Bispecific antibody targeting tumor-associated antigen and CD28, and use of bispecific antibody

Abstract

Provided are a bispecific antibody targeting a tumor-associated antigen and CD28, and a use of the bispecific antibody. Specifically, provided is a bispecific antibody targeting PD-L1 and CD28. The bispecific antibody has good efficacy, specificity, and safety.

Description

Bispecific antibodies targeting tumor-associated antigens and CD28 and their applications Technical Field

[0001] The present invention relates to bispecific antibodies targeting tumor-associated antigens such as PD-L1 and CD28, and uses thereof. Background Art

[0002] CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes and plays a crucial role in T cell activation. It binds to the B7 molecule on antigen-presenting cells (APCs), mediating T cell co-stimulation and promoting their survival, proliferation, and cytokine production. Complete activation of naive T cells typically requires three signals: the first is the binding of antigen to antigen receptor, the second is the co-stimulatory signal, and the third is the cytokine signal. As a co-stimulatory molecule, CD28 participates in the reception of the second signal. The first signal is transmitted when a specific pMHC (peptide-major histocompatibility complex) on the surface of a dendritic cell (DC) binds to a TCR (T cell receptor) expressed on multiple copies of a naive Th cell (helper T cell) or a Tc cell (cytotoxic T cell). Binding of the TCR and pMHC leads to phosphorylation of the ITAM on the CD3 chain, which is in close proximity to the TCR, by the Lck kinase. Other intracellular signaling enzymes are subsequently recruited to this site, where they contact the CD4 or CD8 co-receptors and the cytoplasmic tail of the CD3 chain. Together, these enzymes mediate a cascade of chemical reactions that leads to the activation of multiple other enzymes. When this activation cascade occurs across multiple TCRs, the T cell receives the first signal.

[0003] In most cases, TCR and pMHC binding is insufficient to fully activate a naive Th cell or Tc cell; a second signal in the form of costimulatory signaling is required. For Th cells, receipt of the first signal leads to upregulation of the expression of CD28, a key costimulatory molecule on their surface. However, for CD28 to transmit the second signal to the Th cell nucleus, it must bind to its ligand, B7, on the surface of the DC presenting the activating pMHC. T cell activation is fundamental to continued T cell proliferation and differentiation, and full T cell activation requires both the first and second signals. T cells are recognized by the TCR, which is the first signal of T cell activation. The TCR forms a TCR-CD3 complex with the CD3 molecule, which transduces the activation signal generated by TCR antigen recognition. The second signal (or costimulatory signal) is generated by the interaction of costimulatory molecules on the surface of APCs or target cells with corresponding costimulatory molecules on the T cell surface. CD28 is a crucial costimulatory molecule in T cell activation. The first CD28 super-agonist antibody, Theralizumab (TGN1412, TAB08), was designed to treat autoimmune diseases and began its first clinical trial in 2006. Six volunteers suffered from cytokine release syndrome and multiple organ failure, but fortunately, they were eventually out of danger.

[0004] Summary of the Invention

[0005] The present invention relates to the following aspects.

[0006] 1. A bispecific antibody or an antigen-binding fragment thereof, comprising: a first protein functional domain that targets another antigen, and a second protein functional domain that targets CD28;

[0007] Wherein, the second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1-HCDR3 in the heavy chain variable region shown in any one of SEQ ID NOs: 1-6 and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 7,

[0008] For example, HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs: 12-17, LCDR1 comprises or consists of the sequence shown in SEQ ID NO: 18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO: 19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO: 20;

[0009] wherein the variant has 3, 2 or 1 amino acid differences (preferably conservative amino acid substitutions) with the corresponding CDR sequence, respectively, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively, and the variant retains the original binding affinity.

[0010] In some embodiments, the other antigens are tumor-associated antigens, such as PD-L1, Nectin4, B7-H3 (CD276), EGFR, PSMA, CD38, CD22, CD19, HER2, CEA, and GPC.

[0011] In some embodiments, the additional antigen is PD-L1.

[0012] In some embodiments, the second protein functional region comprises HCDR1-HCDR3 in the heavy chain variable region shown in any one of SEQ ID NOs: 1-6 and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 7.

[0013] In some embodiments, the HCDR1 of the second protein functional region comprises or consists of the sequence shown in SEQ ID NO:10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs:12-17, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20.

[0014] 2. A bispecific antibody or an antigen-binding fragment thereof, comprising:

[0015] Targeting the first protein domain of PD-L1, and

[0016] Targeting the second protein functional region of CD28;

[0017] Wherein, the first protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:40, and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:39,

[0018] For example, HCDR1 comprises or consists of the sequence shown in SEQ ID NO:44, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:45, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:46, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:41, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:42, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:43; and

[0019] The second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1-HCDR3 in the heavy chain variable region shown in any one of SEQ ID NOs: 1-6 and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 7,

[0020] For example, HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs: 12-17, LCDR1 comprises or consists of the sequence shown in SEQ ID NO: 18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO: 19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO: 20;

[0021] wherein the variant has 3, 2 or 1 amino acid differences (preferably conservative amino acid substitutions) with the corresponding CDR sequence, respectively, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively, and the variant retains the original binding affinity.

[0022] In some embodiments, the first protein functional region comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO:44, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:45, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:46, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:41, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:42, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:43; and

[0023] The second protein functional region comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs:12-17, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20.

[0024] 3. The bispecific antibody or antigen-binding fragment thereof according to item 1 or 2, wherein:

[0025] The amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40 or a variant thereof, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:39 or a variant thereof; the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in any one of SEQ ID NOs:1-6 or a variant thereof, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:7 or a variant thereof,

[0026] wherein the sequence of the variant is at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the corresponding sequence.

[0027] In some embodiments, the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:39; the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in any one of SEQ ID NOs:1-6, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:7.

[0028] 4. The bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the bispecific antibody is selected from any one of the following (1) to (6):

[0029] (1) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7;

[0030] (2) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7;

[0031] (3) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7;

[0032] (4) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7;

[0033] (5) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7;

[0034] (6) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7.

[0035] 5. The bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 4, wherein the first protein functional region is an immunoglobulin and the second protein functional region is a single-chain antibody; or, the first protein functional region is a single-chain antibody and the second protein functional region is an immunoglobulin.

[0036] In some embodiments, the first protein functional domain is an immunoglobulin, and the second protein functional domain is a single-chain antibody.

[0037] In some embodiments, the first protein functional domain is a single-chain antibody, and the second protein functional domain is an immunoglobulin.

[0038] In some embodiments, the immunoglobulin is IgG, IgA, IgD, IgE, or IgM. In some embodiments, the immunoglobulin is IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the immunoglobulin is IgG4.

[0039] In some embodiments, the immunoglobulin comprises a heavy chain constant region and a light chain constant region.

[0040] In some embodiments, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and variants thereof, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions and variants thereof. In some embodiments, the heavy chain constant region is a human IgG4 constant region or a variant thereof, and the light chain constant region is a human antibody kappa chain constant region or a variant thereof. In some embodiments, the heavy chain constant region is a human IgG4 constant region or a variant thereof, and the light chain constant region is a human antibody lambda chain constant region or a variant thereof.

[0041] In some embodiments, the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO:23 or SEQ ID NO:47, and the light chain constant region comprises or consists of the sequence shown in SEQ ID NO:8.

