Bispecific antibody targeting PD-1 and 4-1 BB, and application thereof

Abstract

The present invention relates to the field of biomedicine, and provides a bispecific antibody targeting PD-1 and 4-1 BB, and an application thereof, wherein the bispecific antibody comprises a first antigen-binding domain targeting PD-1 and a second antigen-binding domain targeting 4-1 BB. Compared with each individual antibody, the bispecific antibody of the present invention achieves a balance between activity and safety, and has outstanding drug-development potential.

Description

Bispecific antibodies targeting pd-1 and 4-1bb and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a bispecific antibody. BACKGROUND

[0002] The interaction of PD-1 and its ligands PD-L1 (programmed cell death ligand 1) and PD-L2 (programmed cell death ligand 2) generates an inhibitory signal that plays a key role in tumor immune escape, which mainly occurs in the tumor microenvironment. The blockade of the inhibitory signal of PD-1 using anti-PD-1 antibodies or anti-PD-L1 antibodies has achieved great success in the treatment of tumors in the clinic, but many patients still do not respond to this therapy or are resistant, or are intolerant.

[0003] 4-1BB, also known as CD137, belongs to the tumor necrosis factor receptor superfamily, and plays an important role in T cell activation-driven immune response, mainly promoting T cell proliferation, acquiring effector function, promoting immune memory, and inhibiting activation-induced cell death. Agonistic antibodies targeting 4-1BB as monotherapy or combination therapy have shown potential in mouse tumor models, but in the clinic, 4-1BB agonists, whether as monotherapy or combination therapy, have not produced enough response in patients due to toxicity and lack of efficacy, and no 4-1BB agonistic antibody has been approved for marketing. Therefore, there is a great clinical need for additional treatments targeting immune checkpoints.

[0004] Therefore, there is an urgent need in the art for antibodies against these two targets that can be used to optimize the activation of the immune system while avoiding safety problems. SUMMARY

[0005] The object of the present application is to provide new bispecific antibodies targeting PD-1 and 4-1BB, to solve the above problems existing in the prior art, and to provide new related drug options.

[0006] In a first aspect, the present application provides a bispecific antibody comprising a first antigen binding domain targeting PD-1 and a second antigen binding domain targeting 4-1BB.

[0007] In an embodiment, the first antigen binding domain targeting PD-1 comprises a HCDR1 as set forth in SEQ ID NO: 13, a HCDR2 as set forth in SEQ ID NO: 14, and a HCDR3 as set forth in SEQ ID NO: 15, a LCDR1 as set forth in SEQ ID NO: 16, a LCDR2 as set forth in SEQ ID NO: 17, and a LCDR3 as set forth in SEQ ID NO: 18. In an embodiment, the second antigen binding domain targeting 4-1BB comprises a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 11, and a CDR3 as set forth in SEQ ID NO: 12.

[0008] In an embodiment, the first antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the second antigen binding domain comprises a VHH. In an embodiment, the heavy chain variable region comprises a HCDR1 as set forth in SEQ ID NO: 13, a HCDR2 as set forth in SEQ ID NO: 14, and a HCDR3 as set forth in SEQ ID NO: 15. In an embodiment, the light chain variable region comprises a LCDR1 as set forth in SEQ ID NO: 16, a LCDR2 as set forth in SEQ ID NO: 17, and a LCDR3 as set forth in SEQ ID NO: 18. In an embodiment, the VHH comprises a CDR1 as set forth in SEQ ID NO: 10, a CDR2 as set forth in SEQ ID NO: 11, and a CDR3 as set forth in SEQ ID NO: 12.

[0009] In an embodiment, the first antigen binding domain comprises a heavy chain variable region and a light chain variable region, and the second antigen binding domain comprises a VHH; wherein the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 19, a sequence with at least 80% identity to the sequence set forth in SEQ ID NO: 19, or a sequence with one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 19; and / or the light chain variable region comprises a sequence as set forth in SEQ ID NO: 20, a sequence with at least 80% identity to the sequence set forth in SEQ ID NO: 20, or a sequence with one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 20; and / or the VHH comprises a sequence as set forth in SEQ ID NO: 1, a sequence with at least 80% identity to the sequence set forth in SEQ ID NO: 1, or a sequence with one or more conservative amino acid substitutions compared to the sequence set forth in SEQ ID NO: 1.

[0010] In an embodiment, the first antigen binding domain comprises a heavy chain variable region and a light chain variable region, the second antigen binding domain comprises a VHH, wherein the heavy chain variable region comprises a sequence as set forth in SEQ ID NO: 19, the light chain variable region comprises a sequence as set forth in SEQ ID NO: 20, and the VHH comprises a sequence as set forth in SEQ ID NO: 1.

[0011] In an embodiment, the first antigen binding domain further comprises a light chain constant region and / or a heavy chain constant region.

[0012] In an embodiment, the light chain constant region is a kappa or lambda light chain constant region. In an embodiment, the light chain constant region is a kappa light chain constant region.

[0013] In an embodiment, the heavy chain constant region can comprise an amino acid sequence selected from at least a part of a hinge region, CH1, CH2, CH3, or a combination thereof. In an embodiment, the heavy chain constant region is derived from an IgG (e.g., IgG1, IgG2, IgG3, IgG4) heavy chain constant region. In an embodiment, the heavy chain constant region is derived from a human IgG1 heavy chain constant region. In an embodiment, the heavy chain constant region is derived from a human IgG4 heavy chain constant region.

[0014] In an embodiment, when the C-terminal end of the heavy chain constant region is connected to the VHH that specifically binds to 4-1BB, the lysine (K) at the C-terminal end of the heavy chain constant region can be changed to alanine (A). In an embodiment, when the C-terminal end of the heavy chain constant region is connected to the VHH that specifically binds to 4-1BB, the lysine (K) at the C-terminal end of the heavy chain constant region is deleted. In an embodiment, the first antigen binding domain comprises a heavy chain and a light chain, the second antigen binding domain comprises a VHH, wherein the heavy chain comprises a sequence as set forth in SEQ ID NO: 4 or 6, the light chain comprises a sequence as set forth in SEQ ID NO: 3, and the VHH comprises a sequence as set forth in SEQ ID NO: 1.

[0015] In an embodiment, the VHH is connected to the heavy chain variable region, the light chain variable region, the heavy chain, or the light chain of the first antigen binding domain via a linker peptide L1.

[0016] In an embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide.

[0017] In an embodiment, the first polypeptide of the bispecific antibody comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of the first antigen binding domain, a linking peptide L1 and a VHH of the second antigen binding domain, and the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of the first antigen binding domain; wherein the light chain variable region of the first antigen binding domain comprises LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17 and LCDR3 as shown in SEQ ID NO: 18, the heavy chain variable region of the first antigen binding domain comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14 and HCDR3 as shown in SEQ ID NO: 15, and the VHH of the second antigen binding domain comprises CDR1 as shown in SEQ ID NO: 10, CDR2 as shown in SEQ ID NO: 11 and CDR3 as shown in SEQ ID NO: 12.

[0018] In an embodiment, the first polypeptide comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of the first antigen binding domain, a linking peptide L1 and a VHH of the second antigen binding domain, and the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of the first antigen binding domain; wherein the heavy chain variable region of the first antigen binding domain comprises a sequence as shown in SEQ ID NO: 19, or a sequence with at least 80% identity thereto, the light chain variable region of the first antigen binding domain comprises a sequence as shown in SEQ ID NO: 20, or a sequence with at least 80% identity thereto, and the VHH of the second antigen binding domain comprises a sequence as shown in SEQ ID NO: 1, or a sequence with at least 80% identity thereto.

[0019] In an embodiment, the first polypeptide comprises, from N-terminus to C-terminus, a light chain of the first antigen binding domain, a linking peptide L1 and a VHH of the second antigen binding domain, and the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first antigen binding domain; the light chain comprises a sequence as shown in SEQ ID NO: 3, the VHH comprises a sequence as shown in SEQ ID NO: 1, and the heavy chain comprises a sequence as shown in SEQ ID NO: 4 or 6.

[0020] In an embodiment, the linker peptide L1 comprises 4 to 30 amino acids. In an embodiment, the linker peptide L1 comprises 4 to 24 amino acids selected from G and S. In an embodiment, the linker peptide L1 comprises (GGGGS)x, wherein x is 1, 2, 3, 4, 5 or 6. In an embodiment, the amino acid sequence of the linker peptide L1 is set forth in SEQ ID NO: 2. In an embodiment, the amino acid sequence of the linker peptide L1 is set forth in SEQ ID NO: 8.

