispecific or monoclonal antibodies, or antigen-binding fragments thereof, include, but are not limited to, cancer and autoimmune diseases. In some embodiments, the methods provided herein may be used to treat TGF-β-related diseases, GARP-related diseases, or PD-1 / PD-L1-related diseases. TGF-β-related diseases include, but are not limited to, inflammatory diseases, chronic infections, cancer, fibrosis, cardiovascular diseases, cerebrovascular diseases, neurodegenerative diseases, etc. In some embodiments, cancers referred to include, but are not limited to, lung cancer, colon cancer, renal cancer, urothelial cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, blood cancer, etc.

[0097] The present application also provides a use of the bispecific antibody or monoclonal antibody or antigen-binding fragment in the manufacture of a drug or kit. The drug is used to treat cancer. The provided bispecific antibody or monoclonal antibody or antigen-binding fragment can be used to manufacture drugs for treating various diseases. It may also be used to manufacture a kit for use as an immunodiagnostic reagent.

[0098] The technical solutions of the present application are described in detail below with reference to the following examples. These examples are provided to facilitate understanding by those skilled in the art and should not be construed as limiting the scope of protection of the present application. The following examples are provided in detail for the present application. The described examples are illustrative and are intended to aid in the interpretation of the present application and should not be construed as limiting the scope of protection of the present application. The PD-L1 binding moiety derived from the second antigen-binding moiety, and the method for preparing and obtaining the sequence are described in the International Patent Application No. PCT / CN2022 / 110423 (Priority Application No. 202110903293.4). The contents of the International Patent Application No. PCT / CN2022 / 110423 (Priority Application No. 202110903293.4) ​​are incorporated herein in whole or in part. An anti-PD-L1 nanobody library is obtained from PD-L1 nanobodies by immunizing alpacas, followed by screening and identification to obtain candidate nanobodies. A fusion protein composed of human-derived PD-L1 extracellular domain sequences and human immunoglobulin Fc region sequences was mixed and emulsified with Freund's adjuvant and immunized into healthy alpacas to stimulate B cells to express antigen-specific nanobodies. Blood was then collected from the alpacas, lymphocytes were isolated, and total RNA was extracted using the Trizol method. cDNA was then obtained by reverse transcription. VHH antibody gene fragments were then obtained from the reverse-transcribed cDNA by PCR amplification. The VHH gene fragments and a yeast display vector were co-transformed into competent yeast cells, and the fragments were ligated into the vector using yeast homologous recombinase to form complete plasmids. A yeast transformant library with high insertion rates and high diversity was established, and stable passage was maintained in auxotrophic medium. The antibody-expressing yeast cells were then co-incubated with magnetic beads enriched for the target protein antigen. After multiple rounds of enrichment, the magnetic bead-enriched products were identified and selected using a flow cytometer. After multiple rounds of screening, five highly expressing positive clones were screened and named Antibody A to Antibody E, respectively.The amino acid sequence of antibody A 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, whose amino acid sequence is shown in SEQ ID NO: 87, antibody Bhu, whose amino acid sequence is shown in SEQ ID NO: 88, antibody Chu, whose amino acid sequence is shown in SEQ ID NO: 89, antibody D1hu, whose amino acid sequence is shown in SEQ ID NO: 90, and antibody D2hu, whose amino acid sequence is shown in SEQ ID NO: 91.

[0099] These antibodies and their humanized counterparts were confirmed to have high binding activity to hPD-L1 and cynomolgus PD-L1 by FACS and ELISA assays, and also showed blocking activity against PD-1 / PD-L1 and CD80 / PD-L1.

[0100] For example, the binding activity of antibodies to human-derived PD-L1 was detected by FACS. The test procedure was as follows: A flow cytometer was used to detect the affinity of different antibodies to the antigen. 1 × 10 MDA-MB-231 cells, which endogenously express the PD-L1 antigen, were cultured. 5 The cells were added to a 96-well plate at 1000p / well. Different concentrations of samples were then added and incubated at 4°C for 30 minutes. A fluorescently labeled goat anti-human IgG secondary antibody (Abcam) was then added to detect the antibody bound to the cell surface. Geometric values ​​were used to generate antibody-antigen binding dose-response curves, and a four-parameter curve was plotted using the software Graphpad Prism v6.0 to determine the EC of antibody binding to the antigen. 50 The test results were determined. The EC of antibody A, antibody B, antibody C, antibody D and antibody E 50 The EC values ​​were 0.089 nM, 0.031 nM, 0.042 nM, 0.039 nM, and 0.053 nM, respectively, which were similar to the positive control (atezolizumab, 0.059 nM), indicating high binding activity. The binding activity of the humanized antibodies was measured using a similar method, and the EC values ​​of antibody Ahu, antibody Bhu, antibody Chu, antibody D1hu, and antibody D2hu were50 The values ​​were 0.051 nM, 0.18 nM, 0.025 M, 0.18 nM, and 0.29 nM, respectively.

[0101] The blocking activity of antibodies against PD-1 / PD-L1 was also measured, for example, by FACS. The test procedure was as follows: A flow cytometry-based method was used to detect the blocking effect of antibodies against PD-1 and its ligand PD-L1. The 293T-PD-1 cell line (manufacturer: Hongyuan Bochuang Biotechnology (Beijing) Co., Ltd.), which overexpresses PD-1, was resuscitated and 1 × 10 cells were cultured. 5 Cells were plated at 100 μL / well in a 96-well plate, and different concentrations of antibody dilutions (initial concentration 200 nM, 4-fold dilution) and PD-L1-biotin dilutions (2 μg / mL, 50 μL / well) were mixed and incubated at room temperature for 30 minutes. Next, 100 μL / well of the mixture was added to the cells, mixed evenly, and incubated at 4°C for 60 minutes. Then, the cells were washed with FACS buffer (PBS containing 2% FBS), centrifuged at 1200 rpm for 4 minutes, and the supernatant was discarded. 100 μL / well of PE-labeled streptavidin was added and incubated in the dark for 30 minutes. After the incubation, the cells were washed with FACS buffer. 120 μL of FACS buffer was added to resuspend the cells and detected by the instrument. Live cells were gated based on FSC / SSC, and their mean fluorescence intensity was measured. Antibody-antigen blocking reaction curves were generated using geometric values, and four-parameter curves were plotted using the software Graphpad Prism v6.0 to determine IC 50 The test results were as follows: IC values ​​of antibody A, antibody B, antibody C, antibody D, and antibody E were determined. 50 The IC values ​​were 1.024 nM, 2.077 nM, 1.678 nM, 2.103 nM, and 2.536 nM, respectively, indicating that all of the antibodies could block the binding of the antigen to its ligand. A similar method was used to measure the blocking activity of the humanized antibodies, and the IC values ​​of antibody Ahu, antibody Bhu, antibody Chu, antibody D1hu, and antibody D2hu were 1.024 nM, 2.077 nM, 1.678 nM, 2.103 nM, and 2.536 nM, respectively. 50 The values ​​were 4.0 nM, 3.4 nM, 1.0 nM, 2.6 nM, and 12 nM, respectively.

[0102] The blocking activity of antibodies against CD-80 / PD-L1 was measured by FACS. The test procedure was as follows: A flow cytometry-based method was used to detect the blocking effect of antibodies against PD-L1 and CD-80. The 293T-PD-L1 cell line (manufacturer: Hongyuan Bochuang Biotechnology (Beijing) Co., Ltd.), which overexpresses PD-L1, was resuscitated and the cells were cultured at a concentration of 1 × 10 5 Cells were plated in a 96-well plate at 1 / well. Different concentrations of antibody dilutions (initial concentration 200 nM, 4-fold dilutions) were added to the cells and incubated at 4°C for 60 minutes. After incubation, the cells were washed with FACS buffer, centrifuged at 1200 rpm for 4 minutes, and the supernatant was discarded. Next, 100 μL / well of human-CD80-mFc (2 μg / mL) was added and incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed with FACS buffer. 100 μL of a 1:200 dilution of fluorescently labeled goat anti-mouse secondary antibody (manufacturer: Abcam) was added to each well and incubated at 4°C in the dark for 30 minutes. After incubation, the cells were washed with FACS buffer and detected using a flow cytometer. The mean fluorescence values ​​were measured. Antibody-antigen blocking reaction curves were generated using geometrical values, and a four-parameter curve was plotted using the software Graphpad Prism v6.0 to determine the IC. 50 The test results were as follows: IC values ​​of antibody A, antibody B, antibody C, antibody D, and antibody E were determined. 50 The IC values ​​were 3.954 nM, 3.355 nM, 4.146 nM, 4.757 nM, and 2.979 nM, respectively, indicating that all of the antibodies could block the binding of the antigen to its ligand. The blocking activity of the humanized antibodies was measured in a similar manner, and the IC values ​​of antibody Ahu, antibody Bhu, antibody Chu, and antibody D1hu were 50 The values ​​were approximately 1.8 nM, approximately 1.7 nM, approximately 3.2 nM, and approximately 1.5 nM, respectively.