[0042] In some embodiments, the heavy chain constant region of the variant has the following mutations at positions 234, 235, and / or 237 according to the EU numbering system:

[0043] L234A, L235A or G237A;

[0044] L234A and L235A; or

[0045] L234A and G237A; or

[0046] L235A and G237A;

[0047] or

[0048] L234A, L235A and G237A.

[0049] In some embodiments, the heavy chain constant region of the variant further has one or more mutations selected from the following according to the EU numbering system:

[0050] N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A.

[0051] In some embodiments, the heavy chain variable region of the single-chain antibody is directly linked to the light chain variable region of the single-chain antibody or is linked via a linker.

[0052] In some embodiments, the single-chain antibody is connected to the light chain or heavy chain of the immunoglobulin. In some embodiments, the single-chain antibody is connected to the C-terminus of the immunoglobulin light chain or heavy chain. In some embodiments, the N-terminus of the heavy chain variable region of the single-chain antibody is connected to the C-terminus of the immunoglobulin light chain or heavy chain. In some embodiments, the N-terminus of the heavy chain variable region of the single-chain antibody is connected to the C-terminus of the immunoglobulin light chain. In some embodiments, the N-terminus of the heavy chain variable region of the single-chain antibody is connected to the C-terminus of the immunoglobulin heavy chain.

[0053] In some embodiments, the single-chain antibody is connected to the N-terminus of an immunoglobulin light chain or heavy chain. In some embodiments, the C-terminus of the heavy chain variable region of the single-chain antibody is connected to the N-terminus of an immunoglobulin light chain or heavy chain. In some embodiments, the C-terminus of the heavy chain variable region of the single-chain antibody is connected to the N-terminus of an immunoglobulin light chain. In some embodiments, the C-terminus of the heavy chain variable region of the single-chain antibody is connected to the N-terminus of an immunoglobulin heavy chain.

[0054] In some embodiments, the connecting fragment is SGGSTSGSGKPGSGEGSSGS (SEQ ID NO: 48). In some embodiments, the connecting fragment is (GGGGS)n, where n is an integer. In some embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, the sequence of the connecting fragment is as shown in SEQ ID NO: 30.

[0055] In some embodiments, the structural model of the bispecific antibody according to any one of items 1-3 is KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, DVD-Ig, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb.

[0056] 6. The bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein

[0057] The amino acid sequence of the heavy chain of the first protein functional domain is the sequence shown in SEQ ID NO:22 or a variant thereof, and the amino acid sequence of the light chain is the sequence shown in SEQ ID NO:21 or a variant thereof, wherein the sequence of the variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence.

[0058] In some embodiments, the amino acid sequence of the heavy chain of the first protein functional region is the sequence shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is the sequence shown in SEQ ID NO: 21.

[0059] In some embodiments, the heavy chain of the first protein functional region is encoded by the sequence shown in SEQ ID NO:32, and the light chain is encoded by the sequence shown in SEQ ID NO:31.

[0060] 7. The bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the amino acid sequence of the second protein functional region is shown in any one of SEQ ID NOs: 24-29.

[0061] In some embodiments, the amino acid sequence of the second protein functional region is shown in SEQ ID NO:24.

[0062] In some embodiments, the amino acid sequence of the second protein functional region is shown in SEQ ID NO:25.

[0063] In some embodiments, the amino acid sequence of the second protein functional region is shown in SEQ ID NO:26.

[0064] In some embodiments, the amino acid sequence of the second protein functional region is shown in SEQ ID NO:27.

[0065] In some embodiments, the amino acid sequence of the second protein functional region is shown in SEQ ID NO:28.

[0066] In some embodiments, the amino acid sequence of the second protein functional region is shown in SEQ ID NO:29.

[0067] In some embodiments, the single-chain antibody is encoded by the sequence shown in any one of SEQ ID NOs: 33-38.

[0068] 8. The bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the first protein functional region is directly connected to the second protein functional region or is connected via a connecting fragment.

[0069] In some embodiments, the linker fragment is SGGSTSGSGKPGSGEGSSGS (SEQ ID NO: 48).

[0070] In some embodiments, the connecting fragment is (GGGGS)n, where n is an integer. In some embodiments, n is 1, 2, 3, 4, 5, or 6. In some embodiments, the sequence of the connecting fragment is as shown in SEQ ID NO:30.

[0071] 9. The bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 8, wherein the first protein functional domain and the second protein functional domain are independently one or more.

[0072] In some embodiments, the first protein functional region and the second protein functional region are independently two.

[0073] 10. The bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 9, wherein the antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, Fab / c, a complementarity determining region (CDR) fragment, a single-chain antibody, a diabody, or a domain antibody.

[0074] 11. A conjugate comprising the bispecific antibody or antigen-binding fragment thereof according to any one of items 1 to 9 and a conjugated moiety, wherein the conjugated moiety is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof; or the conjugated moiety is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol, or an enzyme.

[0075] 12. A kit or pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof described herein, and the conjugate described in item 11; optionally, the kit further comprises a second antibody that specifically recognizes the first protein functional domain targeting PD-L1 or the second protein functional domain targeting CD28; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or an enzyme; optionally, the kit is used to detect the presence or level of CD28 in a sample; optionally, the kit further comprises antibodies or antigen-binding fragments thereof against other antigens, and / or a cytotoxic agent, and optionally, instructions for use;

[0076] Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; or, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0077] 12. Biomaterials, for

[0078] (1) a nucleic acid molecule encoding the bispecific antibody or antigen-binding fragment thereof described herein;

[0079] (2) a vector comprising a nucleic acid molecule of the bispecific antibody or antigen-binding fragment thereof described herein; or

[0080] (3) A host cell, a nucleic acid molecule of the bispecific antibody or antigen-binding fragment thereof described herein.

[0081] In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence as shown in any one of SEQ ID NOs: 31 to 38. In some embodiments, the bispecific antibody or antigen-binding fragment thereof or nucleic acid molecule described herein is an isolated bispecific antibody or antigen-binding fragment thereof or nucleic acid molecule.

[0082] 13. Use of the bispecific antibody or antigen-binding fragment or conjugate described herein for treating and / or preventing a disease or for preparing a medicament for treating and / or preventing a disease.

[0083] In some embodiments, the disease is a tumor, an autoimmune disease, or an inflammatory disease, such as a disease selected from the group consisting of Merkel cell carcinoma, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, organ transplant rejection, postoperative complications, and Sjogren-Larsson syndrome.

[0084] 14. A method for treating or preventing a disease, comprising administering to a subject a therapeutically effective amount of the bispecific antibody, or antigen-binding fragment or conjugate thereof described herein.

[0085] In some embodiments, the disease is a tumor, an autoimmune disease, or an inflammatory disease, such as a disease selected from the group consisting of Merkel cell carcinoma, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, organ transplant rejection, postoperative complications, and Sjogren-Larsson syndrome.

[0086] Table 1 Variable region and CDR sequences targeting CD28

[0087] Table 2 Amino acid sequences and nucleic acid sequences targeting PD-L1

[0088] Table 3 Anti-CD28 scFv amino acid sequence and nucleic acid sequence (italic bold indicates the connecting fragment)

[0089] Table 4 Amino acid sequences of antibody constant regions and connecting fragments

[0090] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one.

[0091] The terms used in the present invention have their conventional meanings as understood by those skilled in the art. Where a term has two or more definitions as used and / or accepted in the art, the definition of the term used herein is intended to include all of the meanings.