[0021] In an embodiment, the first polypeptide comprises, from N-terminus to C-terminus, a light chain of a first antigen binding domain having an amino acid sequence set forth in SEQ ID NO: 3, a linker peptide L1 having an amino acid sequence set forth in SEQ ID NO: 2, a VHH of a second antigen binding domain having an amino acid sequence set forth in SEQ ID NO: 1; a heavy chain of the first antigen binding domain having an amino acid sequence set forth in SEQ ID NO: 4 or 6.

[0022] In an embodiment, the first polypeptide comprises a sequence set forth in SEQ ID NO: 21 and the second polypeptide comprises a sequence set forth in SEQ ID NO: 4.

[0023] In an embodiment, the first polypeptide comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of a first antigen binding domain, and the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain variable region and CH1 (VH-CH1) of a first antigen binding domain, a linker peptide L1, a VHH of a second antigen binding domain, CH2-CH3 of an IgG.

[0024] In an embodiment, the first polypeptide comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of a first antigen binding domain, and the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain variable region and CH1 of a first antigen binding domain, a linker peptide L1, a VHH of a second antigen binding domain, a linker peptide L2, CH2-CH3 of an IgG.

[0025] In an embodiment, the light chain variable region of the first antigen binding domain comprises a LCDR1 set forth in SEQ ID NO: 16, a LCDR2 set forth in SEQ ID NO: 17 and a LCDR3 set forth in SEQ ID NO: 18; the heavy chain variable region of the first antigen binding domain comprises a HCDR1 set forth in SEQ ID NO: 13, a HCDR2 set forth in SEQ ID NO: 14 and a HCDR3 set forth in SEQ ID NO: 15; the VHH of the second antigen binding domain comprises a CDR1 set forth in SEQ ID NO: 10, a CDR2 set forth in SEQ ID NO: 11 and a CDR3 set forth in SEQ ID NO: 12.

[0026] In an embodiment, the first polypeptide comprises from N- to C-terminus a light chain variable region of a first antigen binding domain and a light chain constant region, the second polypeptide comprises from N- to C-terminus a heavy chain variable region of a first antigen binding domain and CH1, a linker L1, a VHH of a second antigen binding domain, a linker L2, CH2-CH3 of IgG; wherein the heavy chain variable region of the first antigen binding domain comprises a sequence of SEQ ID NO: 19, or a sequence of at least 80% identity thereto, the light chain variable region of the first antigen binding domain comprises a sequence of SEQ ID NO: 20, or a sequence of at least 80% identity thereto, the VHH of the second antigen binding domain comprises a sequence of SEQ ID NO: 1, or a sequence of at least 80% identity thereto.

[0027] In an embodiment, the CH1, CH2, CH3 is derived from an IgG (IgG1, IgG2, IgG3 or IgG4 or a subclass thereof) heavy chain constant region. In an embodiment, the CH1, CH2, CH3 is derived from an IgG1 or IgG4 heavy chain constant region.

[0028] In an embodiment, the first polypeptide comprises from N- to C-terminus a light chain of a first antigen binding domain, the second polypeptide comprises from N- to C-terminus a VH-CH1 of a first antigen binding domain, a linker L1, a VHH of a second antigen binding domain, a linker L2, CH2-CH3 of IgG; wherein the light chain comprises a sequence of SEQ ID NO: 3, the VH-CH1 comprises a sequence of SEQ ID NO: 7, the VHH comprises a sequence of SEQ ID NO: 1, the CH2-CH3 comprises a sequence of SEQ ID NO: 9.

[0029] In an embodiment, the first polypeptide of the bispecific antibody is a light chain of an anti-PD-1 antibody; the second polypeptide comprises from N- to C-terminus a VH-CH1 of an anti-PD-1 antibody, a linker L1, a VHH of an anti-4-1BB, a linker L2, CH2-CH3 of IgG, the amino acid sequence of the light chain of the anti-PD-1 antibody is set forth in SEQ ID NO: 3, the amino acid sequence of the heavy chain VH-CH1 of the anti-PD-1 is set forth in SEQ ID NO: 7, the amino acid sequence of the VHH of the anti-4-1BB is set forth in SEQ ID NO: 1, the amino acid sequence of the CH2-CH3 of IgG is set forth in SEQ ID NO: 9.

[0030] In an embodiment, the linker peptide L1 comprises 4 to 30 amino acids. In an embodiment, the linker peptide L1 comprises 4 to 24 amino acids selected from G and S. In an embodiment, the linker peptide L1 comprises (GGGGS)x, wherein x is 1, 2, 3, 4, 5 or 6. In an embodiment, the amino acid sequence of the linker peptide L1 is set forth in SEQ ID NO: 2. In an embodiment, the amino acid sequence of the linker peptide L1 is set forth in SEQ ID NO: 8.

[0031] In an embodiment, the linker peptide L2 comprises 4 to 30 amino acids. In an embodiment, the linker peptide L2 comprises 4 to 24 amino acids selected from G and S. In an embodiment, the linker peptide L2 comprises (GGGGS)x, wherein x is 1, 2, 3, 4, 5 or 6. In an embodiment, the amino acid sequence of the linker peptide L2 is set forth in SEQ ID NO: 2. In an embodiment, the amino acid sequence of the linker peptide L2 is set forth in SEQ ID NO: 8.

[0032] In an embodiment, the first polypeptide comprises a sequence set forth in SEQ ID NO: 3 and the second polypeptide comprises a sequence set forth in SEQ ID NO: 22.

[0033] In an embodiment, the first polypeptide comprises a sequence set forth in SEQ ID NO: 3 and the second polypeptide comprises a sequence set forth in SEQ ID NO: 22.

[0034] In an embodiment, the first polypeptide comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of the first antigen binding domain, the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of the first antigen binding domain, a linker peptide L1, a VHH of the second antigen binding domain; wherein the heavy chain variable region of the first antigen binding domain comprises a sequence of SEQ ID NO: 19, or an amino acid sequence with at least 80% identity thereof, the light chain variable region of the first antigen binding domain comprises a sequence of SEQ ID NO: 20, or an amino acid sequence with at least 80% identity thereof, the VHH of the second antigen binding domain comprises a sequence of SEQ ID NO: 1, or an amino acid sequence with at least 80% identity thereof.

[0035] In an embodiment, the linker peptide L1 comprises 4 to 30 amino acids. In an embodiment, the linker peptide L1 comprises 4 to 24 amino acids selected from G and S. In an embodiment, the linker peptide L1 comprises (GGGGS)x, wherein x is 1, 2, 3, 4, 5 or 6. In an embodiment, the amino acid sequence of the linker peptide L1 is set forth in SEQ ID NO: 2. In an embodiment, the amino acid sequence of the linker peptide L1 is set forth in SEQ ID NO: 8.

[0036] In an embodiment, the first polypeptide comprises, from N-terminus to C-terminus, a light chain of the first antigen binding domain, the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of the first antigen binding domain, a linker peptide L1, a VHH of the second antigen binding domain; the heavy chain comprises a sequence of SEQ ID NO: 6, the light chain comprises a sequence of SEQ ID NO: 3, the linker peptide L1 comprises a sequence of SEQ ID NO: 2, and the VHH comprises a sequence of SEQ ID NO: 1.

[0037] In an embodiment, the first polypeptide is a light chain of an anti-PD-1 antibody; the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain of an anti-PD-1, a linker peptide L1, a VHH of an anti-4-1BB, the amino acid sequence of the light chain of the anti-PD-1 antibody is set forth in SEQ ID NO: 3, the amino acid sequence of the heavy chain of the anti-PD-1 antibody is set forth in SEQ ID NO: 6, the amino acid sequence of the linker peptide L1 is set forth in SEQ ID NO: 2, and the amino acid sequence of the VHH of the anti-4-1BB is set forth in SEQ ID NO: 1.

[0038] In an embodiment, the first polypeptide comprises a sequence of SEQ ID NO: 3, and the second polypeptide comprises a sequence of SEQ ID NO: 23.

[0039] In one embodiment, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides. In one embodiment, one first polypeptide and one second polypeptide are connected by a disulfide bond, and two second polypeptides are connected by a disulfide bond.

[0040] In one embodiment, the bispecific antibody of the present application can have better safety and tumor inhibition effect. In a second aspect, the present application provides a biological material, which is:

[0041] 1) a nucleic acid molecule encoding the bispecific antibody of the present application or a part thereof;

[0042] 2) an expression vector comprising the nucleic acid molecule of the present application;

[0043] 3) a host cell comprising the nucleic acid molecule or the expression vector of the present application.