[0103] Example 1: Production and acquisition of monoclonal antibodies Monoclonal antibodies were obtained by screening hybridomas. Specific antibodies were produced in mice by immunizing them with human antigens (GARP, TGF-β, and GARP-TGF-β complexes). Mouse myeloma cells were fused with mouse spleen cells, and the fused cells were plated. Screening was then performed using HAT selection medium. The myeloma cells did not survive, and the spleen cells had a very short survival time. Finally, only the myeloma cells fused with lymphocytes survived, i.e., hybridoma cells were screened.

[0104] Next, hybridoma cells capable of producing specific antibodies were selected by limiting dilution of clonal cells. The hybridoma cells were diluted multiple times and seeded into multiwell cell culture plates. They were cultured and grown so that each well contained no more than one cell. Once the clones reached a predetermined size, ELISA was used to detect antibodies secreted by the cells in the supernatant of each well. Culture wells that were capable of binding to specific antigens (GARP, TGF-β, and / or GARP-TGF-β complexes) were positive wells, and the cells were continuously diluted until monoclonal cells were obtained.

[0105] Through screening using the above method, more than 100 positive clones were obtained, from which highly active clones were identified through differential screening. Finally, 15 candidate clones were selected and sequenced, of which 6 clones were selected. The different antibodies were then expressed and purified using mammalian cell lines to obtain different antibodies with purity of at least 90%. These antibodies were named m212 antibody, m305 antibody, m107 antibody, m202 antibody, m301 antibody, and m109 antibody, respectively.

[0106] Example 2 In Example 2, the binding activity of the antibody to GARP and the GARP-TGF-β complex was tested. Plasmids expressing GARP and TGF-β were simultaneously transfected into the 293 cell line to construct a 293-GARP / TGFβ stably transfected cell line overexpressing the GARP-TGFβ complex.

[0107] Add 293-GARP / TGFβ cells at 100 μL / well to a 96-well plate at a cell concentration of 1–2 × 10 6 The cells were washed with FACS buffer (i.e., PBS solution containing 2% FBS), and then 100 μL / well of each test antibody was added (m212 antibody, m305 antibody, m107 antibody, m202 antibody, m301 antibody, and m109 antibody, respectively. Initial antibody concentration: 200 nM, 4-fold gradient dilution). The cells were then incubated at 4°C for 30 minutes. After washing with buffer, a fluorescently labeled goat anti-human secondary antibody (manufacturer: Abcam) was added and incubated at 4°C for 30 minutes. After washing with buffer, the cells were resuspended in buffer, and the fluorescent signal was detected using a flow cytometer. The EC values ​​of the test samples were calculated using the data processing software Graphpad Prism 6.0. 50 The values ​​were calculated and are shown in Table 1 below and in FIG. [Table 1]

[0108] As can be seen from the results in Table 1 and FIG. 5, the m212 antibody, m305 antibody, m107 antibody, m202 antibody, m301 antibody, and m109 antibody can all bind to the GARP-TGF-β complex.

[0109] The GARP-overexpressing CHO-K1-GARP cell line was constructed by transfecting the CHO-K1 cell line with a GARP-expressing plasmid. CHO-K1-GARP cells were added to a 96-well plate at 100 μL / well, with a cell concentration of 1–2 × 10. 6The cells were washed with FACS buffer (i.e., PBS solution containing 2% FBS), and then 100 μL / well of each test antibody was added (m212 antibody, m305 antibody, and m107 antibody were used as examples, with an initial antibody concentration of 200 nM and a 4-fold gradient dilution), followed by incubation at 4°C for 30 minutes. After washing with buffer, a fluorescently labeled goat anti-human secondary antibody (manufacturer: Abcam) was added and incubated at 4°C for 30 minutes. After washing with buffer, the cells were resuspended in buffer, and the fluorescent signal was detected using a flow cytometer. The EC of the test samples was calculated using the data processing software Graphpad Prism 6.0. 50 The values ​​were calculated and are shown in Table 2 below. [Table 2]

[0110] As can be seen from Table 2, the m212 antibody, the m305 antibody, and the m107 antibody are all capable of specifically binding to the GARP protein.

[0111] Example 3 In Example 3, the binding activity of antibodies to TGF-β was identified. Cell culture plates were coated with human transforming growth factor β1 (hTGF-β1, manufacturer: ACRO) at 100 ng / well and incubated overnight at 4°C. After washing with PBST, 200 μL of BSA was added to each well and blocked at 37°C for 1 hour. After washing with PBST, 100 μL / well of the test antibody was added at different concentrations (e.g., m107, m202, m301, and m109 antibodies, respectively; initial antibody concentration: 31.25 nM, 4-fold gradient dilution). The plate was then incubated at 37°C for 2 hours. After washing with PBST, 100 μL / well of goat anti-human secondary antibody (1:10,000 dilution in 1% BSA, manufacturer: Jackson) was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB color development solution was added and allowed to develop for 10-15 minutes, after which 50 μL of 2 M sulfuric acid was added to stop the reaction. OD450 values ​​were read using a microplate reader, and the EC values ​​of the antibodies tested were calculated using the data processing software Graphpad Prism 6.0. 50 The values ​​were calculated and are shown in Table 3 below and in FIG. [Table 3]

[0112] The test results showed that the m107 antibody, m202 antibody, m301 antibody, and m109 antibody were all capable of specifically binding to TGF-β.

[0113] Example 4: TGF-β secretion neutralization test Anti-CD3 antibody can bind to the TCR complex, activate T lymphocytes, and mediate the secretion of TGF-β by T lymphocytes, while anti-CD28 antibody can activate and expand T lymphocytes in vitro in synergistic fashion with anti-CD3 antibody. The use of anti-CD3 and anti-CD28 antibodies can synergistically stimulate and activate immune cells.

[0114] Anti-CD3 antibody and anti-CD28 antibody (manufacturer: Biolegend) were prepared at a concentration of 1 μg / mL and added to a 96-well plate at 100 μL / well for overnight coating at 4°C. Treg cells were then added at 100 μL / well (cell concentration: 5 × 10 5 The cells were incubated in an incubator for 96 hours. Finally, the TGF-β content in the supernatant was detected using a TGF-β ELISA kit (manufacturer: BD). The data were analyzed using the data processing software Graphpad Prism 6.0 to obtain the test results, which are shown in Figure 7.

[0115] As can be seen from FIG. 7, taking the m202 antibody, m301 antibody, m109 antibody and m107 antibody as examples, all of these antibodies can inhibit or neutralize TGF-β produced by Treg cells.

[0116] Example 5 Humanized antibodies were obtained using the CDR-grafting method. The amino acid sequences of the antibody and template were uniformly divided into FR and CDR regions. The CDR regions were then grafted onto the FR regions of the template, combining the CDR regions, which determine antibody specificity, with the framework regions of the human antibody to achieve humanization. To avoid a loss of antibody affinity, critical amino acid residues were identified using computer simulation and other techniques, and the affinity of the humanized antibody was maintained using restorative mutations. The m212 humanized antibody was designated m212-hu, the m305 humanized antibody was designated m305-hu, the m107 humanized antibody was designated m107-hu, the m202 humanized antibody was designated m202-hu, the m301 humanized antibody was designated m301-hu, and the 109 humanized antibody was designated m109-hu. The activity of each of these humanized antibodies was then tested.