[0092] "Antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody ("immunoglobulin") and any antigen-binding fragment thereof or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule that has a biological activity of binding to an antigen. Examples of antibodies, including but not limited to, include heavy chains, light chains, the complementary determining regions (CDRs) of their ligand-binding portions, heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions (CH), light chain constant regions (CL), framework regions (FRs), or any portion thereof, or at least a portion of a binding protein. The CDR region includes the CDR regions (LCDR1-3) of the light chain and the CDR regions (HCDR1-3) of the heavy chain. Those skilled in the art will understand that the categories of immunoglobulin heavy chains include gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), among which there are also some subclasses (such as γ1-γ4), and the properties of the chain determine the "class" of the immunoglobulin, which is IgG, IgM, IgA, IgD or IgE, and the subclass (isotype), for example, IgG can have IgG1, IgG2, IgG3, IgG4 and other subclasses. All immunoglobulin classes are within the scope of protection disclosed in the present invention. In some embodiments, the immunoglobulin molecule is of IgG class, and its two heavy chains and two light chains are connected by disulfide bonds in a "Y" configuration, wherein the light chain starts from the "Y" mouth and continues through the variable region to surround the heavy chain.

[0093] Those of ordinary skill in the art will appreciate that the CDR regions of an antibody are responsible for the binding specificity of the antibody to the antigen. Given the known antibody heavy and light chain variable region sequences, there are currently several methods for determining the CDR regions of an antibody, including Kabat, IMGT, Chothia, and AbM numbering systems. However, each application of the definition of the CDR of an antibody or its variants will fall within the scope of the terms defined and used herein. Given the variable region amino acid sequence of the antibody, those skilled in the art can typically determine which residues are included in a particular CDR, without relying on any experimental data outside the sequence itself.

[0094] In some embodiments, according to the Kabat numbering system, the second protein functional region targeting CD28 of the bispecific antibody of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs: 51-55, LCDR1 comprises or consists of the sequence shown in SEQ ID NO: 18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO: 19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO: 20.

[0095] In some embodiments, according to the Kabat numbering system, the first protein functional region of the bispecific antibody of the present invention targeting PD-L1 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, HCDR1 comprises or consists of the sequence shown in SEQ ID NO:44, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:45, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:46, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:41, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:42, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:43; the second protein functional region targeting CD28 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs:51-55, LCDR1 comprises SEQ LCDR2 comprises or consists of the sequence shown in SEQ ID NO: 19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO: 20.

[0096] The amino acid sequence of SEQ ID NO:51 is HYSPYVRYALFDY, the amino acid sequence of SEQ ID NO:52 is QYSPYVRYALMDY, the amino acid sequence of SEQ ID NO:53 is QYSPYAARALMDY, the amino acid sequence of SEQ ID NO:54 is QYSPYVSSYPLADY, the amino acid sequence of SEQ ID NO:55 is LYSPYLTYGLIDY, and the amino acid sequence of SEQ ID NO:56 is ESYGRYDYAARDWFDY.

[0097] The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not refer to the specific length of the product. Thus, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, and "polypeptide" may be used in place of or interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to products of polypeptides that have been modified after expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but it need not be translated from a specified nucleic acid sequence and may be produced by any means, including chemical synthesis.

[0098] "Amino acids" refer to organic compounds containing both amino and carboxyl groups, such as α-amino acids, which can be encoded by nucleic acids directly or in the form of precursors. Individual amino acids are encoded by nucleic acids consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0099] In the art, substitution with amino acids having similar or similar properties generally does not alter protein function. For another example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. For the purpose of comparing two or more amino acid sequences, the percentage of "sequence homology" (also referred to herein as "amino acid identity") between a first amino acid sequence and a second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], wherein each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence—compared to the first amino acid sequence—is considered a difference at a single amino acid residue (position), i.e., an "amino acid difference" as defined herein. Alternatively, the degree of sequence identity between two amino acid sequences can be calculated using known computer algorithms, such as NCBI Multiple Alignment (https: / / www.ncbi.nlm.nih.gov / tools / cobalt / cobalt.cgi?LINK_LOC=BlastHomeLink). For example, some other techniques, computer algorithms and the settings for determining the degree of sequence identity have been described in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2 357 768-A. Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation method listed above, the amino acid sequence with the maximum number of amino acid residues is considered as "first" amino acid sequence, and another amino acid sequence is considered as "second" amino acid sequence.

[0100] Furthermore, when determining the degree of sequence identity between two amino acid sequences, the skilled person may consider so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue of similar chemical structure and which has little or substantially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art, for example, from WO 04 / 037999, WO 98 / 49185, WO 00 / 46383 and WO 01 / 09300; and the (preferred) type and / or combination of such substitutions may be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and the other references cited therein.

[0101] "Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues of immunoglobulin polypeptides are preferably replaced by other amino acid residues from the same side chain family. In other embodiments, a string of amino acids can be replaced by a structurally similar string of amino acids that differ in sequence and / or in the composition of the side chain family.

[0102] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates a conservative substitution between the two amino acids.

[0103] In some embodiments, the conservative substitutions are preferably substitutions in which an amino acid within the following groups (a)-(e) is replaced by another amino acid residue within the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0104] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0105] Those skilled in the art will appreciate that the CDR region of an antibody is responsible for the binding specificity of the antibody to the antigen. Given the known antibody heavy and light chain variable region sequences, there are currently several methods for determining the CDR region of an antibody, including the Kabat, IMGT, Chothia, and AbM numbering systems.

[0106] Specifically, the AbM numbering system: The AbM CDR definition method is derived from the relevant research of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268–9272). This definition method integrates part of the definitions of Kabat and Chothia.

[0107] Kabat numbering system: See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0108] Chothia numbering system: See, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0109] IMGT numbering system: See, eg, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0110] The term "bispecific antibody" refers to an antibody (including an antibody or its antigen-binding fragment, such as a single-chain antibody) that can specifically bind to two different antigens or two different antigenic epitopes of the same antigen. The prior art has disclosed bispecific antibodies of various structures. According to the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. According to the number of antigen-binding regions, it can be divided into bispecific antibodies of divalent, trivalent, tetravalent or more valence. According to whether the structure is symmetrical, it can be divided into bispecific antibodies of symmetrical structure and bispecific antibodies of asymmetrical structure. Among them, fragment-type bispecific antibodies, such as Fab fragments lacking Fc fragments, form bispecific antibodies by combining two or more Fab fragments into one molecule. It has low immunogenicity, small molecular weight, and high tumor tissue permeability. Typical antibody structures of this type include F(ab)2, scFv-Fab, (scFv)2-Fab, etc. IgG-like bispecific antibodies (e.g., with an Fc fragment) have a relatively large molecular weight. The Fc fragment helps purify the antibody and improve its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody serum half-life. Typical bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, F(ab)4-CrossMAb and other bispecific antibodies (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019); Chen S1 et al., J Immunol Res. 2019 Feb 11;2019:4516041).