[0044] In a third aspect, a method for preparing the bispecific antibody of the present application is provided, which is:

[0045] 1) chemical synthesis: according to the amino acid sequence of the bispecific antibody provided by the present application, the bispecific antibody is prepared by chemical synthesis;

[0046] 2) biological synthesis: the host cell provided by the present application is cultured to express the bispecific antibody of the present application;

[0047] In one embodiment, it further comprises isolating the antibody from the obtained reaction or culture; and purifying the antibody.

[0048] In a fourth aspect, a pharmaceutical composition comprising the bispecific antibody of the present application is provided; preferably, it further comprises a pharmaceutically acceptable adjuvant, such as a pharmaceutically acceptable excipient, diluent or carrier.

[0049] In a fifth aspect, the bispecific antibody or the biological material or the pharmaceutical composition of the present application is used in the preparation of a medicament for treating or preventing tumors or viral infections.

[0050] In a sixth aspect, the bispecific antibody or the biological material or the pharmaceutical composition of the present application is used in the treatment or prevention of tumors or viral infections.

[0051] In a seventh aspect, a method for treating or preventing tumors or viral infections is provided, which comprises administering the bispecific antibody or the biological material or the pharmaceutical composition of the present application to a patient in need thereof.

[0052] In aspects of the present application, the tumor includes melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colon cancer, prostate cancer, gastric cancer, kidney cancer, bladder cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, thyroid cancer, hepatocellular carcinoma, mesothelioma, etc., and the viral infection includes hepatitis C, hepatitis B, etc.

[0053] In one embodiment, the bispecific antibody in the present application is an IgG-like structure. The bispecific antibody adopts the structure of Fab-VHH-Fc, the Fab targets PD-1, the VHH targets 4-1BB, and the Fc is a natural IgG4 sequence, which maximally retains the natural antibody structure while weakening the binding with FcyR and Clq, and weakening the ADCC and CDC effects. At the same time, the binding domain with PD-1 in this structure is not changed compared with the parent, which maximally retains the activity of the bispecific antibody in binding with PD-1. In addition, the bispecific antibody in the present application only contains one heavy chain and one light chain, which greatly reduces the possibility of mismatch.

[0054] The bispecific antibody targeting PD-1 and 4-1BB provided in the present application has an affinity to PD-1 which is at least 10 times, and at most 100 times, the affinity to 4-1BB, achieving the balance of optimal activity and safety, and having very excellent drug development potential. BRIEF DESCRIPTION OF DRAWINGS

[0055] Fig. 1 is a schematic diagram of the structure of the bispecific antibody;

[0056] Fig. 2 is the PAGE electrophoresis result of the bispecific antibody;

[0057] Fig. 3 is the detection result of the PD-1 activity reporter system of the bispecific antibody;

[0058] Fig. 4A is the detection result of the 4-1BB activity reporter system dependent on PD-1 of the bispecific antibody;

[0059] Fig. 4B is the detection result of the 4-1BB activity reporter system independent of PD-1 of the bispecific antibody;

[0060] Figs. 5A to J are the results of the in vitro cytokine release test;

[0061] Fig. 6 is the detection result of the efficacy of the bispecific antibody on Balb / c tumor-bearing mice;

[0062] Fig. 7 is the treatment effect experiment of the antibody on the subcutaneous tumor formation of MC38 colon cancer cells in C57 mice;

[0063] Fig. 8 is the detection result of the binding affinity of the antibody to PD-1;

[0064] Figure 9 is the result of a binding affinity assay of the antibody to 4-1BB;

[0065] Figure 10 is the result of a binding assay of the antibody to PD-1 and 4-1BB;

[0066] Figure 11 is the result of a binding assay of the antibody to PD-1 of different species origin;

[0067] Figure 12 is the result of a binding assay of the antibody to 4-1BB of different species origin. DETAILED DESCRIPTION

[0068] TERMS

[0069] Unless otherwise indicated, each of the terms below shall have the meaning set forth herein.

[0070] DEFINITIONS

[0071] The term "a" or "an" entity refers to one or more of that entity; for example, "an antibody" shall be understood to mean one or more antibodies, and, alternatively, "one or more" or "at least one" can be used interchangeably with "a" or "an" in the present disclosure.

[0072] As used herein the terms "comprising" or "including," or "having" mean that the antibody, composition or method includes the recited elements, for example, components or steps, but not excluding others. "Consisting essentially of means that the antibody, composition or method excludes other elements of any essential influence on the character of the combination, but does not exclude elements that do not materially affect the character of the antibody, composition or method. "Consisting of means excluding elements not specifically recited. Embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0073] An "antibody" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be an intact antibody ("immunoglobulin, IgG") and any antigen-binding fragment or single chain thereof, such as an antigen-binding domain. Thus, the term "antibody" includes any protein or peptide that contains at least one portion of an immunoglobulin molecule that has a biological activity of binding to an antigen. Antibodies include, but are not limited to, examples include a heavy chain, a light chain, a complementarity determining region (CDR) of a ligand binding portion thereof, a variable region of a heavy chain (VH), a variable region of a light chain (VL), a constant region of a heavy chain (CH), a constant region of a light chain (CL), a framework region (FR), or any portion thereof, or at least a portion of a binding protein. CDR regions include CDR regions of a light chain (LCDR1-3) and CDR regions of a heavy chain (HCDR1-3). Those skilled in the art will understand that the class of an immunoglobulin heavy chain includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses thereof (e.g., γ1-γ4), the

[0074] It is understood by one of ordinary skill in the art that the CDR regions of an antibody are responsible for the binding specificity of the antibody to an antigen. In the case of known antibody heavy and light chain variable region sequences, there are currently several methods of determining the CDR regions of an antibody, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, the application of each definition of CDRs to an antibody or variant thereof will be within the scope of the terms defined and used herein. One of ordinary skill in the art can generally determine which residues are included in a particular CDR without relying on any experimental data other than the variable region amino acid sequence of the antibody given, if any.

[0075] A "heavy chain constant region" includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of an antibody can include a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4.

[0076] A "light chain constant region" consists of one domain, CL. In some embodiments, the light chain constant region can be derived from a kappa constant region domain or a lambda constant region domain.

[0077] The antibodies disclosed herein can be derived from any animal, including but not limited to fish, birds, and mammals. Preferably, the antibodies are human, murine, equine, rabbit, goat, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be condricthoid in origin (e.g., from a shark).

[0078] The term "bispecific antibody" refers to an antibody (including an antibody or antigen-binding fragment thereof, such as a single-chain antibody, nanobody) capable of specifically binding to two different antigens or two different epitopes of the same antigen. 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 divalent, trivalent, tetravalent or more valent bispecific antibodies. According to whether the structure is symmetrical, it can be divided into symmetrical structure bispecific antibodies and asymmetrical structure bispecific antibodies. Among them, the fragment type bispecific antibody, such as Fab fragment lacking Fc fragment, forms a bispecific antibody by combining 2 or more Fab fragments in one molecule, which has lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration. The typical antibody structure of this type is F(ab)2, scFv-Fab, (scFv)2-Fab, etc. IgG-like bispecific antibodies (e.g., with Fc fragments), such antibodies have relatively large molecular weights, Fc fragments help purify antibodies and improve their solubility and stability, and Fc portions can also bind to the receptor FcRn, increasing the serum half-life of antibodies. Bispecific antibody structure models such as KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 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 (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).

[0079] VHH or "single domain antibody" or "sdAb" refers to an antigen binding fragment that comprises only a single antibody variable region. A single sdAb is capable of binding to an antigen. In some cases, sdAbs are engineered from camelid HcAbs, whose heavy chain variable domains are referred to herein as "VHH" (heavy chain variable domain of a heavy chain antibody). Some VHHs are also referred to as nanobodies. A VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0080] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Ed., E. S. Golub and D. R. Green, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), unnatural amino acids such as a-, a-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids can also be components of polypeptides useful in the present disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamate, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, sigma-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide representation methods used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxy-terminal direction, consistent with standard usage and convention. The conventional (or natural) amino acids are L-amino acids, including alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (lie, 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), valine (Val, V), and the like.

[0081] A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has "identity" or "sequence identity" to another sequence to the extent the sequences are the same, with a certain percentage (e.g., 90%, 95%, 98%, or 99%) of the bases (or amino acids) identical, when compared and aligned for maximum correspondence over a specified comparison window. The determination of percent identity or sequence identity can use visual inspection or software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, the alignment is performed using default parameters. One such alignment program is BLAST, e.g., BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant; GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss Protein + SPupdate + PIR. A biologically equivalent polynucleotide is one which has the specified percentage identity and which encodes a polypeptide having the same or similar biological activity.