[0117] 293-GARP / TGFβ cell line and CHO-K1-GARP cell line were added to a 96-well cell plate at 100 μL / well, with the cell concentration being 1–2 × 10 6 The cells were collected at a concentration of 100 μL / well, and the concentration of the antibody to be measured was 100 μL / well (initial concentration 200 nM, 4-5 fold gradient dilution). The cells were then incubated at 4°C for 30 minutes. After washing with FACS buffer, a fluorescently labeled goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were resuspended in 150-200 μL of FACS buffer, and the fluorescent signal was detected using a flow cytometer. The EC of the sample to be measured was calculated using the data processing software Graphpad Prism 6.0. 50 The values ​​were calculated and are shown in Tables 4 and 5 below. [Table 4]

[0118] Taking the above-mentioned antibody as an example, the humanized antibody exhibits strong specific binding activity to the GARP-TGF-β complex. [Table 5]

[0119] The test results showed that the above antibody, for example, exhibited strong binding activity with the GARP protein.

[0120] ELISA microplates were coated with hTGF-β1 (manufacturer: ACRO) at 100 ng / well and incubated overnight at 4°C. After washing with PBST, 200 μL of BSA was added to each well and blocked at 37°C for 1 hour. After washing with PBST, 100 μL / well of different concentrations of the test antibody (initial concentration 31.25 nM, 4-fold gradient dilution with 1% BSA) was added and incubated at 37°C for 2 hours. After washing with PBST, 100 μL / well of goat anti-human secondary antibody (1:10,000, diluted with 1% BSA) was added and incubated at 37°C for 1 hour. After washing with PBST, 100 μL of TMB color development solution was added for 10–15 minutes, and the reaction was stopped by adding 50 μL of 2 M sulfuric acid. OD450 values ​​were read using a microplate reader, and the EC values ​​of the test antibodies were calculated using the data processing software Graphpad Prism 6.0. 50 The values ​​were calculated and are shown in Table 6 below. [Table 6]

[0121] The test results showed that the humanized antibodies also exhibited specific binding activity to TGF-β. Furthermore, the neutralizing activity of the humanized antibodies against TGF-β was detected using the method described in Example 4. The results showed that each antibody could specifically bind to and neutralize TGF-β.

[0122] Example 6 This Example mainly describes the construction of bispecific antibodies expressing anti-GARP and PD-L1, or anti-TGF-β, GARP-TGF-β complex, and PD-L1, by genetically engineering anti-human GARP or anti-GARP-TGF-β complex antibodies and anti-PD-L1 antibodies. Bispecific antibodies are named starting with "b" to distinguish them from monoclonal antibodies, which start with "m." Bispecific antibodies are linked by a (G4S)4 linker. As shown in Figure 1, during the linking process, an anti-PD-L1 nanobody and an IgG heavy chain constant domain third domain are linked. The two antigen-binding moieties are named with an H (separator), and the previous numbers are retained for the two antigen-binding moieties. For example, the bispecific antibody designated "b212-hu-H-D2hu" is constructed by linking the antibody sequence of m212-hu with the antibody sequence of anti-PD-L1 nanobody D2hu, with the nanobody linked to the heavy chain constant domain third domain of an IgG structure. When a Nanobody is attached to an IgG-type heavy chain third constant domain, if the Nanobody moiety is represented by only one number, it indicates that only one Nanobody attached to the carboxy terminus is the same, and that one Nanobody attached to the two symmetric carboxy termini of the heavy chain third constant domain is the same; if the attached Nanobodies are different, the numbers of all attached Nanobodies must be indicated. For example, a bispecific antibody represented by "b301-H-Bhu-D2hu" is constructed by attaching the antibody sequence m301-hu to the antibody sequences of the anti-PD-L1 Nanobodies Bhu and D2hu, where Bhu and D2hu are attached to the carboxy termini of the IgG-type heavy chain third constant domain, respectively.

[0123] As shown in Figure 3, during the conjugation process, an anti-PD-L1 nanobody is attached to each light chain constant domain of an IgG structure (different from Figure 3 in that one anti-PD-L1 nanobody is attached to the C-terminus of each light chain constant domain), and the two antigen-binding moieties are connected by an L (delimiter) when naming them. As shown in Figure 2, during the conjugation process, two anti-PD-L1 nanobodies can be attached symmetrically to the carboxy termini of each heavy chain constant domain of an IgG structure. For example, if the two nanobodies attached to the carboxy terminus of one of the heavy chain constant domains are different, the two nanobodies can be named by connecting them with a "-" and the two nanobodies can be designated by their respective nanobody numbers, and the two nanobodies can be connected by a (G4S)3.

[0124] Next, bispecific antibody proteins were produced by transient transfection into CHO-S cells, the supernatant was collected, and the antibodies were purified using a protein A affinity column. The purity of the purified antibodies was determined by HPLC and SDS-PAGE, and the purity of the bispecific antibodies was found to be 90% or higher.

[0125] Example 7: Binding activity of bispecific antibodies 293-GARP / TGFβ cell line, CHO-K1-GARP cell line, and MC38-hPD-L1 (manufacturer: Kangyuan Bochuo Biotechnology (Beijing) Co., Ltd.) cell line were cultured, and the cell concentration was adjusted to 1 to 2 × 10 6 The cells were adjusted to a concentration of 100 cells / mL and added to a 96-well cell plate at 100 μL per well. Different concentrations of the antibody to be tested were then added at 100 μL per well (initial concentration 200 nM, 4-5 fold gradient dilution) and incubated at 4°C for 30 minutes. After washing with FACS buffer, a fluorescently labeled goat anti-human secondary antibody was added and incubated at 4°C for 30 minutes. After washing with FACS buffer, the cells were resuspended in 150-200 μL of FACS buffer and the fluorescent signal was detected using a flow cytometer. The EC values ​​of the antibody to be tested were calculated using the data processing software Graphpad Prism 6.0. 50 The values ​​were calculated and are shown in Tables 7 to 9 below. [Table 7] [Table 8] [Table 9]

[0126] Example 8: TGFβ reporter gene blocking activity Culture 293F-GAPR / TGFβ / αvβ6 cells at a cell density of 0.6 × 10 6 Add 50 μL / well of 293-SBE-Luciferase cells (cell density 1.8 × 10 cells / mL) to the cell culture plate. 6 50 μL / well of a solution of 133.33 nM of antibody (cells / mL) was added to the cell culture plate. Different concentrations of the antibody to be tested (initial concentration 400 nM or 133.33 nM, 3-fold gradient dilution) were then added to the cell culture plate at 100 μL / well. After uniform mixing, the plate was placed in an incubator and cultured for 18 to 24 hours. 100 μL of the luciferase substrate ONE-Glo™ Luciferase Assay system was added to each well and incubated in the dark for 5 minutes. The luminescence signal was then read using a microplate reader. The IC of the test sample was calculated using the data processing software Graphpad Prism 6.0. 50 The values ​​were calculated and are shown in Table 10 below. [Table 10]

[0127] As can be seen from the results using the above bispecific antibodies as an example, the above bispecific antibodies exhibit blocking activity against TGF-β.

[0128] Example 9: PD-L1 antibody reporter gene blocking system In Example 9, the blocking effect of antibodies on the PD-L1 / PD-1 pathway was investigated using a reporter gene assay. This test was performed using two cell lines. The Jurkat-PD1-CD3ζ-NFAT-Luc2 cell line (manufacturer: Hongyuan Bochuang Bio-Technology (Beijing) Co., Ltd.) was a Jurkat cell line stably expressing a fusion protein consisting of PD-1 ECD and CD3ζ and harboring an NFAT-driven luciferase reporter gene. The 293T-hPD-L1 cells (manufacturer: Hongyuan Bochuang Bio-Technology (Beijing) Co., Ltd.) were 293T cells expressing human PD-L1. When the two cells were co-cultured, PD-1 / PD-L1 interaction served as the first signal, and the intracellular CD3ζ chain served as the second signal, transmitting an internal activation signal, resulting in the expression of the NFAT-driven luciferase reporter gene and emitting fluorescence. When PD-L1 antibodies are added, PD-1 / PD-L1 binding is blocked, the transmission of activation signals is inhibited, and the luciferase reporter gene cannot be expressed.