[0111] The term "antibody fragment" or "antigen-binding fragment" includes, but is not limited to, F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, Fab / c, complementarity determining region (CDR) fragments, single-chain Fvs (scFv), disulfide-stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), diabodies, disulfide-stabilized diabodies (ds-Diabodies), scFv multimers (such as scFv dimers, scFv trimers), multispecific antibodies formed from a portion of an antibody comprising one or more CDRs, nanobodies, single domain antibodies (sdAbs), domain antibodies (dAbs), bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure. Regardless of the structure, an antigen-binding fragment includes any polypeptide or polypeptide complex that is capable of binding to the same antigen as the parent antibody or parent antibody fragment. Mao CS et al. "Disulfide stabilized Fv Fragments (dsFv): a New Type of Engineering Antibody Fragments". Progress in Biochemistry and Biophysics, 1998, 25(6): 525-526 introduces the structure of dsFv. Holt et al. "Domain antibodies: proteins for therapy" Trends in Biotechnology (2003): Vol. 21, No. 11: 484-490 review antigen-binding fragments called "domain antibodies" or dAbs, which contain only the VH or VL domain of an antibody and are therefore smaller than, for example, Fab and scFv. Fab / c fragments contain the Fc and Fab determinants, wherein the "Fc" region contains two heavy chain fragments, which include the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term "antibody fragment" includes aptamers, aptamer enantiomers (spiegelmers) and diabodies. The term "antibody fragment" also includes any synthetic or genetically modified protein that, like an antibody, can bind to a specific antigen to form a complex. Generally, an antibody fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the present invention.

[0112] The term "single chain antibody (ScFv)" refers to a molecule comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL) connected by a linker. The VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain (see, for example, Bird RE et al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (e.g., Holliger et al, Proc. Natl. Acad. Sci. USA 1993; 90: 6444-6448). Other linkers can also be used in the present invention, such as those described by Alfthan et al, Protein Eng. 1995; 8: 725-731, Choi et al, Eur. J. Immunol. 2001; 31: 94-106, Hu et al, Cancer Res. 1996; 56: 3055-3061, Kipriyanov et al, J. Mol. Biol. 1999; 293: 41-56, and Roovers et al, Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59. For example, the linker is a linker well known in the art for linking polypeptides, such as (G4S). n , wherein n is an integer, preferably 1, 2, 3 or 4; or SGGSTSGSGKPGSGEGSSGS (SEQ ID NO: 48) (Fan Y, Geren IN, Dong J, et al. Plos one. 2015; 10(8): e0135306).

[0113] The antibodies and antigen-binding fragments disclosed herein include modified derivatives, i.e., modified by covalent attachment of any type of molecule to the antibody or antigen-binding fragment, wherein the covalent attachment does not prevent the antibody or antigen-binding fragment from binding to the epitope. The antibody or antigen-binding fragment can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized with known protecting / blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

[0114] In some embodiments, the antibody or antigen-binding fragment can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, oligonucleotide (e.g., siRNA), or PEG, among others.

[0115] The antibodies described herein can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse or chicken antibodies.

[0116] In the present invention, the CD28 antigen may be derived from mammals, such as humans, rats, mice, and monkeys.

[0117] As used herein with respect to cells, nucleic acids, polypeptides, antibodies, and the like, the term "isolated" refers to molecules that have been separated from one or more of the other components of a cell's natural environment, such as DNA or RNA. The term "isolated" also refers to nucleic acids or peptides that are substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not exist in their natural state and do not exist in their natural state. "Isolated" is also used to refer to cells or polypeptides that have been separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, and the like are typically prepared by at least one purification step. In some embodiments, the purity of an isolated nucleic acid, polypeptide, antibody, and the like is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including the endpoints) between any two of these values, or any value therein.

[0118] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide and that, in its native state or when manipulated by methods well known to those skilled in the art, can produce the polypeptide and / or its fragments via transcription and / or translation.

[0119] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow, ameliorate or halt an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following results, whether detectable or undetectable, relief of symptoms, reduction in the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, alleviation, reduction or elimination of the disease state (whether partial or complete), and prolongation of life expectancy compared to that expected in the absence of treatment. Those in need of treatment include patients already suffering from a condition or disorder, patients susceptible to a condition or disorder, or patients in need of prevention of the condition or disorder, and patients who can or are expected to benefit from the administration of the antibodies, antigen-binding fragments or pharmaceutical compositions disclosed herein for detection, diagnostic procedures and / or treatment.

[0120] The effective dose and treatment regimen for treating a particular patient will depend on various factors, including the specific antibody, Fab or derivative used, the patient's age and weight, general health, sex and diet, as well as the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are judged by a medical caregiver included within the scope of those of ordinary skill in the art. The dosage used can be determined by pharmacology and pharmacokinetic principles well known in the art. In some embodiments, the dosage of the antibody of the present invention administered to the patient is 0.01 mg / kg to 100 mg / kg patient weight each time. In some embodiments, administration is weekly or monthly.

[0121] "Pharmaceutically acceptable" refers to materials listed in the pharmacopoeia for use in animals, particularly for human medicine. In addition, "pharmaceutically acceptable carriers and / or excipients" will generally be any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary.

[0122] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle that can be administered to a patient together with the active ingredient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions, aqueous glucose solutions, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If necessary, the composition can also contain a small amount of wetting agent or emulsifier, or pH buffer. Antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulfite are also foreseeable. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Said composition can be formulated into suppositories with traditional adhesives and carriers such as triglycerides. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate, etc. The example of suitable pharmaceutical carriers is described in Remington's Pharmaceutical Sciences by EW Martin, which is incorporated herein by reference. Such compositions will contain clinically effective doses of antibodies or antigen-binding fragments, preferably in purified form, together with an appropriate number of carriers and / or excipients, to provide a dosage form suitable for the patient. Said preparation should be suitable for administration mode. The parent preparation can be packaged in an ampoule, disposable syringe, or a multidose bottle made of glass or plastic.

[0123] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into human body according to conventional steps. The composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. The composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally speaking, the active ingredient is supplied alone or mixed together in a unit dose form, such as in a sealed container (such as an ampoule or a pouch) that indicates the amount of active agent in the form of a dry lyophilized powder or anhydrous concentrate. In the case of administering the composition by infusion, the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water for injection or saline can be used so that the active ingredient can be mixed before administration.

[0124] The antibodies or antigen-binding fragments of the present invention include neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions derived from, for example, hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

[0125] "About" refers to the normal error range of the corresponding numerical value that is easily known to those skilled in the relevant art. In some embodiments, "about" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0126] “EC 50 The concentration for 50% of maximal effect refers to the concentration that can cause 50% of the maximum effect.

[0127] Antibody preparation

[0128] Various methods for preparing antibodies are known in the art, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, and phage display library methods.

[0129] Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors and cell lines can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary content, are incorporated by reference in their entirety.

[0130] In some embodiments, DNA encoding the antibody can be designed and synthesized according to conventional methods based on the antibody amino acid sequence described herein, inserted into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector comprises at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on either side of the inserted sequence. Efficient transcription can be achieved using the early and late promoters of SV40, and the early promoters of long terminal repeats from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus. Other cellular promoters, such as the actin promoter, can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian host cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

[0131] In some embodiments, the inserted gene fragment must contain a selection marker. Common selection markers include genes for dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and the like, to facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking these genes. After culture in a selective medium, the successfully transfected cells grow in large quantities and produce the desired target protein. The resulting antibodies can be isolated or purified using conventional techniques, such as protein A-Sepharose, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1. Exemplary structures of anti-CD28 × anti-PD-L1 bispecific antibodies. Panel a) shows an anti-CD28 single-chain antibody linked to the C-terminus of the light chain of an anti-PD-L1 antibody; Panel b) shows an anti-CD28 single-chain antibody linked to the C-terminus of the heavy chain of an anti-PD-L1 antibody.