[0082] Minor variations to the amino acid sequence of the antibody or immunoglobulin molecule are encompassed by the present disclosure provided that the identity of the amino acid sequence remains at least 90%, such as at least 92%, 95%, 98%, or 99%. In some embodiments, the variations are conservative amino acid substitutions. Conservative amino acid substitutions are substitutions occurring within a family of amino acids that are related in their side chains. Genetically encoded amino acids are broadly grouped as follows: (1) acidic amino acids as aspartate, glutamate; (2) basic amino acids as lysine, arginine, histidine; (3) non-polar amino acids as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and (4) uncharged polar amino acids as glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Other families of amino acids include (i) serine and threonine of the aliphatic-hydroxyl family; (ii) asparagine and glutamine of the amide family; (iii) alanine, valine, leucine and isoleucine of the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine of the aromatic family. In some embodiments, the conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate- aspartate, and asparagine-glutamine. For example, it is reasonable to predict that a substitution of leucine for isoleucine or valine, of glutamate for aspartate, of serine for threonine, or of one aliphatic amino acid for another, will not have a major effect on the properties of the resulting molecule, especially if the substitution occurs at a position remote from the active site of the molecule. Whether a change in an amino acid results in a functional peptide can be readily determined by assaying the specific activity of the polypeptide derivative. Such assays are described in detail herein. Fragments or analogs of the antibody or immunoglobulin molecule can be readily prepared by one of ordinary skill in the art.

[0083] In some embodiments, the amino acid substitution has the effect of: (1) decreasing susceptibility to proteolysis, (2) decreasing susceptibility to oxidation, (3) altering binding affinity for forming a protein complex, (4) altering binding affinity, or (5) conferring or improving other physicochemical or functional properties of such analogs. The analogs can include various muteins having sequences that differ from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in naturally occurring sequences, preferably in portions of the polypeptide other than those forming intermolecular contacts. Conservative amino acid substitutions should not significantly alter the structural properties of the parent sequence (e.g., the substituted amino acid should not tend to disrupt a helical structure present in the parent sequence, or other types of secondary structure characteristic of the parent sequence). Examples of secondary and tertiary structures of polypeptides that are recognized by the art are described in Proteins, Structures and Molecular Principles (Creighton, ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature 354:105 (1991).

[0084] The term "polypeptide" is intended to encompass both singular "polypeptide" as well as plural "polypeptides" and refers to a molecule composed of linear sequence of amino acid monomers joined by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids and does not relate to a particular length of the product. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, and "polypeptide" can be used in place of or alternatively to any of the above terms. The term "polypeptide" is also intended to refer to products of polypeptide expression which have been modified after expression, including but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic processing, or non-naturally occurring amino acid modifications. The polypeptide can be derived from a natural biological source or produced by recombinant techniques, but it need not necessarily be translated from a specified nucleic acid sequence, it can be produced in any manner including chemical synthesis.

[0085] Antibodies, 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 an epitope. The antibody or antigen-binding fragment can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting / blocking groups, proteolytic cleavage, attachment to a cell ligand or other protein, etc. Any of numerous chemical modifications can be introduced into an antibody or antigen-binding fragment of the application by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0086] 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, oligonucleotide (e.g., siRNA), or PEG, etc.

[0087] As used herein, the term "specifically binds" or "immunoreacts" refers to the noncovalent interaction between an immunoglobulin molecule and one or more antigenic determinants of its target antigen. The strength or affinity of an immunological binding interaction can be expressed in terms of the equilibrium dissociation constant (K D ) of the interaction, where a smaller K D represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method measures the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentrations of the complex partners, the affinity of the interaction, and other geometric parameters that similarly affect the rates. Both the "on rate constant" (kon) and the "off rate constant" (koff) can be determined by measuring the concentrations and actual association and dissociation rates (see Malmqvist, M., Nature 361 : 186-87 (1993)). The koff / kon ratio eliminates parameters that are independent of affinity, and is equal to the equilibrium dissociation constant K D (see Davies et al. (1990) Annual Rev Biochem 59: 439-473). Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to one of skill in the art.

[0088] The term "isolated" as used herein in connection with cells, nucleic acids, polypeptides, antibodies, etc. of the application, e.g., "isolated" DNA, RNA, polypeptide, means a molecule separated from at least one other component with which it is normally associated in nature. The term "isolated" as used herein also means a nucleic acid or polypeptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques or chemical synthesis. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to a cell or polypeptide that is separated from other cellular proteins or tissues. An isolated polypeptide is intended to include a purified and recombinant polypeptide. An isolated polypeptide, etc. is typically produced by at least one purification step. In one or more embodiments, an isolated nucleic acid, polypeptide, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or a range between any two of these values, inclusive of the endpoints, or any value therein.

[0089] The term "encodes" applied to a polynucleotide refers to a polynucleotide that is referred to as "encoding" a polypeptide, which, when transcribed and / or translated in its natural state or when manipulated by methods known to those skilled in the art, can produce the polypeptide and / or fragments thereof.

[0090] "About" refers to the conventional error range for a given technical field or technical school of engineers, which is readily known by those skilled in the art. In some embodiments, "about" refers to a range of ±10%, ±5%, or ±1% of a value stated.

[0091] "EC50" or "half maximal effective concentration" refers to the concentration that produces 50% of the maximal effect.

[0092] "Treatment" refers to any action providing a therapeutic benefit or providing a prophylactic benefit. Therapeutic benefit is indicated by, for example, the alleviation of symptoms making up a disease or condition, or the slowing of disease progression. Prophylactic benefit is indicated by, for example, the prevention of a disease or condition, or the slowing of disease progression. A "treatment" is a therapeutic treatment and a prophylactic or preventative measure, which aims to prevent, slow down, ameliorate or stop an adverse physiological change or disorder, such as the progression of a disease, including but not limited to alleviation of symptoms, whether or not detectable, reduction in extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, amelioration of disease state, remission, whether partial or total, prolonging of survival, and / or quality of life, as compared to expected survival without treatment, etc. Patients in need of treatment include those already with a condition or disorder, those likely to develop a condition or disorder, or those in need of prevention of a condition or disorder, who can or expect to benefit from administration of an antibody or pharmaceutical composition of the present disclosure for detection, diagnostic procedures, and / or treatment.

[0093] The term "tumor" means or is intended to describe the physiological state of a mammal that is typically characterized by uncontrolled cell growth, including benign and malignant tumors such as cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, or leukemia. More particular examples of such cancers include, but are not limited to, colon cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, colon cancer, salivary gland cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell cancer, nasopharyngeal cancer, laryngeal cancer, lung adenocarcinoma, lung squamous carcinoma, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, glioblastoma, breast cancer, brain cancer, renal cancer, renal cell carcinoma, rectal cancer, prostate cancer, vulvar cancer, testicular cancer, squamous cell cancer, small cell lung cancer, cervical cancer, bladder cancer, retinoblastoma, neurogliocytoma, mesothelioma, oral epithelioid cancer, choriocarcinoma, and head and neck cancer.

[0094] The effective dosage and treatment regimen for treating a particular patient will depend on a variety of factors, including the particular antibody, antigen-binding fragment, or derivative used, the age, and body weight, general health, sex, and diet of the patient, the time of administration, the frequency of administration, the combination with other drugs, and the severity of the particular disease being treated. These factors are within the judgment of the medical caregiver, which is within the purview of one of ordinary skill in the art. The dosage used can be determined by principles of pharmacology and pharmacokinetics well known in the art. In some embodiments, the antibody of the present application is administered to a patient at a dosage of 0.01 mg / kg to 100 mg / kg of patient body weight per administration. In some embodiments, the administration is once a week, or once a month.

[0095] The term "patient" refers to any mammal in need of diagnosis, prognosis, or treatment, including, but not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, and the like.

[0096] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In addition, "pharmaceutically acceptable carrier" generally refers to any type of nontoxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation auxiliary of the like.