[0129] 2 × 10 293T-hPD-L1 cells 4 10 cells / well in a 96-well plate, followed by 2 × 10 Jurkat-PD1-CD3ζ-NFAT-Luc2 effector cells. 4 The 96-well plate was then added at 100 μL / well, followed by different concentrations of the antibody to be tested (initial concentration 60 μg / mL, 4-fold gradient dilution), and incubated at 37°C for 18-24 hours. 100 μL of luciferase substrate ONE-Glo™ Luciferase Assay system detection reagent was added to each well and incubated in the dark for 5 minutes. The fluorescent signal from the 96-well plate was read using a microplate reader. A four-parameter curve was plotted with the relative fluorescence value on the y-axis and the antibody sample concentration on the x-axis. The curve was analyzed using GraphPad Prism 6.0 software to determine the IC of the bispecific antibody. 50 The values ​​were obtained and are shown in Table 11 below. Taking the antibodies shown as examples, the monoclonal antibody does not exhibit PD-L1 blocking activity, while the bispecific antibody does. Also, referring to the method in Example 5, these antibodies also exhibit TGF-β neutralizing activity. [Table 11]

[0130] Example 10: In vivo activity evaluation MC38-hPD-L1KL#3 cells were constructed, in which murine PD-L1 was knocked out and the human PD-L1 gene was inserted. In the study, MC-38-hPD-L1 KL#3 cells were subcutaneously inoculated into the right scapula of C57BL / 6-hGARP transgenic female mice (manufacturer: Chuo Pharmaceutical Co., Ltd.). The inoculation volume per mouse was 0.1 mL, and the cell density was 1 x 10 6 / animal, tumor volume 75-100mm 3 Once the tumor volume reached 100 mg / kg, the mice were divided into groups as shown in Table 12. The test mice were divided into 5 groups, each consisting of 6 mice, and the drug was administered intraperitoneally 4 times, twice a week. After administration, the body weight and tumor volume of the mice were measured every 3 to 4 days. [Table 12]

[0131] The results of the animal studies are shown in Figure 8. The results show that all of the antibodies shown exhibited antitumor effects, suppressing tumor size and volume. For example, the tumor growth inhibition rate (TGI) was 45-60%. Furthermore, the antitumor effect of bispecific antibodies was clearly superior to that of monoclonal antibodies, with tumor growth inhibition rates reaching 75% or more.

[0132] In the present description, "plurality" means at least two, e.g., two, three, etc., unless otherwise expressly and specifically limited.

[0133] In the description herein, when the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" are used, it means that the specific feature, structure, material, or characteristic described in the embodiment or example is included in at least one embodiment or example of the present application. Exemplary descriptions using the above terms in the present specification do not necessarily refer to the same embodiment or example. Furthermore, the specific feature, structure, material, or characteristic described may be combined in an appropriate manner in any one or more embodiments or examples. Furthermore, unless mutually inconsistent, a person skilled in the art may combine different embodiments or examples and features of different embodiments or examples described herein.

[0134] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are merely illustrative and should not be construed as limitations on the present application, and that those skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present application. The present disclosure relates, for example, to the following: [1] comprising a first antigen-binding portion and a second antigen-binding portion; The first antigen-binding portion comprises a heavy chain variable region, the heavy chain variable region comprising: The HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or comprising the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or comprising the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or comprising the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12, or comprising the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15, or comprising the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, or sequences having one or two amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, The first antigen-binding portion further comprises a light chain variable region, the light chain variable region comprising: The LCDR sequences shown in SEQ ID NOs: 19, 20 and 21, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 19, 20 and 21, or comprising the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24, or comprising the LCDR sequences shown in SEQ ID NOs: 25, 20 and 26, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 25, 20 and 26, or comprising the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29, or comprising the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32, or comprising the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35, or sequences having one or two amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35; the first antigen-binding portion is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1; A bispecific antibody, wherein the HCDR sequence and the LCDR sequence are obtained based on the IMGT scheme. [2] the first antigen-binding portion is selected from: (1) having an HCDR sequence shown in SEQ ID NOs: 1, 2, and 3, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NOs: 1, 2, and 3, and an LCDR sequence shown in SEQ ID NOs: 19, 20, and 21, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NOs: 19, 20, and 21; or (2) having an HCDR sequence shown in SEQ ID NOs: 4, 5, and 6, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NOs: 4, 5, and 6, and an LCDR sequence shown in SEQ ID NOs: 22, 23, and 24, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NOs: 22, 23, and 24; or (3) having an HCDR sequence shown in SEQ ID NOs: 7, 8, and 9, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NOs: 7, 8, and 9, and an LCDR sequence shown in SEQ ID NOs: 25, 20, and 26, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NOs: 25, 20, and 26; or (4) Having an HCDR sequence shown in SEQ ID NOs: 10, 11, and 12, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NOs: 10, 11, and 12, and an LCDR sequence shown in SEQ ID NOs: 27, 28, and 29, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NOs: 27, 28, and 29, or (5) Having an HCDR sequence shown in SEQ ID NOs: 13, 14, and 15, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NOs: 13, 14, and 15, and an LCDR sequence shown in SEQ ID NOs: 30, 31, and 32, or a sequence having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NOs: 30, 31, and 32; or (6) The bispecific antibody according to [1] above, characterized in that it has the HCDR sequences shown in SEQ ID NOs: 16, 17, and 18, or sequences having one or two amino acid substitutions, deletions, or additions relative to 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, or sequences having one or two amino acid substitutions, deletions, or additions relative to the LCDR sequences shown in SEQ ID NOs: 33, 34, and 35. [3] comprising a first antigen-binding portion and a second antigen-binding portion; The first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, with the proviso that: HCDR1 comprises the sequence set forth in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16; HCDR2 comprises the sequence set forth in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17; HCDR3 comprises the sequence set forth in SEQ ID NO: 3 or 6 or 9 or 12 or 15 or 18; LCDR1 comprises the sequence set forth in SEQ ID NO: 19, 22, 25, 27, 30, or 33; LCDR2 comprises the sequence set forth in SEQ ID NO: 20 or 23 or 28 or 31 or 34; LCDR3 comprises the sequence set forth in SEQ ID NO: 21 or 24 or 26 or 29 or 32 or 35; the first antigen-binding portion is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1; A bispecific antibody, wherein the HCDR1, HCDR2, and HCDR3 sequences and the LCDR1, LCDR2, and LCDR3 sequences are obtained based on the IMGT scheme. [4] the first antigen-binding portion, a heavy 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% or 99% sequence identity to the sequence set forth 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; The bispecific antibody according to any one of [1] to [3] above, comprising a light chain variable region having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59. [5] the first antigen-binding portion, (1) A heavy chain variable region represented by SEQ ID NO: 36, 37, 38, 39, 40, or 41 and a light chain variable region represented by SEQ ID NO: 48, 49, 50, 51, 52, or 53; or (2) The bispecific antibody according to [4] above, comprising a heavy chain variable region shown in SEQ ID NO: 42, 43, 44, 45, 46, or 47, and a light chain variable region shown in SEQ ID NO: 54, 55, 56, 57, 58, or 59. [6] the second antigen-binding portion is a single domain antibody; The bispecific antibody according to any one of [1] to [5], wherein the IgG antibody and the second antigen-binding portion are connected via a linker, with the proviso that the IgG antibody contains the first antigen-binding portion. [7] the second antigen-binding portion, (a) a CDR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 60, 63 and 66, or a sequence having one or two amino acid substitutions, deletions or additions compared to SEQ ID NOs: 60, 63 and 66; (b) a CDR2 comprising a sequence selected from the following group: SEQ ID NOs: 61, 64, and 67, or a sequence having one or two amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 61, 64, and 67; (c) a CDR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 62, 65, 68 and 69, or a sequence having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NOs: 62, 65, 68 and 69; The bispecific antibody according to any one of [1] to [5] above, wherein the CDR1, CDR2, and CDR3 are obtained based on the IMGT scheme. [8] the second antigen-binding portion is selected from: (1) having 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: 62, or (2) having a CDR1 sequence shown in SEQ ID NO: 63, a CDR2 sequence shown in SEQ ID NO: 64, and a CDR3 sequence shown in SEQ ID NO: 65, or (3) having a CDR1 sequence shown in SEQ ID NO: 66, a CDR2 sequence shown in SEQ ID NO: 67, and a CDR3 sequence shown in SEQ ID NO: 68, or (4) The bispecific antibody according to [7] above, characterized in that it 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: 69. [9] the second antigen-binding portion further comprises a framework region, the framework region comprising FR1, FR2, FR3, and FR4, wherein FR1, FR2, FR3, and FR4 each comprise the following amino acid sequences: (1) FR1 is selected from the sequence shown in SEQ ID NO: 70, 74, 77, or 80, or a sequence having one, two, or three conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 70, 74, 77, or 80; (2) FR2 is selected from the sequence set forth in SEQ ID NO: 71, 75, or 78, or a sequence having one, two, or three conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 71, 75, or 78; (3) FR3 is selected from the sequence set forth in SEQ ID NO: 72, 76, 79, or 81, or a sequence having one, two, three, or four conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 72, 76, 79, or 81; (4) The bispecific antibody according to [7] or [8], wherein FR4 is selected from the sequence shown in SEQ ID NO: 73 or a sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 73.