[0133] Figure 2. SDS-PAGE of the αPDL1-LC-CD28 scFv bispecific antibody. Lane 1: αPDL1-LC-CD28 scFv (1B11); Lane 2: αPDL1-LC-CD28 scFv (2D8); Lane 3: αPDL1-LC-CD28 scFv (1H3); Lane 4: αPDL1-LC-CD28 scFv (1G12); Lane 5: αPDL1-LC-CD28 scFv (2D5); Lane 6: αPDL1-LC-CD28 scFv (2D6).

[0134] Figure 3. SDS-PAGE of the αPDL1-HC-CD28 scFv bispecific antibody. Lane 1: αPDL1-HC-CD28 scFv (1H3); Lane 2: αPDL1-HC-CD28 scFv (2D8); Lane 3: αPDL1-HC-CD28 scFv (2D5); Lane 4: αPDL1-HC-CD28 scFv (2D6); Lane 5: αPDL1-HC-CD28 scFv (1G12); Lane 6: αPDL1-HC-CD28 scFv (1B11).

[0135] Figure 4. Biological activity detection of αPDL1-LC-CD28scFv.

[0136] Figure 5. αPDL1-HC-CD28scFv biological activity detection

[0137] Figure 6. Comparison of the biological activities of two anti-CD28 × anti-PD-L1 bispecific antibodies with different structures, as well as anti-CD28 and anti-PD-L1 antibodies. Panel a) shows the biological activity of the 2D6-related bispecific antibody; Panel b) shows the biological activity of the 2D8-related bispecific antibody.

[0138] Figure 7. Bispecific antibody-induced cytokine release from PBMCs under dry-pack conditions. LPS = lipopolysaccharide; medium = culture medium; anti-CD3 = anti-CD3 antibody; IgG4 isotype control = IgG4 isotype control.

[0139] Figure 8. The activity of the anti-CD28 × anti-PD-L1 bispecific antibody requires both the primary signal and PD-L1. Medium = culture medium; anti-CD3 = anti-CD3 antibody; IgG4 isotype control = IgG4 isotype control.

[0140] Figure 9. Tumor volume growth trend after in vivo administration of anti-CD28 × anti-PD-L1 bispecific antibody.

[0141] Figure 10. Tumor weights of mice in each group after in vivo administration of anti-CD28 × anti-PD-L1 bispecific antibody. DETAILED DESCRIPTION

[0142] The present invention is described in detail below by way of examples. Those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art (e.g., reference to "Molecular Cloning Experiment Guide" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press) or the product instructions are used. Where the manufacturer of the reagents or instruments used is not specified, they are conventional products that can be purchased on the market.

[0143] Example 1: Preparation of anti-CD28 antibodies and anti-PD-L1 antibodies

[0144] Nucleic acids encoding the variable regions of the anti-CD28 antibodies shown in Table 5 were transferred into expression vectors containing a human IgG kappa light chain constant region framework (SEQ ID NO: 8) and a human IgG1 heavy chain constant region framework (SEQ ID NO: 9). The anti-CD28 monoclonal antibodies were expressed by transient transfection in HEK293F host cells. The amino acid sequence compositions of the CDRs of the heavy and light chain variable regions of the anti-CD28 antibodies are shown in Table 5.

[0145] The anti-PD-L1 antibody used as a control had a heavy chain amino acid sequence consisting of the heavy chain variable region set forth in SEQ ID NO:40 and the human IgG4 heavy chain constant region set forth in SEQ ID NO:23, and a light chain amino acid sequence as set forth in SEQ ID NO:21. The anti-PD-L1 antibody was expressed and purified in HEK293F host cells, and its variable region amino acid sequence composition is shown in Table 6. The amino acid sequence of the control antibody TGN1412 is described in US Patent No. 8709414B2, and it was prepared using conventional antibody preparation methods.

[0146] Table 5 Sequence composition of anti-CD28 antibody variable region

[0147] Table 6 Sequence composition of anti-PD-L1 antibodies

[0148] Example 2: Preparation of anti-CD28 × anti-PD-L1 bispecific antibody

[0149] Figure 1 shows two exemplary anti-CD28 × anti-PD-L1 bispecific antibody (BsAb) structures. The construction strategy involves converting the anti-CD28 monoclonal antibody into a single-chain Fv (scFv) format, which is then linked to the C-terminus of the light chain of the anti-PD-L1 antibody via a linker fragment L1 (Figure 1a). This BsAb is designated αPDL1-LC-CD28 scFv. Alternatively, the anti-CD28 single-chain Fv is linked to the C-terminus of the heavy chain of the anti-PD-L1 antibody via a linker fragment L1 (Figure 1b). This BsAb is designated αPDL1-HC-CD28 scFv. The anti-CD28 scFv is constructed in the VH-linker fragment L2-VL format. In this example, the heavy chain amino acid sequence of the anti-PD-L1 antibody is shown in SEQ ID NO:22, and the light chain amino acid sequence is shown in SEQ ID NO:21; the amino acid sequence of the anti-CD28 scFv is shown in Table 7; and the linker fragment L1 is (G4S)4. For example, the variable region amino acid sequence of the 1B11 anti-CD28 scFv is the variable region amino acid sequence of the P64-1B11 antibody (i.e., the 1B11 anti-CD28 scFv is composed of the heavy chain variable region of P64-1B11-(G4S)4-anti-CD28 antibody light chain variable region sequence VL). Other anti-CD28 scFv amino acid sequences are similarly composed. For example, in the αPDL1-LC-CD28 scFv (1B11), the N-terminus of the 1B11 anti-CD28 scFv is positioned at the C-terminus of the light chain of the anti-PD-L1 antibody. The nucleic acid encoding the bispecific antibody was transferred into an expression vector, expressed in HEK293F host cells, and purified to obtain the bispecific antibody. The polyacrylamide gel electrophoresis (SDS-PAGE) results for the αPDL1-LC-CD28scFv are shown in Figure 2, and the polyacrylamide gel electrophoresis for the αPDL1-HC-CD28scFv is shown in Figure 3. The SDS-PAGE results shown in Figures 2 and 3 demonstrate that both bispecific antibody formats were correctly assembled.

[0150] Table 7 Anti-CD28 scFv amino acid and nucleic acid sequence composition

[0151] Example 3: Evaluation of the biological activity of anti-CD28 × anti-PD-L1 bispecific antibodies

[0152] Because CD28 is highly expressed on Jurkat cells, the activity of anti-CD28 × anti-PD-L1 bispecific antibodies can be assessed using the PD-1 / PD-L1 signaling pathway blocking assay system (Promega, Cat. No. J1252). This assay system contains two cell types: PD-L1 aAPC / CHO-K1 cells (CHO-K1 cells genetically modified to express human PD-L1 and CD3 antibodies) and PD-1 effector cells (Jurkat T cells expressing human PD-1 and CD28, and a luciferase reporter driven by an NFAT response element). The day before the assay, 40,000 PD-L1 aAPC / CHO-K1 cells were plated onto white cell culture plates (Costar, Cat. No. 3917). The next day, the medium was discarded and PD-1 effector cells (50,000 cells per well) and the test sample were added. After 6 hours of incubation, luciferase substrate (Promega, Cat. No. E6120) was added, and the fluorescence value (RLU) was read on a microplate reader. Figures 4 and 5 show that the ability of the αPDL1-LC-CD28scFv or αPDL1-HC-CD28scFv bispecific antibodies to activate Jurkat T cells is far superior to that of the anti-PD-L1 antibody.