[0097] The term "excipient" refers to a diluent, adjuvant, vehicle, or carrier with which the active ingredient is administered to a patient. Such pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred vehicle when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, lactose, dextrose, glycerol, propylene glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Antibacterial agents, such as benzyl alcohol or methyl parabens, antioxidants, such as ascorbic acid, chelating agents, and agents for the adjustment of tonicity, such as sodium chloride or dextrose, are also envisioned. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated into a suppository for rectal administration with traditional binders and vehicles such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the antibody or antigen-binding fragment or fusion protein, preferably in purified form, together with a suitable amount of excipient to provide the desired form for administration. The formulation should suit the mode of administration. Parenteral formulations can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0098] In some embodiments, the compositions are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous injection. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. Compositions can also contain solubilizers and local anesthetics such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0099] The antibody or antigen-binding fragment or fusion protein of the present application can be in neutral or salt form. Pharmaceutically acceptable salts include those formed from acetic acid, phosphoric acid, hydrochloric acid, oxalic acid, tartaric acid, etc., and those formed from such bases as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

[0100] Preparation of antibodies

[0101] A variety of methods for preparing antibodies are known in the art, such as the hybridoma technique, recombinant DNA technology, transgenic mouse technology, and phage display library, etc.

[0102] Antibodies can be prepared by using conventional recombinant DNA technology. Techniques for selecting, constructing, and cultivating vectors and cell lines that produce antibodies can be selected, constructed, and cultivated 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, D. L. Hacker, F. M. Wurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplements, are incorporated herein by reference.

[0103] In some embodiments, DNA encoding the antibodies can be designed and synthesized according to the amino acid sequences of the antibodies described herein in a conventional manner, inserted into an expression vector, and then transfected into host cells, and the transfected host cells are cultured in a culture medium to produce monoclonal antibodies. In some embodiments, the expression vector of the antibody includes at least one promoter element, antibody coding sequence, transcription termination signal and polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites of RNA splicing flanking the insertion sequence. Efficient transcription can be obtained by the early and late promoters of SV40, the long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIVI, and cytomegalovirus early promoters, and other cell promoters such as actin promoter. Suitable expression vectors can 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, murine L cells, and CHO cells, etc.

[0104] In some embodiments, the inserted gene fragment needs to contain a screening marker, common screening markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance and other screening genes, so as to facilitate the screening and separation of successfully transfected cells. The constructed plasmid is transfected into a host cell without the above-mentioned gene, and after selective medium culture, the successfully transfected cells grow in large quantities to produce the desired target protein. The obtained antibody can be isolated or purified by conventional technical means, such as protein A-sepharose, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0105] The sequence information related to the present application is as follows:

[0106] The technical solutions of the present application will be further illustrated by specific examples below. The specific examples do not represent a limitation on the scope of protection of the present application, and some non-essential modifications or adjustments made by others according to the concept of the present application still fall within the scope of protection of the present application.

[0107] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0108] hPD-1 full-length sequence (SEQ ID NO: 24)

[0109] h4-1BB full-length sequence (SEQ ID NO: 25)

[0110] Anti-4-1BB monoclonal antibody sequence (SEQ ID NO: 26)

[0111] Example 1: Construction, expression and biophysical characterization of PD-1 / 4-1BB bispecific antibodies

[0112] This example describes exemplary PD-1 / 4-1BB bispecific antibody (BsAb) vector construction and expression. Three structures of bispecific antibodies were designed and expressed, each containing the following polypeptide chains, and the diagram of the three structures is shown in Figure 1.

[0113] Structure 1: contains two identical first polypeptides and two identical second polypeptides, one first polypeptide and one second polypeptide are connected by a disulfide bond, and two second polypeptides are connected by a disulfide bond. Among them, the first polypeptide contains, from N-terminus to C-terminus: light chain of anti-PD-1 antibody, connecting peptide L1, VHH of anti-4-1BB; the second polypeptide is the heavy chain of anti-PD-1 antibody in IgG4 form.

[0114] Representative BsAb is 2-15, sequence composition is shown in Table 1-1.

[0115] Table 1-1: Sequence composition of mode 1 BsAb

[0116] Mode 2 structure: containing two identical first polypeptides and two identical second polypeptides, one first polypeptide and one second polypeptide are connected by disulfide bond, two second polypeptides are connected by disulfide bond. Among them, the first polypeptide is the light chain of anti-PD-1 antibody; the second polypeptide comprises, from N-terminus to C-terminus: heavy chain VH-CH1 of anti-PD-1 antibody, connecting peptide L1, VHH of anti-4-1BB, connecting peptide L2, CH2-CH3 of IgG4.

[0117] Representative BsAb is 5-15, sequence composition is shown in Table 1-2.

[0118] Table 1-2: Sequence composition of mode 2 BsAb

[0119] Mode 3 structure: containing two identical first polypeptides and two identical second polypeptides, one first polypeptide and one second polypeptide are connected by disulfide bond, two second polypeptides are connected by disulfide bond. Among them, the first polypeptide is the light chain of anti-PD-1 antibody; the second polypeptide comprises, from N-terminus to C-terminus: heavy chain of anti-PD-1, connecting peptide L1, VHH of anti-4-1BB.

[0120] Representative BsAb is 7-15, sequence composition is shown in Table 1-3.

[0121] Table 1-3: Sequence composition of mode 3 BsAb

[0122] Plasmids expressing the above three structures of BsAb were prepared and transiently expressed in 293F cells. Purified by one-step protein A. As shown in Figure 2, under reducing and non-reducing conditions, the composition and purity of the purified BsAb were analyzed by SDS-PAGE, the size of the polypeptide chain was consistent with the molecular weight calculated based on the amino acid sequence of the full-length molecule.

[0123] Example 2: PD-1 activity reporter system detection of BsAb

[0124] A variety of bioassays can be used to investigate the blockade of the PD-1 pathway by the BsAb proteins, and the bioassay described in this example is accomplished by monitoring the expression of a reporter gene driven by signaling in the PD-1 pathway. The PD-1 / PD-L1 Blockade Bioassay system constructed by Promega is suitable for detecting the cell biological activity of anti-PD-1 monoclonal antibodies, and has the advantages of simple operation and stable system. Its main principle is: the CHO-K1 cell strain has a stable expression of PD-L1 and T cell receptor activator (TCR activator) on the cell membrane, simulating antigen presenting cells (APCs); Jurkat is a human T lymphocyte cell line, with PD-1 expression on the cell membrane, and transfected with a luciferase reporter gene vector with an NFAT promoter. When the two cells are co-incubated, the TCR activator of CHO-K1 cells can activate the reporter gene expression of Jurkat cells, but at the same time the interaction of PD-1 / PD-L1 will block this activation; after adding anti-PD-1 monoclonal antibodies, the interaction of PD-1 / PD-L1 is blocked, and the Luciferase reporter gene restores expression, making the substrate produce a fluorescent signal, and the signal value is positively correlated with the amount of anti-PD-1 monoclonal antibodies added.

[0125] Figure 3 shows data on the biological activity of anti-PD-L1 neutralizing antibodies in a PD-1 / PD-L1 cell-based assay using PD-1 / NFAT reporter-Jurkat cells. Briefly, CHO-K1 cells stably expressing human PD-L1 and engineered T cell receptor (TCR) activator (Promega, CS187108) were used. PD-1 / NFAT reporter-Jurkat cells (Promega, CS187102) were pre-incubated with serially diluted PD-1 mAb CL1 (CN111808190B) or the BsAb of the present application (10 pg / ml starting concentration, 3-fold dilution, 10 consecutive gradients) for 30 minutes, and then co-cultured with engineered CHO-K1 cells (Promega, CS187108). After about 6 hours of stimulation, Bio-Lite TM Luciferase Assay System luciferase reagent (Novagen) was added to the cells to measure NFAT activity. Using nonlinear regression, data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA), and EC50 values were calculated and shown in Figure 3. The reporter assay showed similar activity of 2-15, 5-15, 7-15 compared to PD-1 mAb CL1.

[0126] Example 3: 4-1BB activity reporter system assay for BsAbs

[0127] A variety of bioassay methods can be used to investigate the activation of the 4-1BB pathway by BsAb samples, which monitor T cell proliferation, IFN-g release, IL-2 secretion, or expression of a reporter gene driven by signaling in the 4-1BB pathway.

[0128] PD-1 / 4-1BB / NF-κB reporter-HEK-293T cells were used for in vitro bioactivity evaluation of BsAbs, which were constructed by transfecting HEK-293T with a luciferase reporter vector for the NF-κB promoter, and a stable cell line was obtained by screening with hygromycin resistance (100 μg / ml). On this basis, a vector containing the full-length sequences of hPD-1 (Uniprot No.: Q15116, SEQ ID NO: 24) and h4-1BB (Uniprot No.: Q07011, SEQ ID NO: 25) was transfected, and a PD-1 / 4-1BB / NF-κB reporter-HEK-293T stable cell line was obtained by screening with hygromycin resistance (400 μg / ml) and puromycin (0.3 μg / ml).