[10] The second antigen portion further comprises a framework region FR, wherein the framework region FR comprises: (1) 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, or (2) 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, or (3) 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, or (4) 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, or (5) The bispecific antibody according to [7] or [8], characterized in that the FR1 sequence is selected from the group consisting of 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.

[11] the second antigen-binding portion, (a) an amino acid sequence represented by SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91; (b) The bispecific antibody according to [7] or [8] above, characterized in that the bispecific antibody is selected from amino acid sequences having sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more compared to (a).

[12] comprising a first antigen-binding portion and a second antigen-binding portion; the first antigen-binding portion is capable of binding to GARP, TGF-β, and / or the GAPR-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1; the second antigen-binding portion comprises CDR1, CDR2, and CDR3, wherein: (a) the CDR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 60, 63 and 66, or a sequence having one or two amino acid substitutions, deletions or additions compared to SEQ ID NOs: 60, 63 and 66; (b) the CDR2 comprises a sequence selected from the following group: SEQ ID NOs: 61, 64, and 67, or a sequence having one or two amino acid substitutions, deletions, or additions compared to SEQ ID NOs: 61, 64, and 67; (c) the CDR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 62, 65, 68 and 69, or a sequence having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NOs: 62, 65, 68 and 69; A bispecific antibody, wherein the CDR1, CDR2 and CDR3 sequences are obtained based on the IMGT scheme.

[13] the second antigen-binding portion, (1) having 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: 62, or (2) having a CDR1 sequence shown in SEQ ID NO: 63, a CDR2 sequence shown in SEQ ID NO: 64, and a CDR3 sequence shown in SEQ ID NO: 65, or (3) having a CDR1 sequence shown in SEQ ID NO: 66, a CDR2 sequence shown in SEQ ID NO: 67, and a CDR3 sequence shown in SEQ ID NO: 68, or (4) The bispecific antibody according to

[12] above, characterized in that it 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: 69.

[14] the second antigen-binding portion further comprises a framework region, the framework region comprising FR1, FR2, FR3, and FR4, wherein FR1, FR2, FR3, and FR4 each comprise the following amino acid sequences: (1) FR1 is selected from the sequence shown in SEQ ID NO: 70, 74, 77, or 80, or a sequence having one, two, or three conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 70, 74, 77, or 80; (2) FR2 is selected from the sequence set forth in SEQ ID NO: 71, 75, or 78, or a sequence having one, two, or three conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 71, 75, or 78; (3) FR3 is selected from the sequence set forth in SEQ ID NO: 72, 76, 79, or 81, or a sequence having one, two, three, or four conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 72, 76, 79, or 81; (4) The bispecific antibody according to

[12] or

[13] , wherein FR4 is selected from the sequence shown in SEQ ID NO: 73 or a sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 73.

[15] The second antigen portion further comprises a framework region FR, wherein the framework regions FR each comprise: (1) FR1 is the amino acid sequence shown in SEQ ID NO: 70, FR2 is the amino acid sequence shown in SEQ ID NO: 71, FR3 is the amino acid sequence shown in SEQ ID NO: 72, and FR4 is the amino acid sequence shown in SEQ ID NO: 73, or (2) FR1 is the amino acid sequence shown in SEQ ID NO: 74, FR2 is the amino acid sequence shown in SEQ ID NO: 75, FR3 is the amino acid sequence shown in SEQ ID NO: 76, and FR4 is the amino acid sequence shown in SEQ ID NO: 73, or (3) FR1 is the amino acid sequence shown in SEQ ID NO: 77, FR2 is the amino acid sequence shown in SEQ ID NO: 78, FR3 is the amino acid sequence shown in SEQ ID NO: 79, and FR4 is the amino acid sequence shown in SEQ ID NO: 73, or (4) FR1 is the amino acid sequence shown in SEQ ID NO: 80, FR2 is the amino acid sequence shown in SEQ ID NO: 78, FR3 is the amino acid sequence shown in SEQ ID NO: 79, and FR4 is the amino acid sequence shown in SEQ ID NO: 73, or (5) The bispecific antibody according to

[12] or

[13] , wherein FR1 is the amino acid sequence shown in SEQ ID NO: 70, FR2 is the amino acid sequence shown in SEQ ID NO: 71, FR3 is the amino acid sequence shown in SEQ ID NO: 81, and FR4 is the amino acid sequence shown in SEQ ID NO: 73.

[16] the second antigen-binding portion, (a) an amino acid sequence represented by SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91; (b) The bispecific antibody according to any one of

[12] to

[15] above, characterized in that the bispecific antibody is selected from amino acid sequences having sequence identity of 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more compared to (a).

[17] the first antigen-binding portion forms an IgG-type structure with a heavy chain first constant domain CH1, a light chain constant domain VL, and an Fc region; The bispecific antibody according to any one of

[12] to

[16] above, wherein the second antigen-binding portion is connected to the IgG-type structure via a linker.

[18] a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1; The first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises: comprising the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or comprising the HCDR sequences shown in SEQ ID NOs: 4, 5 and 6, or comprising the HCDR sequences shown in SEQ ID NOs: 7, 8 and 9, or comprising the HCDR sequences shown in SEQ ID NOs: 10, 11 and 12; or comprising the HCDR sequences shown in SEQ ID NOs: 13, 14 and 15; or comprising the HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, The light chain variable region comprising LCDR sequences shown in SEQ ID NOs: 19, 20 and 21; or comprising the LCDR sequences shown in SEQ ID NOs: 22, 23 and 24, or comprising the LCDR sequences shown in SEQ ID NOs: 25, 20 and 26, or comprising the LCDR sequences shown in SEQ ID NOs: 27, 28 and 29; or comprising the LCDR sequences shown in SEQ ID NOs: 30, 31 and 32; or comprising the LCDR sequences shown in SEQ ID NOs: 33, 34 and 35; the second antigen-binding portion comprises CDR1, CDR2, and CDR3; the CDR1 comprises the sequence set forth in SEQ ID NO: 60, 63 or 66; the CDR2 comprises the sequence set forth in SEQ ID NO: 61 or 64 or 67; the CDR3 comprises the sequence set forth in SEQ ID NO: 62 or 65 or 68 or 69; A bispecific antibody, wherein the HCDR sequence, the LCDR sequence, and the CDR1, CDR2, and CDR3 sequences are obtained based on the IMGT scheme.

[19] the first antigen-binding portion is selected from: (1) having the HCDR sequences shown in SEQ ID NOs: 1, 2, and 3, and the LCDR sequences shown in SEQ ID NOs: 19, 20, and 21; or (2) having 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; or (3) having 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; or (4) having 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; or (5) having 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; or (6) having HCDR sequences shown in SEQ ID NOs: 16, 17, and 18, and LCDR sequences shown in SEQ ID NOs: 33, 34, and 35; the second antigen-binding portion is selected from: (1) CDR1 is the amino acid sequence shown in SEQ ID NO: 60, CDR2 is the amino acid sequence shown in SEQ ID NO: 61, and CDR3 is the amino acid sequence shown in SEQ ID NO: 62, or (2) CDR1 is the amino acid sequence shown in SEQ ID NO: 63, CDR2 is the amino acid sequence shown in SEQ ID NO: 64, and CDR3 is the amino acid sequence shown in SEQ ID NO: 65, or (3) CDR1 is the amino acid sequence shown in SEQ ID NO: 66, CDR2 is the amino acid sequence shown in SEQ ID NO: 67, and CDR3 is the amino acid sequence shown in SEQ ID NO: 68, or (4) CDR1 is the amino acid sequence represented by SEQ ID NO: 60, CDR2 is the amino acid sequence represented by SEQ ID NO: 61, and CDR3 is the amino acid sequence represented by SEQ ID NO: 69; The bispecific antibody according to

[18] , wherein the first antigen-binding portion forms an IgG-type structure together with the heavy chain first constant domain CH1, the light chain constant domain VL, and the Fc region, and the second antigen-binding portion is connected to the IgG-type structure via a linker.