[0153] Both bispecific antibodies with different structures showed superior biological activity to their corresponding anti-PD-L1 and anti-CD28 antibodies. As shown in Figure 6, which uses the bispecific antibodies corresponding to anti-CD28 antibody clones 2D6 and 2D8 as an example, similar to Figures 4 and 5, the anti-PD-L1 antibody alone has limited ability to activate Jurkat T cells, and the anti-CD28 antibody alone has almost no ability to activate Jurkat T cells. However, both αPDL1-LC-CD28scFv and αPDL1-HC-CD28scFv can better activate Jurkat T cells. In fact, in addition to the bispecific antibodies corresponding to anti-CD28 antibody clones 2D6 and 2D8, the same situation also exists for the bispecific antibodies corresponding to other anti-CD28 antibody clones.

[0154] Example 4: Safety Evaluation of Anti-CD28 × Anti-PD-L1 Bispecific Antibody

[0155] The emergence of monoclonal antibody drugs has provided a new method for treating a variety of diseases. However, monoclonal antibody drugs still have side effects, a large part of which are "clinical infusion reactions". Depending on the antibody, the reaction may occur in a large part of patients, usually during the first or second infusion, and some may be severe enough to constitute the so-called "cytokine release syndrome" (Findlay, Eastwood et al. 2011, Journal of Immunological Methods 371 (1-2): 134-142.). In vitro tests using antibody-mediated human PBMC (peripheral blood mononuclear cell) cytokine release tests can effectively predict the clinical risks of drugs (Stebbings, Findlay et al. 2007, The Journal of Immunology 179 (5): 3325-3331.; Stebbings, Eastwood et al. 2013, Journal of immunotoxicology 10 (1): 75-82.), thereby avoiding the occurrence of cytokine release syndrome.

[0156] Anti-CD28 × anti-PD-L1 bispecific antibody, IgG4 isotype control (Sino Biological, Cat. No. HG4K), anti-CD3 antibody (BD Biosciences, Cat. No. 555336), and TGN1412 were diluted in sterile phosphate-buffered saline (PBS) at 1 μg / well. 40 μl was added to each well of the cell culture plate and then air-dried overnight in a clean bench. The next day, the coated wells were washed once with sterile PBS to remove uncoated proteins and salts. The treated plates were then set aside.

[0157] On the second day, human PBMCs were resuspended in fresh RPMI-1640 medium (containing 10% FBS and 1% double-antibody, purchased from Gibco) and then added to the corresponding test wells of the above-mentioned spare cell culture plate. The final volume per well was 250 mL, and the number of PBMCs per well was approximately 10-12.5×10 5 / mL. In addition, positive and negative controls were set up, that is, the wells corresponding to the positive control group contained LPS (final concentration 200ng / mL) and PBMC, and the wells corresponding to the negative control group contained only PBMC suspension. The cell culture plate was placed in a CO2 cell culture incubator and cultured for about 48 hours. After the culture was completed, the cell culture plate was centrifuged at 2500rpm for 5 minutes (min). Then, about 200μl of the culture supernatant was aspirated and stored in another clean cell culture plate. The release of IL-2, IFN-γ and TNF-α cytokines was detected using a kit (Mabtech, IL-2 detection kit item number: 3445-1H-20, IFN-γ detection kit item number: 3420-1H-20, TNF-α detection kit item number: 3512-1H-20). The experiment was repeated 3 times, and the PBMCs used came from 3 different healthy human donors (i.e., donor 1, donor 2 and donor 3), and other experimental conditions remained consistent.

[0158] As can be seen from Figure 7, overall, the anti-CD28×anti-PD-L1 bispecific antibody produces fewer cytokines compared with the positive antibody TGN1412, that is, overall, the anti-CD28×anti-PD-L1 bispecific antibody has a stronger safety profile and is expected to be less likely to produce a cytokine storm in clinical practice.

[0159] Example 5: The activity of anti-CD28 × anti-PD-L1 bispecific antibody requires the simultaneous participation of the first signal and PD-L1

[0160] Since the anti-CD28 antibodies in the present invention are immune agonist antibodies, verifying their specificity is particularly important to avoid nonspecific activation and serious adverse reactions in clinical practice. A biological activity assay similar to that in Example 3 was used, using CHO-K1 cells that do not express human PD-L1 as a negative control cell line, PD-L1-MC38 cells that overexpress human PD-L1 (Southern Model Organisms Science, Inc.), and anti-CD3 antibodies purchased from Tongli Haiyuan Biotechnology (Cat. No. GMP-TL101).

[0161] The results are shown in Figure 8. In the absence of anti-CD3 antibody stimulation and in the presence of CHO-K1, none of the three bispecific antibodies effectively activated CD28-NF-κB-Jurkat (in Jurkat cells expressing human CD28 and transduced with an NF-κB response element). This was also true in the presence of PD-L1-MC38. Furthermore, under anti-CD3 antibody stimulation and in the presence of CHO-K1, CD28-NF-κB-Jurkat cells were not fully activated compared to the IgG4 isotype control and the medium control. Strong activation of CD28-NF-κB-Jurkat was observed only under anti-CD3 antibody stimulation and in the presence of PD-L1-MC38. These results demonstrate that the αPDL1-HC-CD28scFv requires both the primary signal and PD-L1 for its full activity, demonstrating excellent specificity and potential clinical safety. The results of αPDL1-LC-CD28scFv were similar to those of αPDL1-HC-CD28scFv.

[0162] Example 6: In vivo efficacy test of anti-CD28×anti-PD-L1 bispecific antibody

[0163] To test the efficacy of the anti-CD28 × anti-PD-L1 bispecific antibody, the huCD28-C57BL / 6J humanized mouse MC38-PDL1 11# colorectal cancer model was used to evaluate the therapeutic effect of subcutaneous tumor formation in mice. To reduce the frequency and duration of anti-drug antibody (ADA) production by bispecific antibodies in mice, the IgG4 subtype constant region of the anti-CD28 × anti-PD-L1 bispecific antibodies (i.e., 2D5 and 2D6) was replaced with a mouse IgG1 constant region (mIgG1, amino acid sequence shown in SEQ ID NO:49, nucleotide sequence shown in SEQ ID NO:50). The new bispecific antibodies were designated CD28 / PDL1-mIgG1 2D5 and CD28 / PDL1-mIgG1 2D6. The constant region of the control anti-PD-L1 antibody was also replaced with a mouse IgG1 constant region (mIgG1, amino acid sequence shown in SEQ ID NO:49, nucleotide sequence shown in SEQ ID NO:50). The variable regions of the control anti-PD-L1 antibody were the heavy chain variable region (SEQ ID NO:40) and light chain variable region (SEQ ID NO:39) of the anti-PD-L1 antibody. The new chimeric antibody was designated 6006-mIgG1.