[0129] The characterization of the biological activity of 4-1BB agonism of BsAbs using PD-1 / 4-1BB / NF-κB reporter-HEK-293T cells is shown in FIG. 4A. In this case, the BsAb samples were prepared (100 nM starting concentration, 3-fold serial dilution, with 10 concentrations), and the PD-1 / 4-1BB / NF-κB reporter-HEK-293T cells were mixed with the serially diluted samples and placed in a cell incubator for culture. After about 6 hours of stimulation, the Bio-Lite TM Luciferase Assay System luciferase reagent (Promega) was added to the cells to measure NF-κB activity. Data were analyzed using nonlinear regression with GraphPad PRISM 8 (GraphPad Software, San Diego, CA), and EC 50 values were calculated. The FIG. 4A reporter assay showed that all BsAbs effectively activated the NF-κB signal in the presence of PD-1, and the EC50 was comparable.

[0130] In addition, in order to evaluate the activation of 4-1BB by BsAb 5-15 in the absence of PD-1, 4-1BB / NF-κB reporter-HEK-293T cells were used, which were constructed by transfecting HEK-293T with a luciferase reporter vector of the NF-κB promoter, and screening a stable cell line with hygromycin resistance (100 μg / ml), and then transfecting a vector containing the full-length sequence of h4-1BB (Uniprot No.: Q07011, SEQ ID NO: 25) and screening a 4-1BB / NF-κB reporter-HEK-293T stable cell line with puromycin (0.3 μg / ml).

[0131] The characterization of the biological activity of 4-1BB agonism of BsAb in the 4-1BB / NF-κB reporter-HEK-293T cell-based assay is shown in FIG. 4B. 5-15 samples, i.e., 5-87 and 5-160 in FIG. 4B (two batches, 100 nM starting concentration, 3-fold serial dilution, with 10 concentrations), were prepared, and PD-1 control antibody CL1 (anti-PD-1 antibody, sequence derived from CL1 of patent CN111808190B) and 4-1BB control antibody Urelumab (CN1867585B) were diluted in the same way, and 4-1BB / NF-κB reporter-HEK-293T cells were mixed with serially diluted samples and incubated in a cell incubator. After about 6 hours of stimulation, Bio-Lite TM Luciferase Assay System luciferase reagent (Promega) was added to the cells to measure NF-κB activity. Data were analyzed using nonlinear regression with GraphPad PRISM 8 (GraphPad Software San Diego, CA), and EC 50 values were calculated. FIG. 4B reporter assay shows that Urelumab can still effectively activate NF-κB signaling in the absence of PD-1, while the two samples of 5-15 and CL1 cannot activate NF-κB signaling, indicating that the BsAb activates 4-1BB signaling specifically dependent on the presence of PD1.

[0132] Example 4: Cytokine release experiment of PD-1 / 4-1BB bispecific antibody

[0133] Cytokine release syndrome (CRS) is a group of clinical syndromes caused by excessive activation of immune cells and rapid release of pro-inflammatory factors, which is a common adverse reaction of immunomodulatory drugs. For immunomodulatory drugs, the complete nonclinical evaluation needs to integrate all available in vivo and in vitro test data. In addition to cytokine-related detection in routine animal in vivo toxicity tests, in vitro cytokine release tests should also be conducted. The use of human whole blood or human peripheral blood mononuclear cells (PBMCs) in vitro to evaluate cell activation and cytokine release can to some extent make up for the defects that animal models cannot completely simulate the human immune stimulation process due to species differences. When the in vivo test results are negative, positive results of in vitro tests can indicate potential clinical safety risks. It is recommended to design appropriate in vitro cytokine release tests based on the cytokine release mechanism. Liquid and solid phase incubation systems should be considered in general.

[0134] In this example, the cytokine release of BsAb was evaluated using liquid and solid phase incubation systems. The dry pack method, i.e. the solid phase incubation system, coated the sample to be tested at 25 μg / mL, 40 μL volume, and dried overnight in a clean bench at 2 air speed. Then 1×10 5 PBMC cells (Redbiotech, Guangzhou) from different donors were added to each well, with a volume of 200 μl. The wet pack method, i.e. the liquid phase incubation system, used the sample to be tested at 25 μg / mL, 100 μL, and added 1×10 5 PBMC cells to each well. Anti-CD3 antibody (Jin'an Biotech, GMP-A018), TGN1412 (CD28 agonistic antibody, sequence derived from patent US8709414B2, prepared by Baiota Biopharmaceutical Co., Ltd.), and CL1 (anti-PD-1 antibody, sequence derived from patent CN111808190B CL1) were selected as positive control antibodies. After three days of incubation, the supernatant was collected for detection of IL-2, IL-6, IL-10, IFN-γ and TNFα (Neobioscience, China). As shown in FIGS. 5A-J, TGN1412 and anti-CD3 antibody can very obviously activate PBMC in both liquid and solid phase incubation systems, with the release of detected cytokines much higher than other test samples, indicating that these two antibodies can cause excessive activation leading to serious safety problems. However, BsAb 5-15 sample cannot significantly activate PBMC in both cases, indicating that 5-15 will not cause excessive activation leading to serious safety problems under the condition of non-activation of PBMC, and has less side effects.

[0135] Example 5: In vivo anti-tumor efficacy of PD-1 / 4-1BB bispecific antibody

[0136] This example describes in vivo experiments of PD-1 / 4-1BB bispecific antibodies on the functional blockade of PD-1 and the functional activation of 4-1BB. Anti-tumor efficacy was evaluated in a tumor model developed in human 4-1BB, PD-1 and PD-L1 knock-in mice.

[0137] 1) Mouse tumor-bearing model was prepared by implanting tumor cells into Balb / c-hPD1hPDL1hCD137 KI mice (Jiangsu Jicui Yekang Biotechnology Co., Ltd., Nanjing). In this assay, the murine breast cancer cell line EMT6-hPDL1 (Jiangsu Jicui Yekang Biotechnology Co., Ltd., Nanjing) was used, and the tumor cells were injected subcutaneously into 8-week-old Balb / c-hPD1hPDL1hCD137 KI mice to obtain Balb / c-hPD1hPDL1hCD137-EMT6.hPDL1 tumor-bearing mice. The tumor size was measured with a caliper, and the tumor volume was calculated by a modified ellipsoid formula: length x (width) 2 / 2. The tumor inhibition effect was evaluated by tumor inhibition rate (TGI), which was calculated as follows:

[0138] TGI = [1-(TV t -TV initial ) / (CV t -CV initial )] x 100% wherein TV t represents the tumor volume of the treatment group at each measurement; TV initial represents the tumor volume of the treatment group at the time of grouping; CV t represents the tumor volume of the control group at each measurement; CV initial represents the tumor volume of the control group at the time of grouping. On day 7 after inoculation, when the average tumor volume reached about 82.56 mm 3 , 48 mice were selected and randomly divided into 6 groups according to the tumor volume, with 8 mice in each group. The grouping day was defined as D0 day, and the drug administration was started on the grouping day (D0 day), and PBS, anti-PD-1 antibody (CL1), urelumab and each BsAb were administered to the mice by intraperitoneal injection, twice a week. The dosages of CL1 and urelumab were both 5 mg / kg, and the dosages of 2-15, 5-15 and 7-15 were all 6 mg / kg, and the tumor volume was measured twice a week. The efficacy of BsAb was evaluated by assessing the inhibition of tumor size. As can be seen from Figure 6 and Table 2, in this model, the tumor inhibition effect of 5-15 and 7-15 was significantly higher than that of anti-PD-1 control antibody CL1 and 4-1BB control antibody urelumab.

[0139] Table 2: Tumor inhibition rate (TGI) of antibodies in Balb / c-hPD1hPDL1hCD137-EMT6.hPDL1 tumor-bearing mouse model

[0140] 2) The efficacy of antibody 5-15 was evaluated by subcutaneously inoculating C57 humanized PD-1 / PD-L1 / 4-1BB mice with mouse colorectal cancer MC38 tumor cells (Shanghai Psitrain Biomedical Technology Co., Ltd.) to evaluate the efficacy of antibody 5-15. C57 mice were subcutaneously inoculated with mouse colon cancer MC38 tumor cells, 8 mice per group. When the average tumor volume reached about 200 mm 3 , the treatment was started, intraperitoneal injection, antibody 5-15 was administered at a dose of 12 mg / kg per administration, twice a week, for a total of 18 administrations. The human IgG4 control antibody was the negative control group, the anti-PD-1 antibody was CL1, and the anti-4-1BB antibody was anti-4-1BB mAb (the variable region sequence of the anti-4-1BB single-domain antibody of antibody 5-15 was the same as that of the antibody, and the sequence was shown in SEQ ID NO: 26). All samples had the same molar amount.