[20] the first antigen-binding portion, (1) A heavy chain variable region represented by SEQ ID NO: 36, 37, 38, 39, 40, or 41 and a light chain variable region represented by SEQ ID NO: 48, 49, 50, 51, 52, or 53; or (2) A heavy chain variable region represented by SEQ ID NO: 42, 43, 44, 45, 46, or 47 and a light chain variable region represented by SEQ ID NO: 54, 55, 56, 57, 58, or 59; the second antigen-binding portion, The bispecific antibody according to

[19] above, comprising the amino acid sequence shown in SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. [twenty one] comprising a first antigen-binding portion and a second antigen-binding portion; the first antigen-binding portion a heavy chain variable region HCDR1, HCDR2 and HCDR3 sequence shown in SEQ ID NO: 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47, and a light chain variable region LCDR1, LCDR2 and LCDR3 sequence shown in SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59; a bispecific antibody, wherein the first antigen-binding portion is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1. [twenty two] the second antigen-binding portion, The bispecific antibody according to

[21] above, comprising the CDR1, CDR2 and CDR3 sequences of the amino acids shown in SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91. [twenty three] a first antigen-binding moiety that specifically binds to TGF-β, GARP, and / or a GARP-TGF-β complex, and a second antigen-binding moiety that specifically binds to PD-L1; the first antigen-binding portion further comprises a heavy chain first constant domain CH1 and a light chain constant domain CL, and the first antigen-binding portion is covalently linked to the heavy chain first constant domain CH1 and the light chain constant domain CL, respectively; a bispecific antibody further comprising an Fc region, wherein the Fc region is connected to a first heavy chain constant domain, CH1, by a hinge region, and the second antigen-binding portion is connected to the Fc region and / or to a light chain constant domain, CL, by a linker. [twenty four] (i) the second antigen-binding portion is connected to the carboxy terminus (C terminus) of the Fc region by a linker; (ii) the second antigen-binding portion is connected to the carboxy terminus (C-terminus) of the light chain constant domain by a linker; (iii) The bispecific antibody according to

[23] , characterized in that at least a portion of the second antigen-binding portion is connected to the carboxy terminus (C-terminus) of the Fc domain via a first linker, and at least a portion of the second antigen-binding portion is connected to the carboxy terminus (C-terminus) of the light chain constant domain via a second linker. [twenty five] At least the following characteristics: (a) specifically binds to PD-L1; (b) specifically binds to glycoprotein A repeat dominant sequences (GARPs); (c) specifically binds to the GARP-TGF-β complex; (d) specifically binds to TGF-β; (e) having inhibitory activity against the immunosuppressive function of regulatory T cells; (f) having antitumor activity The bispecific antibody according to

[23] above, characterized in that it comprises one of the following:

[26] a heavy chain variable region and a light chain variable region, The HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequences shown in SEQ ID NOs: 1, 2 and 3, and the LCDR sequences shown in SEQ ID NOs: 19, 20 and 21, or sequences having one, two or three amino acid substitutions, deletions or additions relative to the LCDR sequences shown in SEQ ID NOs: 19, 20 and 21, or comprising an HCDR sequence shown in SEQ ID NOs: 4, 5 and 6, or a sequence having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequence shown in SEQ ID NOs: 4, 5 and 6, and an LCDR sequence shown in SEQ ID NOs: 22, 23 and 24, or a sequence having one, two or three amino acid substitutions, deletions or additions relative to the LCDR sequence shown in SEQ ID NOs: 22, 23 and 24, or comprising an HCDR sequence shown in SEQ ID NOs: 7, 8 and 9, or a sequence having one, two or three amino acid substitutions, deletions or additions relative to the HCDR sequence shown in SEQ ID NOs: 7, 8 and 9, and a sequence having one, two or three amino acid substitutions, deletions or additions relative to the LCDR sequence shown in SEQ ID NOs: 25, 20 and 26, or comprising an HCDR sequence shown in SEQ ID NO: 10, 11, or 12, or a sequence having one, two, or three amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NO: 10, 11, or 12, and an LCDR sequence shown in SEQ ID NO: 27, 28, or 29, or a sequence having one, two, or three amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NO: 27, 28, or 29, or comprising an HCDR sequence shown in SEQ ID NO: 13, 14, or 15, or a sequence having one, two, or three amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NO: 13, 14, or 15, and an LCDR sequence shown in SEQ ID NO: 30, 31, or 32, or a sequence having one, two, or three amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NO: 30, 31, or 32, or comprising an HCDR sequence shown in SEQ ID NO: 16, 17, or 18, or a sequence having one, two, or three amino acid substitutions, deletions, or additions relative to the HCDR sequence shown in SEQ ID NO: 16, 17, or 18, and an LCDR sequence shown in SEQ ID NO: 33, 34, or 35, or a sequence having one, two, or three amino acid substitutions, deletions, or additions relative to the LCDR sequence shown in SEQ ID NO: 33, 34, or 35, A monoclonal antibody or antigen-binding fragment, wherein the HCDR sequence and the LCDR sequence are obtained based on the IMGT scheme.

[27] a heavy chain variable region and a light chain variable region, (1) having the HCDR sequences shown in SEQ ID NOs: 1, 2, and 3, and the LCDR sequences shown in SEQ ID NOs: 19, 20, and 21; or (2) having 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; or (3) having 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; or (4) having 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; or (5) having 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; or (6) A monoclonal antibody or antigen-binding fragment characterized by having HCDR sequences shown in SEQ ID NOs: 16, 17 and 18, and LCDR sequences shown in SEQ ID NOs: 33, 34 and 35.

[28] a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises complementarity determining regions LCDR1, LCDR2, and LCDR3, with the proviso that: HCDR1 comprises the sequence set forth in SEQ ID NO: 1 or 4 or 7 or 10 or 13 or 16; HCDR2 comprises the sequence set forth in SEQ ID NO: 2 or 5 or 8 or 11 or 14 or 17; HCDR3 comprises the sequence set forth in SEQ ID NO: 3 or 6 or 9 or 12 or 15 or 18; LCDR1 comprises the sequence set forth in SEQ ID NO: 19, 22, 25, 27, 30, or 33; LCDR2 comprises the sequence set forth in SEQ ID NO: 20 or 23 or 28 or 31 or 34; LCDR3 is a monoclonal antibody or antigen-binding fragment comprising the sequence shown in SEQ ID NO: 21, 24, 26, 29, 32, or 35.

[29] A monoclonal antibody or antigen-binding fragment characterized by comprising the HCDR1, HCDR2 and HCDR3 sequences of a heavy chain variable region 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 a light chain variable region 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.

[30] a heavy 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% or 99% sequence identity to the sequence set forth 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; The monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[29] above, characterized in that it comprises 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%, or 99% sequence identity to the sequence shown in SEQ ID NO: 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59.

[31] a heavy chain variable region set forth in SEQ ID NO: 36 and a light chain variable region set forth in SEQ ID NO: 48, or a heavy chain variable region set forth in SEQ ID NO: 37 and a light chain variable region set forth in SEQ ID NO: 49, or a heavy chain variable region set forth in SEQ ID NO: 38 and a light chain variable region set forth in SEQ ID NO: 50, or a heavy chain variable region set forth in SEQ ID NO: 39 and a light chain variable region set forth in SEQ ID NO: 51; or a heavy chain variable region set forth in SEQ ID NO: 40 and a light chain variable region set forth in SEQ ID NO: 52, or a heavy chain variable region set forth in SEQ ID NO: 41 and a light chain variable region set forth in SEQ ID NO: 53, or a heavy chain variable region set forth in SEQ ID NO: 42 and a light chain variable region set forth in SEQ ID NO: 54, or a heavy chain variable region set forth in SEQ ID NO: 43 and a light chain variable region set forth in SEQ ID NO: 55, or a heavy chain variable region set forth in SEQ ID NO: 44 and a light chain variable region set forth in SEQ ID NO: 56, or a heavy chain variable region set forth in SEQ ID NO: 45 and a light chain variable region set forth in SEQ ID NO: 57, or a heavy chain variable region set forth in SEQ ID NO: 46 and a light chain variable region set forth in SEQ ID NO: 58, or The monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[30] above, characterized in that it is selected from the heavy chain variable region shown in SEQ ID NO: 47 and the light chain variable region shown in SEQ ID NO: 59.