[0164] MC38-hPD-L1 tumor cells were subcutaneously transplanted into CD28 humanized mice to establish an MC38-hPD-L1 xenograft tumor model (Shanghai Model Organisms Science Co., Ltd.). On day 8 after inoculation, the average tumor volume was approximately 240 mm 3 Tumor-bearing mice were randomly divided into three groups, with 10 mice in each group. Each group received intraperitoneal administration (ip) twice weekly for three consecutive weeks, for a total of seven doses. Group 1: 6006-mlIgG1, 3.75 mg / kg, ip, BIW*7 times (hereinafter referred to as Group 1); Group 2: CD28 / PDL1-mIgG1 2D5, 5 mg / kg, ip, BIW*7 times (hereinafter referred to as Group 2); and Group 3: CD28 / PDL1-mIgG1 2D6, 5 mg / kg, ip, BIW*7 times (hereinafter referred to as Group 3).

[0165] On the 22nd day after the start of administration, the tumor volumes of Group 1, Group 2, and Group 3 were 948.87±250.88mm, respectively. 3 、925.34±249.06mm 3 and 694.92±229.29mm 3 From a biological perspective, CD28 / PDL1-mIgG1 2D6 has better efficacy than the control 6006-mIgG1 (Figure 9). On the 22nd day after the start of administration, the body weight growth rates of mice in Group 1, Group 2, and Group 3 were 11.0%, 10.7%, and 10.2%, respectively, with no abnormal changes in body weight compared to the control group. After the experiment, the animals were euthanized, and the tumor masses were weighed. The tumor weights of Group 1, Group 2, and Group 3 were 0.8229±0.2198g, 0.6355±0.1657g, and 0.5258±0.1877g, respectively. CD28 / PDL1-mIgG12D6 has better efficacy than the control 6006-mIgG1 (Figure 10).

Claims

1. A bispecific antibody or an antigen-binding fragment thereof, wherein the bispecific antibody comprises: a first protein functional region targeting other antigens, and a second protein functional region targeting CD28; in, The second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or their variants: HCDR1-HCDR3 in the heavy chain variable region shown in any one of SEQ ID NOs: 1-6 and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 7, wherein the variants have 3, 2 or 1 amino acid differences (preferably conservative amino acid substitutions) with the corresponding CDR sequences, respectively, or have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively, and the variants retain the original binding affinity.

2. A bispecific antibody or an antigen-binding fragment thereof, wherein the bispecific antibody comprises: a first protein functional region targeting other antigens, and a second protein functional region targeting CD28; wherein, The second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1 comprises or consists of the sequence shown in SEQ ID NO: 10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO: 11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs: 12-17, LCDR1 comprises or consists of the sequence shown in SEQ ID NO: 18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO: 19, LCDR3 comprises or consists of the sequence shown in SEQ ID NO: 20, Wherein the variant has 3, 2 or 1 amino acid differences (preferably conservative amino acid substitutions) with the corresponding CDR sequence, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively, and the variant retains the original binding affinity.

3. The bispecific antibody or antigen-binding fragment thereof according to claim 2, wherein: The second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: HCDR1 comprises the sequence shown in SEQ ID NO:10, HCDR2 comprises the sequence shown in SEQ ID NO:11, HCDR3 comprises the sequence shown in any one of SEQ ID NOs:12-17, LCDR1 comprises the sequence shown in SEQ ID NO:18, LCDR2 comprises the sequence shown in SEQ ID NO:19, and LCDR3 comprises the sequence shown in SEQ ID NO:

20.

4. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the bispecific antibody comprises: a first protein functional region targeting other antigens, and a second protein functional region targeting CD28; in, The second protein functional region comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: HCDR1 comprises the sequence shown in SEQ ID NO:10, HCDR2 comprises the sequence shown in SEQ ID NO:11, HCDR3 comprises the sequence shown in any one of SEQ ID NOs:12-17, LCDR1 comprises the sequence shown in SEQ ID NO:18, LCDR2 comprises the sequence shown in SEQ ID NO:19, and LCDR3 comprises the sequence shown in SEQ ID NO:

20.

5. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the other antigen is a tumor-associated antigen, such as PD-L1, Nectin4, B7-H3 (CD276), EGFR, PSMA, CD38, CD22, CD19, HER2, CEA, GPC.

6. A bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody comprising: Targeting the first protein domain of PD-L1, and Targeting the second protein domain of CD28; in, The first protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1-HCDR3 in the heavy chain variable region shown in SEQ ID NO:40, and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO:39; and The second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1-HCDR3 in the heavy chain variable region shown in any one of SEQ ID NOs: 1-6 and LCDR1-LCDR3 in the light chain variable region shown in SEQ ID NO: 7, Wherein the variant has 3, 2 or 1 amino acid differences (preferably conservative amino acid substitutions) with the corresponding CDR sequence, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively, and the variant retains the original binding affinity.

7. A bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody comprising: Targeting the first protein domain of PD-L1, and Targeting the second protein domain of CD28; in, The first protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1 comprises SEQ ID NO: 44 44 or consisting of the sequence shown in SEQ ID NO:43; HCDR2 comprises or consists of the sequence shown in SEQ ID NO:45, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:46, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:41, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:42, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:43; and The second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or variants thereof: HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs:12-17, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:20; Wherein the variant has 3, 2 or 1 amino acid differences (preferably conservative amino acid substitutions) with the corresponding CDR sequence, or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, respectively, and the variant retains the original binding affinity.

8. The bispecific antibody or antigen-binding fragment thereof according to claim 6 or 7, wherein: The first protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: HCDR1 comprises or consists of the sequence shown in SEQ ID NO:44, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:45, HCDR3 comprises or consists of the sequence shown in SEQ ID NO:46, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:41, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:42, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:43; and The second protein functional region comprises one or more of the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: HCDR1 comprises or consists of the sequence shown in SEQ ID NO:10, HCDR2 comprises or consists of the sequence shown in SEQ ID NO:11, HCDR3 comprises or consists of the sequence shown in any one of SEQ ID NOs:12-17, LCDR1 comprises or consists of the sequence shown in SEQ ID NO:18, LCDR2 comprises or consists of the sequence shown in SEQ ID NO:19, and LCDR3 comprises or consists of the sequence shown in SEQ ID NO:

20.

9. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 6 to 8, wherein: The first protein functional region comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: HCDR1 comprises the sequence shown in SEQ ID NO:44, HCDR2 comprises the sequence shown in SEQ ID NO:45, HCDR3 comprises the sequence shown in SEQ ID NO:46, LCDR1 comprises the sequence shown in SEQ ID NO:41, LCDR2 comprises the sequence shown in SEQ ID NO:42, and LCDR3 comprises the sequence shown in SEQ ID NO:43; and The second protein functional region comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3: HCDR1 comprises the sequence shown in SEQ ID NO:10, HCDR2 comprises the sequence shown in SEQ ID NO:11, HCDR3 comprises the sequence shown in any one of SEQ ID NOs:12-17, LCDR1 comprises the sequence shown in SEQ ID NO:18, LCDR2 comprises the sequence shown in SEQ ID NO:19, and LCDR3 comprises the sequence shown in SEQ ID NO:

20.

10. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein The amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40 or a variant thereof, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:39 or a variant thereof; the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in any one of SEQ ID NOs:1-6 or a variant thereof, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO:7 or a variant thereof, The sequence of the variant is at least 60%, at least 70%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the corresponding sequence.