[0141] The results are shown in Figure 7. Before the fourth administration on the tenth day, the tumor volume of the mice in the IgG4 control group and the anti-PD-1 antibody CL1 group reached 2000 mm 3 , and euthanasia was performed, so only the day was counted for these two groups. The relative tumor inhibition rates of the groups on the tenth day are shown in Table 3. Compared with the control group, the 5-15 group had a significant difference (P < 0.05*), while the anti-PD-1 antibody group or the anti-4-1BB antibody group had no significant difference. The tumor volumes of the remaining 2 groups of mice gradually decreased with the increase in the number of administrations, and the average tumor volume of the antibody 5-15 group was reduced to nearly 0 mm 3 , while one mouse in the anti-4-1BB mAb group had tumor recurrence in the later stage. After 18 administrations, subsequent administration was stopped, and by the 58th day, only 8 mice in the antibody 5-15 group survived, and 7 mice in the anti-4-1BB mAb group survived. Antibody 5-15 showed good antitumor efficacy.

[0142] Table 3: Tumor inhibition rate (TGI) of antibodies in C57 humanized tumor-bearing mouse model

[0143] Example 6: Affinity identification

[0144] The specific binding of antibody 5-15 to human PD-1-His antigen (Yiqiao God State, 10377-H08H) or 4-1BB-His recombinant protein (Acro, 41B-H52Hc) was detected by ELISA. The ELISA experiment was performed according to the known method in the art. The anti-PD-1 antibody and the anti-4-1BB antibody were the same as in Example 5. The operation steps of the experiment of binding to PD-1 were as follows: 100 μl of PD-1-His antigen coating solution (Yiqiao God State, 10377-H08H) with a concentration of 2 μg / ml was added to each reaction well, and was coated at 4°C overnight, washed with PBST for 3 times, 200 μl of PBST containing 3% BSA was added to each reaction well, the cover plate was covered, and was incubated in a 37°C incubator for 2 hours in the dark. The prepared antibody sample (according to the highest final concentration of 50 nM, 1:6 dilution, 100 μl / well preparation, and the sample diluent was PBST containing 1% BSA) was added to the reaction well, and was incubated in a 37°C incubator for 1 hour in the dark. 300 μl of PBST was added to each well, and was washed for 5 times. 100 μl / well of secondary antibody diluent Peroxidase AffiniPure Goat Anti-Human IgG, Fcy fragment specific, 1:10000, Jackson ImmunoResearch, 109-035-008) was added to the reaction well, the cover plate was covered, and was incubated in a 37°C incubator for 1 hour in the dark. 300 μl of PBST was added to each well, and was washed for 5 times. According to 100 μl / well, TMB (Biopanda, TMB-S-001) was added, and was incubated in a 37°C incubator for 10±5 min in the dark. The enzymatic color development reaction was terminated according to the color development, and the detection wavelength of the microplate reader was set to 450 nm for reading. The experimental operation of the experiment of binding to 4-1BB was similar, the antigen coating solution was 4-1BB-His recombinant protein (Acro, 41B-H52Hc), and the antibody sample was prepared according to the highest final concentration of 200 nM, 1:3 dilution, 100 μl / well.

[0145] The results of binding to PD-1 are shown in Figure 8: the binding affinity of antibody 5-15 to PD-1 was equivalent to that of anti-PD-1 antibody CL1 (i.e. aPD-1), and the EC50 was 0.007817 and 0.005658 nM, respectively.

[0146] The binding to 4-1BB is shown in Figure 9, Urelumab has the strongest ability to bind 4-1BB, with an EC50 of about 0.235 nM, antibody 5-15 has a suitable decrease in the ability to bind 4-1BB antigen compared to anti-4-1BB mAb (i.e. a4-1BB), with EC50 of 4.12 and 0.446 nM, respectively, indicating that the structure after making a bispecific antibody further weakens the binding ability of 4-1BB, which may be a shielding effect caused by steric hindrance. Compared with the high binding ability of PD-1, the binding of antibody 5-15 to 4-1BB is significantly reduced.

[0147] The experimental operation steps for simultaneous binding to PD-1 and 4-1BB are as follows: add 100 μL of PD-1-His antigen coating solution with a concentration of 2 μg / mL to each reaction well, wash 3 times with PBST, add 200 μL of PBST containing 3% BSA to the reaction well, and incubate in a 37°C incubator for 2 hours in the dark; add the prepared antibody sample to be tested (according to the highest final concentration of 100 nM, 1:3 dilution, 100 μl / well preparation, sample diluent is PBST containing 1% BSA) to the coated plate, incubate in a 37°C incubator for 1 hour in the dark, wash 5 times with PBST, add 4-1BB antigen diluent (1 μg / ml Bio-4-1BB protein, Acro, 41B-H82E6) to the reaction well at 100 μl / well, cover the cover plate, incubate in a 37°C incubator for 1 hour in the dark, wash 5 times, add secondary antibody diluent (SA-HRP, 1:10000, Jackson Immuno Research, 016-030-084) to the reaction well at 100 μl / well, cover the cover plate, incubate in a 37°C incubator for 1 hour in the dark, add TMB (Biopanda, TMB-S-001) for color development according to 100 μL / well, place in a 37°C incubator for 10±5 min in the dark, stop the enzymatic color development reaction according to the color development, set the detection wavelength of the enzyme label instrument to 450 nm, and read the results. As shown in Figure 10, only antibody 5-15 can bind to PD-1 and 4-1BB simultaneously, with an EC50 of 1.211 nM, indicating that the construction of Fab-VHH-Fc does not hinder the binding of the bispecific antibody to the two antigens.

[0148] Further, the binding specificity and affinity of antibody 5-15 to PD-1 and 4-1BB recombinant proteins of four species of human, cynomolgus monkey, rat and mouse were detected. The sources and item numbers are as follows: human PD-1 / PDCD1, His tag recombinant protein (Yi Qiao Shenzhou, 10377-H08H); cynomolgus monkey PD-1 / PDCD1, His Tag recombinant protein (Acro, PD1-C52H5); mouse PD-1 / PDCD1, His Tag recombinant protein (Acro, PD1-M5228); rat PD-1 / PDCD1, His Tag recombinant protein (Acro, PD1-R52H2); human 4-1BB / TNFRSF9, His tag recombinant protein (Acro, 41B-H52Hc); mouse 4-1BB / TNFRSF9, His tag recombinant protein (Acro, 41B-M52H7); rat 4-1BB / TNFRSF9, His tag recombinant protein (Acro, 41B-R52H3); cynomolgus monkey / macaque 4-1BB / TNFRSF9, His tag recombinant protein (Acro, 41B-C52H4).

[0149] The results of binding to PD-1 are shown in Figure 11. Antibody 5-15 specifically binds to human and cynomolgus monkey PD-1 with comparable affinity, with EC50 values of 0.02333 and 0.02241 nM, respectively, and does not bind to mouse and rat PD-1.

[0150] The results of binding to 4-1BB are shown in Figure 12. Antibody 5-15 specifically binds to human and cynomolgus monkey 4-1BB, with EC50 values of 15.87 nM for human and 0.3768 nM for cynomolgus monkey, respectively, and does not bind to mouse and rat 4-1BB.

Claims

1. A bispecific antibody comprising a first antigen binding domain targeting PD-1 and a second antigen binding domain targeting 4-1BB, wherein, the first antigen binding domain comprises a heavy chain variable region and a light chain variable region, and the second antigen binding domain comprises a VHH; wherein the heavy chain variable region comprises a HCDR1 of SEQ ID NO: 13, a HCDR2 of SEQ ID NO: 14, and a HCDR3 of SEQ ID NO: 15, and the light chain variable region comprises a LCDR1 of SEQ ID NO: 16, a LCDR2 of SEQ ID NO: 17, and a LCDR3 of SEQ ID NO: 18; and the VHH comprises a CDR1 of SEQ ID NO: 10, a CDR2 of SEQ ID NO: 11, and a CDR3 of SEQ ID NO:

12.