[32] The monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[31] above, further comprising an Fc region.

[33] A polynucleotide encoding the bispecific antibody according to any one of [1] to

[25] above, or encoding the monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[32] above.

[34] A construct comprising the polynucleotide according to

[33] .

[35] A host cell characterized by containing the polynucleotide according to

[33] above or the construct according to

[34] above.

[36] The bispecific antibody according to any one of [1] to

[25] above, or the monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[32] above; and a pharmaceutically acceptable carrier.

[37] The bispecific antibody according to any one of [1] to

[25] above, or the monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[32] above; and a functional small molecule linked to the bispecific antibody or the monoclonal antibody or antigen-binding fragment.

[38] A kit comprising the bispecific antibody according to any one of [1] to

[25] above or the monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[32] above.

[39] Culturing the host cell according to

[35] above; and recovering the bispecific antibody or the monoclonal antibody or antigen-binding fragment thereof from the culture.

[40] A method for preventing and / or treating a disease, comprising administering to a subject in need thereof an effective amount of the bispecific antibody according to any one of [1] to

[25] above, or the monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[32] above, or the pharmaceutical composition according to

[36] above, or the antibody complex according to

[37] above.

[41] The method according to

[40] , wherein the disease is selected from cancer and autoimmune diseases.

[42] Use of the bispecific antibody according to any one of [1] to

[25] above, or the monoclonal antibody or antigen-binding fragment according to any one of

[26] to

[32] above, in the manufacture of a drug, a kit, or an antibody complex.

Claims

1. comprising a first antigen-binding portion and a second antigen-binding portion; the first antigen-binding portion is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1; the first antigen-binding portion comprises a heavy chain variable region comprising an HCDR1, HCDR2, and HCDR3 sequence and a light chain variable region comprising an LCDR1, LCDR2, and LCDR3 sequence; and The HCDR1, HCDR2 and HCDR3 sequences and the LCDR1, LCDR2 and LCDR3 sequences are obtained based on an IMGT scheme; (1) HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 30, 31 and 32, respectively; (2) HCDR1, HCDR2 and HCDR3 sequences represented by SEQ ID NOs: 10, 11 and 12, respectively, and LCDR1, LCDR2 and LCDR3 sequences represented by SEQ ID NOs: 27, 28 and 29, respectively; (3) HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 19, 20, and 21, respectively; (4) HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 4, 5, and 6, respectively, and LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; (5) HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 7, 8, and 9, respectively, and LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 25, 20, and 26, respectively; or (6) A bispecific antibody characterized in that the HCDR1, HCDR2, and HCDR3 sequences are selected from the HCDR1, HCDR2, and HCDR3 sequences shown in SEQ ID NOs: 16, 17, and 18, respectively, and the LCDR1, LCDR2, and LCDR3 sequences shown in SEQ ID NOs: 33, 34, and 35, respectively.

2. the first antigen-binding portion, a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 39 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 51; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 45 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 57; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 40 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 52; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 46 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 58; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 36 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 48; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 37 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 49; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 38 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 50; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 41 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 53; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 42 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 54; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 43 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 55; a heavy chain variable region having at least 90% sequence identity to the sequence set forth in SEQ ID NO:44 and a light chain variable region having at least 90% sequence identity to the sequence set forth in SEQ ID NO:56; or a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 47 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 59; The bispecific antibody of claim 1, comprising:

3. the second antigen-binding portion is a single domain antibody; The bispecific antibody according to claim 1, wherein the bispecific antibody has a structure in which an IgG antibody and a second antigen-binding portion are connected by a linker, and the IgG antibody contains a first antigen-binding portion.

4. the second antigen-binding portion is selected from: (1) a sequence having a CDR1 sequence represented by SEQ ID NO: 60, a CDR2 sequence represented by SEQ ID NO: 61, and a CDR3 sequence represented by SEQ ID NO: 62; (2) a sequence having a CDR1 sequence represented by SEQ ID NO: 63, a CDR2 sequence represented by SEQ ID NO: 64, and a CDR3 sequence represented by SEQ ID NO: 65; (3) A sequence having a CDR1 sequence represented by SEQ ID NO: 66, a CDR2 sequence represented by SEQ ID NO: 67, and a CDR3 sequence represented by SEQ ID NO: 68; and (4) a sequence having a CDR1 sequence represented by SEQ ID NO: 60, a CDR2 sequence represented by SEQ ID NO: 61, and a CDR3 sequence represented by SEQ ID NO: 69; The bispecific antibody of claim 1, wherein the CDR1, CDR2 and CDR3 are obtained based on the IMGT scheme.

5. The second antigen-binding portion further comprises a framework region, wherein the framework region comprises FR1, FR2, FR3, and FR4, and the FR1, FR2, FR3, and FR4 each comprise the following amino acid sequences: (1) FR1 is selected from the sequence set forth in SEQ ID NO: 70, 74, 77, or 80, or a sequence having one, two, or three conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 70, 74, 77, or 80; (2) FR2 is selected from the sequence set forth in SEQ ID NO: 71, 75, or 78, or a sequence having one, two, or three conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 71, 75, or 78; (3) FR3 is selected from the group consisting of a sequence represented by SEQ ID NO: 72, 76, 79, or 81, or a sequence having one, two, three, or four conservative amino acid substitutions compared to the sequence represented by SEQ ID NO: 72, 76, 79, or 81; (4) The bispecific antibody according to claim 4, characterized in that FR4 is selected from the sequence shown in SEQ ID NO: 73 or a sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:

73.

6. The second antigen-binding portion further comprises a framework region FR, wherein the framework region FR comprises: (1) the FR1 sequence represented by SEQ ID NO: 70, the FR2 sequence represented by SEQ ID NO: 71, the FR3 sequence represented by SEQ ID NO: 72, and the FR4 sequence represented by SEQ ID NO: 73; (2) the FR1 sequence represented by SEQ ID NO: 74, the FR2 sequence represented by SEQ ID NO: 75, the FR3 sequence represented by SEQ ID NO: 76, and the FR4 sequence represented by SEQ ID NO: 73; (3) the FR1 sequence represented by SEQ ID NO: 77, the FR2 sequence represented by SEQ ID NO: 78, the FR3 sequence represented by SEQ ID NO: 79, and the FR4 sequence represented by SEQ ID NO: 73; (4) The FR1 sequence represented by SEQ ID NO: 80, the FR2 sequence represented by SEQ ID NO: 78, the FR3 sequence represented by SEQ ID NO: 79, and the FR4 sequence represented by SEQ ID NO: 73, and (5) The bispecific antibody according to claim 5, characterized in that the FR1 sequence is selected from the group consisting of 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.

7. The second antigen-binding portion, (a) an amino acid sequence represented by SEQ ID NO: 82 or 83 or 84 or 85 or 86 or 87 or 88 or 89 or 90 or 91; (b) the amino acid sequence of (a) is selected from amino acid sequences having sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more compared to (a).