11. A bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody comprising: Targeting the first protein domain of PD-L1, and Targeting the second protein domain of CD28; in, The bispecific antibody is selected from any one of the following (1)-(6): (1) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7; (2) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO: 40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO: NO:39; and, the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7; (3) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7; (4) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7; (5) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:7; (6) the amino acid sequence of the heavy chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:40, and the amino acid sequence of the light chain variable region of the first protein functional region is the sequence shown in SEQ ID NO:39; and the amino acid sequence of the heavy chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:6, and the amino acid sequence of the light chain variable region of the second protein functional region is the sequence shown in SEQ ID NO:

7.

12. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein: The first protein functional region is an immunoglobulin, and the second protein functional region is a single-chain antibody.

13. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein: The first protein functional region is a single-chain antibody, and the second protein functional region is an immunoglobulin. 14 . The bispecific antibody or antigen-binding fragment thereof according to claim 12 , wherein the heavy chain variable region of the single-chain antibody is directly connected to the light chain variable region of the single-chain antibody or is connected via a connecting fragment.

15. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 12 to 14, wherein the connecting fragment is SEQ ID NO: 48, 16. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 12 to 14, wherein the connecting fragment is (GGGGS)n, where n is an integer. 17 . The bispecific antibody or antigen-binding fragment thereof according to claim 16 , wherein n is 1, 2, 3, 4, 5 or 6.

18. The bispecific antibody or antigen-binding fragment thereof according to claim 16, wherein the sequence of the connecting fragment is shown in SEQ ID NO:

30.

19. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 12 to 18, wherein: The immunoglobulin comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is a human IgG4 constant region or a variant thereof, and the light chain constant region is a human κ chain constant region or a variant thereof.

20. The bispecific antibody or antigen-binding fragment thereof according to claim 19, wherein the heavy chain constant region of the variant has the following mutations at positions 234, 235 and / or 237 according to the EU numbering system: L234A, L235A or G237A; Such as L234A and L235A; or L234A and G237A; or L235A and G237A; or L234A, L235A and G237A.

21. The bispecific antibody or antigen-binding fragment thereof according to claim 19, wherein the heavy chain constant region of the variant further has one or more mutations selected from the following according to the EU numbering system: N297A, D265A, D270A, P238D, L328E, E233D, H268D, P271G, A330R, C226S, C229S, E233P, P331S, S267E, L328F, A330L, M252Y, S254T, T256E, N297Q, P238S, P238A, A327Q, A327G, P329A, K322A, T394D, G236R, G236A, L328R, A330S, P331S, H268A, E318A, and K320A.

22. The bispecific antibody or antigen-binding fragment thereof according to claim 19, wherein the heavy chain constant region comprises or consists of the sequence shown in SEQ ID NO: 23 or SEQ ID NO: 47, and the light chain constant region comprises or consists of the sequence shown in SEQ ID NO:

8.

23. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 22, wherein: The amino acid sequence of the heavy chain of the first protein functional domain is the sequence shown in SEQ ID NO:22 or a variant thereof, and the amino acid sequence of the light chain is the sequence shown in SEQ ID NO:21 or a variant thereof, wherein the sequence of the variant has at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% homology with the corresponding sequence.

24. A bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody comprising: Targeting the first protein domain of PD-L1, and Targeting the second protein domain of CD28; in, The first protein functional region comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is the sequence shown in SEQ ID NO:22, and the amino acid sequence of the light chain is the sequence shown in SEQ ID NO:

21.

25. The bispecific antibody or antigen-binding fragment thereof according to claim 24, wherein: The heavy chain of the first protein functional region is encoded by the sequence shown in SEQ ID NO:32, and the light chain is encoded by the sequence shown in SEQ ID NO:

31.

26. A bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody comprising: Targeting the first protein domain of PD-L1, and Targeting the second protein domain of CD28; in, The amino acid sequence of the second protein functional region is the sequence shown in any one of SEQ ID NOs: 24-29.

27. A bispecific antibody or an antigen-binding fragment thereof, the bispecific antibody comprising: Targeting the first protein domain of PD-L1, and Targeting the second protein domain of CD28; in, The first protein functional region comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is the sequence shown in SEQ ID NO: 22, and the amino acid sequence of the light chain is the sequence shown in SEQ ID NO: 21, and the amino acid sequence of the second protein functional region is any one of SEQ ID NOs: 24-29. sequence shown.

28. The bispecific antibody or antigen-binding fragment thereof according to claim 26, wherein the second protein functional region is encoded by a sequence shown in any one of SEQ ID NOs: 33-38.

29. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, wherein: The first protein functional region is directly connected to the second protein functional region or connected via a connecting fragment.

30. The bispecific antibody or antigen-binding fragment thereof according to claim 29, wherein the connecting fragment is SEQ ID NO:

48.

31. The bispecific antibody or antigen-binding fragment thereof according to claim 29, wherein the connecting fragment is (GGGGS)n, where n is an integer; for example, 1, 2, 3, 4, 5 or 6.

32. The bispecific antibody or antigen-binding fragment thereof according to claim 31, wherein the sequence of the connecting fragment is shown in SEQ ID NO:

30.

33. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 32, wherein: The first protein functional region and the second protein functional region are independently one or more.

34. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 12 to 33, wherein: The single-chain antibody (such as the heavy chain variable region thereof) is linked to the light chain or heavy chain of an immunoglobulin, such as linked to the C-terminus of the light chain or heavy chain of an immunoglobulin.

35. The bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, wherein: The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, Fab / c, a complementarity determining region (CDR) fragment, a single chain antibody, a diabody or a domain antibody.

36. A conjugate comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 35 and a conjugated moiety, wherein: The coupling part is a purification tag (such as a His tag), a detectable label, a drug, a toxin, a cytokine, an enzyme, or a combination thereof; or, the coupling part is a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, a chemotherapeutic agent, a biotoxin, polyethylene glycol or an enzyme.

37. A kit or pharmaceutical composition, comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, and the conjugate according to claim 36; optionally, the kit further comprises a second antibody that specifically recognizes the first protein functional region targeting PD-L1 or the second protein functional region targeting CD28; optionally, the second antibody further comprises a detectable label, such as a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance or an enzyme; optionally, the kit is used to detect the presence or level of CD28 in a sample; optionally, the kit further comprises antibodies or antigen-binding fragments thereof against other antigens, and / or a cytotoxic agent, and optionally, instructions for use; Optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient; or, the pharmaceutical composition is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

38. Biomaterials, for (1) a nucleic acid molecule encoding the bispecific antibody or antigen-binding fragment thereof or a portion thereof according to any one of claims 1 to 35; (2) a vector comprising a nucleic acid molecule encoding the bispecific antibody or antigen-binding fragment thereof or a portion thereof according to any one of claims 1 to 35; or (3) A host cell comprising a nucleic acid molecule encoding the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, or a portion thereof.

39. The nucleic acid molecule of claim 38, comprising the nucleic acid sequence shown in any one of SEQ ID NOs: 31-38.

40. Use of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 35, or the conjugate according to claim 36 for treating and / or preventing a disease or for preparing a medicament for treating and / or preventing a disease, wherein the disease is a tumor, an autoimmune disease or an inflammatory disease, such as selected from the following diseases: Merkel cell carcinoma, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, organ transplant rejection, postoperative complications, and Sjogren-Larsson syndrome.

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