2. The bispecific antibody of claim 1, the first antigen binding domain comprising a heavy chain variable region and a light chain variable region, the second antigen binding domain comprising a VHH; wherein, the heavy chain variable region comprises a sequence of SEQ ID NO: 19, a sequence with at least 80% identity to the sequence of SEQ ID NO: 19, or a sequence with one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO:

19.

3. The bispecific antibody of claim 1 or 2, the light chain variable region comprises a sequence of SEQ ID NO: 20, a sequence with at least 80% identity to the sequence of SEQ ID NO: 20, or a sequence with one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO:

20.

4. The bispecific antibody of any one of claims 1-3, the VHH comprises a sequence of SEQ ID NO: 1, a sequence with at least 80% identity to the sequence of SEQ ID NO: 1, or a sequence with one or more conservative amino acid substitutions compared to the sequence of SEQ ID NO:

1.

5. The bispecific antibody of any one of claims 1-4, wherein, the first antigen binding domain further comprises a light chain constant region and / or a heavy chain constant region.

6. The bispecific antibody of claim 5, the light chain constant region is a kappa or lambda light chain constant region.

7. The bispecific antibody of claim 5, the heavy chain constant region comprises an amino acid sequence selected from at least a part of a hinge region, CH1, CH2, CH3, or a combination thereof.

8. The bispecific antibody of claim 5, the heavy chain constant region is derived from an IgG heavy chain constant region.

9. The bispecific antibody of claim 5, the heavy chain constant region is derived from a human IgG1 heavy chain constant region or a human IgG4 heavy chain constant region.

10. A bispecific antibody comprising a first antigen binding domain targeting PD-1 and a second antigen binding domain targeting 4-1BB, the bispecific antibody comprising a first polypeptide and a second polypeptide, wherein, the first polypeptide comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant region of the first antigen binding domain, a linker peptide L1, and a VHH of the second antigen binding domain, and the second polypeptide comprises, from N-terminus to C-terminus, a heavy chain variable region and a heavy chain constant region of the first antigen binding domain; or the first polypeptide comprises, from N- to C-terminus, a light chain variable region and a light chain constant region of the first antigen binding domain, and the second polypeptide comprises, from N- to C-terminus, a heavy chain variable region of the first antigen binding domain and CH1, a linker L1, a VHH of the second antigen binding domain, a linker L2, CH2-CH3 of IgG; or the first polypeptide comprises, from N- to C-terminus, a light chain variable region and a light chain constant region of the first antigen binding domain, and the second polypeptide comprises, from N- to C-terminus, a heavy chain variable region of the first antigen binding domain and CH1, a linker L1, a VHH of the second antigen binding domain, a linker L2, CH2-CH3 of IgG; or the light chain variable region of the first antigen binding domain comprises LCDR1 as shown in SEQ ID NO: 16, LCDR2 as shown in SEQ ID NO: 17, and LCDR3 as shown in SEQ ID NO: 18, the heavy chain variable region of the first antigen binding domain comprises HCDR1 as shown in SEQ ID NO: 13, HCDR2 as shown in SEQ ID NO: 14, and HCDR3 as shown in SEQ ID NO: 15, and the VHH of the second antigen binding domain comprises CDR1 as shown in SEQ ID NO: 10, CDR2 as shown in SEQ ID NO: 11, and CDR3 as shown in SEQ ID NO: 12; each of the linkers L1 and L2 comprises (GGGGS)x, wherein x is 1, 2, 3, 4, 5, or 6; or the amino acid sequence of the linker L1 is as shown in SEQ ID NO: 2 or 8, and the amino acid sequence of the linker L2 is as shown in SEQ ID NO: 2 or 8.

11. The bispecific antibody of claim 10, the heavy chain variable region of the first antigen binding domain comprises a sequence as shown in SEQ ID NO: 19, or a sequence with at least 80% identity thereto, the light chain variable region of the first antigen binding domain comprises a sequence as shown in SEQ ID NO: 20, or a sequence with at least 80% identity thereto, the VHH of the second antigen binding domain comprises a sequence as shown in SEQ ID NO: 1, or a sequence with at least 80% identity thereto; the linker L1 comprises a sequence as shown in SEQ ID NO: 2; and the linker L2 comprises a sequence as shown in SEQ ID NO:

8.

12. A bispecific antibody comprising a first antigen binding domain targeting PD-1 and a second antigen binding domain targeting 4-1BB, the bispecific antibody comprising a first polypeptide and a second polypeptide, wherein, the first polypeptide comprises, from N- to C-terminus, a light chain of the first antigen binding domain, a linker L1, and a VHH of the second antigen binding domain, and the second polypeptide comprises, from N- to C-terminus, a heavy chain of the first antigen binding domain; or the first polypeptide comprises, from N- to C-terminus, a light chain of the first antigen binding domain, and the second polypeptide comprises, from N- to C-terminus, VH-CH1 of the first antigen binding domain, a linker L1, a VHH of the second antigen binding domain, a linker L2, CH2-CH3 of IgG; or the first polypeptide comprises, from N- to C-terminus, a light chain of the first antigen binding domain, and the second polypeptide comprises, from N- to C-terminus, VH-CH1 of the first antigen binding domain, a linker L1, a VHH of the second antigen binding domain, a linker L2, CH2-CH3 of IgG; or the first polypeptide comprises a light chain of the first antigen binding domain, the second polypeptide comprises a heavy chain of the first antigen binding domain, a connecting peptide L1, a VHH of the second antigen binding domain from N-terminus to C-terminus; the heavy chain of the first antigen binding domain comprises a sequence as set forth in SEQ ID NO: 4 or 6, the light chain of the first antigen binding domain comprises a sequence as set forth in SEQ ID NO: 3, the VH-CH1 comprises a sequence as set forth in SEQ ID NO: 7, the VHH comprises a sequence as set forth in SEQ ID NO: 1; the CH2-CH3 of the IgG comprises a sequence as set forth in SEQ ID NO: 9; each of the connecting peptides L1 and L2 comprises (GGGGS)x, wherein x is 1, 2, 3, 4, 5 or 6; preferably, the amino acid sequence of the connecting peptide L1 is as set forth in SEQ ID NO: 2 or 8, the amino acid sequence of the connecting peptide L2 is as set forth in SEQ ID NO: 2 or 8.

13. A bispecific antibody comprising a first antigen binding domain targeting PD-1 and a second antigen binding domain targeting 4-1BB, the bispecific antibody comprising a first polypeptide and a second polypeptide, wherein, the first polypeptide comprises a sequence as set forth in SEQ ID NO: 21, the second polypeptide comprises a sequence as set forth in SEQ ID NO: 4; or the first polypeptide comprises a sequence as set forth in SEQ ID NO: 3, the second polypeptide comprises a sequence as set forth in SEQ ID NO: 22; or the first polypeptide comprises a sequence as set forth in SEQ ID NO: 3, the second polypeptide comprises a sequence as set forth in SEQ ID NO:

23.

14. The bispecific antibody of any one of claims 1-13, wherein, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are connected by a disulfide bond, and two second polypeptides are connected by a disulfide bond.

15. A biological material, being: 1) a nucleic acid molecule encoding the bispecific antibody or a portion thereof according to any one of claims 1-14; 2) an expression vector comprising the nucleic acid molecule of 1); 3) a host cell comprising the nucleic acid molecule of 1) or the expression vector of 2).

16. A method for preparing the bispecific antibody according to any one of claims 1-14, being: 1) a chemical synthesis method: preparing according to the amino acid sequence of the bispecific antibody according to any one of claims 1-14 by chemical synthesis; 2) a biological synthesis method: culturing a host cell containing a nucleic acid encoding the bispecific antibody according to any one of claims 1-14 to express the specific antibody according to any one of claims 1-14.

17. The method for preparing according to claim 16, further comprising isolating the antibody from the obtained reactants or culture; and purifying the antibody.

18. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1-14.

19. The pharmaceutical composition according to claim 18, further comprising a pharmaceutically acceptable excipient, such as a pharmaceutically acceptable excipient, diluent or carrier.

20. Use of the bispecific antibody according to any one of claims 1 to 14 or of the biological material according to claim 15 or of the pharmaceutical composition according to claim 18 or 19 for the manufacture of a medicament.

21. Use according to claim 20, wherein the medicament is for the treatment or prevention of a tumor or a viral infection.

22. Use according to claim 21, wherein the tumor is melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colon cancer, prostate cancer, gastric cancer, renal cancer, bladder cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, thyroid cancer, hepatocellular carcinoma or mesothelioma, and wherein the viral infection is a hepatitis C or hepatitis B infection.

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