8. a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, and the second antigen-binding portion is capable of binding to PD-L1; the first antigen-binding portion comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences; the second antigen-binding portion comprises CDR1, CDR2, and CDR3; the first antigen-binding portion, (1) HCDR1, HCDR2 and HCDR3 sequences represented by SEQ ID NOs: 13, 14 and 15, respectively, and LCDR1, LCDR2 and LCDR3 sequences represented by SEQ ID NOs: 30, 31 and 32, respectively; (2) HCDR1, HCDR2 and HCDR3 sequences represented by SEQ ID NOs: 10, 11 and 12, respectively, and LCDR1, LCDR2 and LCDR3 sequences represented by SEQ ID NOs: 27, 28 and 29, respectively; (3) HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 1, 2, and 3, respectively, and LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 19, 20, and 21, respectively; (4) HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 4, 5, and 6, respectively, and LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 22, 23, and 24, respectively; (5) HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 7, 8, and 9, respectively, and LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 25, 20, and 26, respectively; or (6) HCDR1, HCDR2, and HCDR3 sequences represented by SEQ ID NOs: 16, 17, and 18, respectively, and LCDR1, LCDR2, and LCDR3 sequences represented by SEQ ID NOs: 33, 34, and 35, respectively; the second antigen-binding portion is selected from: (1) a sequence in which CDR1 is the amino acid sequence represented by SEQ ID NO: 60, CDR2 is the amino acid sequence represented by SEQ ID NO: 61, and CDR3 is the amino acid sequence represented by SEQ ID NO: 62; (2) a sequence in which CDR1 is the amino acid sequence represented by SEQ ID NO: 63, CDR2 is the amino acid sequence represented by SEQ ID NO: 64, and CDR3 is the amino acid sequence represented by SEQ ID NO: 65; (3) A sequence in which CDR1 is the amino acid sequence shown in SEQ ID NO: 66, CDR2 is the amino acid sequence shown in SEQ ID NO: 67, and CDR3 is the amino acid sequence shown in SEQ ID NO: 68, or (4) A sequence in which CDR1 is the amino acid sequence represented by SEQ ID NO: 60, CDR2 is the amino acid sequence represented by SEQ ID NO: 61, and CDR3 is the amino acid sequence represented by SEQ ID NO: 69; The HCDR1, HCDR2 and HCDR3 sequences and the LCDR1, LCDR2 and LCDR3 sequences, and the CDR1, CDR2 and CDR3 sequences are obtained based on an IMGT scheme; a first antigen-binding portion forming an IgG-type structure together with a first heavy chain constant domain CH1, a light chain constant domain VL, and an Fc region, and a second antigen-binding portion connected to the IgG-type structure via a linker.

9. the first antigen-binding portion, A heavy chain variable region represented by SEQ ID NO: 39 and a light chain variable region represented by SEQ ID NO: 51; A heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region represented by SEQ ID NO: 57; A heavy chain variable region represented by SEQ ID NO: 40 and a light chain variable region represented by SEQ ID NO: 52; A heavy chain variable region represented by SEQ ID NO: 46 and a light chain variable region represented by SEQ ID NO: 58; A heavy chain variable region represented by SEQ ID NO: 36 and a light chain variable region represented by SEQ ID NO: 48; A heavy chain variable region represented by SEQ ID NO: 37 and a light chain variable region represented by SEQ ID NO: 49; A heavy chain variable region represented by SEQ ID NO: 38 and a light chain variable region represented by SEQ ID NO: 50; A heavy chain variable region represented by SEQ ID NO: 41 and a light chain variable region represented by SEQ ID NO: 53; A heavy chain variable region represented by SEQ ID NO: 42 and a light chain variable region represented by SEQ ID NO: 54; A heavy chain variable region represented by SEQ ID NO: 43 and a light chain variable region represented by SEQ ID NO: 55; a heavy chain variable region set forth in SEQ ID NO: 44 and a light chain variable region set forth in SEQ ID NO: 56, or a heavy chain variable region represented by SEQ ID NO: 47 and a light chain variable region represented by SEQ ID NO: 59; the second antigen-binding portion, The bispecific antibody of claim 8, comprising the amino acid sequence set forth in SEQ ID NO: 82, 83, 84, 85, 86, 87, 88, 89, 90 or 91.

10. A compound capable of binding to TGF-β, GARP, or a GARP-TGF-β complex, The heavy chain variable region and the light chain variable region include: HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 13, 14 and 15, respectively, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 30, 31 and 32, respectively; HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 10, 11 and 12, respectively, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 27, 28 and 29, respectively; HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 19, 20 and 21, respectively; HCDR1, HCDR2 and HCDR3 sequences shown in SEQ ID NOs: 4, 5 and 6, respectively, and LCDR1, LCDR2 and LCDR3 sequences shown in SEQ ID NOs: 22, 23 and 24, respectively; HCDR1, HCDR2 and HCDR3 sequences set forth in SEQ ID NOs: 7, 8 and 9, respectively, and LCDR1, LCDR2 and LCDR3 sequences set forth in SEQ ID NOs: 25, 20 and 26, respectively; or comprising HCDR1, HCDR2 and HCDR3 sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively, and LCDR1, LCDR2 and LCDR3 sequences set forth in SEQ ID NOs: 33, 34 and 35, respectively; A monoclonal antibody or antigen-binding fragment, wherein the HCDR1, HCDR2 and HCDR3 sequences and the LCDR1, LCDR2 and LCDR3 sequences are obtained based on the IMGT scheme.

11. The monoclonal antibody or antigen-binding fragment thereof, a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 39 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 51; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 45 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 57; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 40 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 52; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 46 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 58; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 36 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 48; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 37 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 49; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 38 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 50; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 41 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 53; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 42 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 54; a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 43 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 55; a heavy chain variable region having at least 90% sequence identity to the sequence set forth in SEQ ID NO:44 and a light chain variable region having at least 90% sequence identity to the sequence set forth in SEQ ID NO:56; or a heavy chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 47 and a light chain variable region having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 59; The monoclonal antibody or antigen-binding fragment of claim 10, comprising:

12. The monoclonal antibody or antigen-binding fragment thereof, A heavy chain variable region represented by SEQ ID NO: 39 and a light chain variable region represented by SEQ ID NO: 51; A heavy chain variable region represented by SEQ ID NO: 45 and a light chain variable region represented by SEQ ID NO: 57; A heavy chain variable region represented by SEQ ID NO: 40 and a light chain variable region represented by SEQ ID NO: 52; A heavy chain variable region represented by SEQ ID NO: 46 and a light chain variable region represented by SEQ ID NO: 58; A heavy chain variable region represented by SEQ ID NO: 36 and a light chain variable region represented by SEQ ID NO: 48; A heavy chain variable region represented by SEQ ID NO: 37 and a light chain variable region represented by SEQ ID NO: 49; A heavy chain variable region represented by SEQ ID NO: 38 and a light chain variable region represented by SEQ ID NO: 50; A heavy chain variable region represented by SEQ ID NO: 41 and a light chain variable region represented by SEQ ID NO: 53; A heavy chain variable region represented by SEQ ID NO: 42 and a light chain variable region represented by SEQ ID NO: 54; A heavy chain variable region represented by SEQ ID NO: 43 and a light chain variable region represented by SEQ ID NO: 55; a heavy chain variable region set forth in SEQ ID NO: 44 and a light chain variable region set forth in SEQ ID NO: 56, or The monoclonal antibody or antigen-binding fragment of claim 11, characterized in that it is selected from the heavy chain variable region shown in SEQ ID NO: 47 and the light chain variable region shown in SEQ ID NO:

59.

13. 13. The monoclonal antibody or antigen-binding fragment of claim 10, 11 or 12, further comprising an Fc region.

14. A polynucleotide encoding the bispecific antibody of any one of claims 1 to 9 or encoding the monoclonal antibody or antigen-binding fragment of any one of claims 10 to 13.

15. A construct comprising the polynucleotide of claim 14.

16. A host cell comprising a polynucleotide encoding the bispecific antibody of any one of claims 1 to 9 or encoding the monoclonal antibody or antigen-binding fragment of any one of claims 10 to 13, or a construct comprising said polynucleotide.

17. A bispecific antibody according to any one of claims 1 to 9, or a monoclonal antibody or antigen-binding fragment according to any one of claims 10 to 13; and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17 for preventing and / or treating cancer or an autoimmune disease.

19. A bispecific antibody according to any one of claims 1 to 9, or a monoclonal antibody or antigen-binding fragment according to any one of claims 10 to 13; and a functional small molecule linked to the bispecific antibody or the monoclonal antibody or antigen-binding fragment.

20. A kit comprising the bispecific antibody of any one of claims 1 to 9 or the monoclonal antibody or antigen-binding fragment of any one of claims 10 to 13.

21. Culturing the host cell of claim 16; and recovering the bispecific antibody or the monoclonal antibody or antigen-binding fragment thereof from the culture.

22. Use of a bispecific antibody according to any one of claims 1 to 9, or a monoclonal antibody or antigen-binding fragment according to any one of claims 10 to 13, in the manufacture of an antibody complex.

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