Anti-PD-L1 and 4-1BB Bispecific Antibodies and Their Applications

Abstract

This invention belongs to the field of biomedicine and provides a bispecific antibody and its applications. The bispecific antibody comprises a first antigen-binding domain targeting PD-L1 and a second antigen-binding domain targeting 4-1BB. The antibody of this invention retains the activity of anti-PD-L1 antibodies, achieving a balance between activity and safety, and possesses excellent drug development potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to anti-PD-L1 and 4-1BB bispecific antibodies, their preparation, composition and application. Background Technology

[0002] The interaction between PD-L1 and its receptor PD-1 generates inhibitory signals that play a crucial role in tumor immune escape. This immunosuppression mainly occurs in the tumor microenvironment. Blocking the inhibitory signal of PD-1 using PD-1 antibodies or PD-L1 antibodies has achieved great success in the clinical treatment of tumors. However, many patients still do not respond to this therapy, develop resistance, or are intolerant to it.

[0003] 4-1BB, also known as CD137, is composed of four cysteine-rich domains (CRDs) and belongs to the tumor necrosis factor receptor superfamily. It plays a crucial role in T cell activation-driven immune responses, primarily promoting T cell proliferation, gaining effector function, enhancing immune memory, and inhibiting activation-induced cell death. Agonistic antibodies targeting 4-1BB have shown potential as monotherapy or in combination therapy in mouse tumor models. However, due to toxicity and lack of efficacy, 4-1BB agonists, whether used as monotherapy or in combination therapy, have failed to elicit sufficient responses in clinical practice. Currently, no 4-1BB agonist antibodies have been approved for marketing.

[0004] The combination of two antibodies, a 4-1BB agonist and a PD-L1 antagonist, such as the combination of Utomilumab (a 4-1BB agonist) and Avelumab (a PD-L1 antagonist), has been used in clinical trials for the treatment of solid tumors. Nevertheless, there remains a huge demand for multispecific antibodies that simultaneously bind PD-L1 and 4-1BB. Summary of the Invention

[0005] This invention provides a bispecific antibody targeting PD-L1 and 4-1BB, offering new options for related drugs.

[0006] In a first aspect, the present invention provides a bispecific antibody comprising a first antigen-binding domain targeting PD-L1 and a second antigen-binding domain targeting 4-1BB.

[0007] In one embodiment, the first antigen-binding domain targeting PD-L1 includes LCDR1 as shown in SEQ ID NO: 31, LCDR2 as shown in SEQ ID NO: 32, LCDR3 as shown in SEQ ID NO: 33, HCDR1 as shown in SEQ ID NO: 34, HCDR2 as shown in SEQ ID NO: 35, and HCDR3 as shown in SEQ ID NO: 36; the second antigen-binding domain targeting 4-1BB includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in any one of SEQ ID NO: 22-26, and CDR3 as shown in any one of SEQ ID NO: 27-30.

[0008] In one 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 VHH. In one embodiment, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO: 34, HCDR2 as shown in SEQ ID NO: 35, and HCDR3 as shown in SEQ ID NO: 36; the light chain variable region comprises LCDR1 as shown in SEQ ID NO: 31, LCDR2 as shown in SEQ ID NO: 32, and LCDR3 as shown in SEQ ID NO: 33; and the VHH comprises CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in any one of SEQ ID NO: 22-26, and CDR3 as shown in any one of SEQ ID NO: 27-30.

[0009] In one embodiment, the VHH includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in any one of SEQ ID NO: 22-24, and CDR3 as shown in SEQ ID NO: 27.

[0010] In one embodiment, the VHH includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in SEQ ID NO: 22, and CDR3 as shown in SEQ ID NO: 28.

[0011] In one embodiment, the VHH includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in SEQ ID NO: 25, and CDR3 as shown in SEQ ID NO: 29.

[0012] In one embodiment, the VHH includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in SEQ ID NO: 26, and CDR3 as shown in SEQ ID NO: 30.

[0013] In one 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 shown in SEQ ID NO: 7, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 7; and / or the light chain variable region comprises a sequence as shown in SEQ ID NO: 9, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 9, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 9; and / or the VHH comprises a sequence as shown in any one of SEQ ID NO: 1-6, or a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO: 1-6, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 1-6.

[0014] In one embodiment, the heavy chain variable region comprises the sequence shown in SEQ ID NO:7, the light chain variable region comprises the sequence shown in SEQ ID NO:9, and the VHH comprises the sequence shown in any one of SEQ ID NO:1-6. In one embodiment, the VHH comprises the sequence shown in SEQ ID NO:3 or 6.

[0015] In one embodiment, the first antigen-binding domain and / or the second antigen-binding domain further include a light chain constant region and / or a heavy chain constant region.

[0016] In one embodiment, the first antigen-binding domain and / or the second antigen-binding domain further include an Fc region, or further include a light chain constant region.

[0017] In one embodiment, the VHH is linked to the heavy chain variable region, light chain variable region, light chain constant region, heavy chain constant region, or Fc region via a linker peptide.

[0018] In one embodiment, the bispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide includes a light chain variable region and a light chain constant region of a first antigen-binding domain from its N-terminus to its C-terminus. The second polypeptide includes a heavy chain variable region and a heavy chain constant region of the first antigen-binding domain from its N-terminus to its C-terminus. The third polypeptide includes the VHH region of the second antigen-binding domain, a linker peptide, and the Fc region of IgG from its N-terminus to its C-terminus. Alternatively, The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide, from its N-terminus to its C-terminus, comprises a light chain variable region, a light chain constant region, a linker peptide, and a VHH region of the second antigen-binding domain. The second polypeptide, from its N-terminus to its C-terminus, comprises a heavy chain variable region and a heavy chain constant region of the first antigen-binding domain. Alternatively... The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide includes a light chain variable region and a light chain constant region of a first antigen-binding domain from the N-terminus to the C-terminus. The second polypeptide includes a heavy chain variable region, a heavy chain constant region, a linker peptide, and a VHH of the second antigen-binding domain from the N-terminus to the C-terminus. Alternatively... The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide includes a light chain variable region and a light chain constant region of a first antigen-binding domain from the N-terminus to the C-terminus. The second polypeptide includes a heavy chain variable region of the first antigen-binding domain and CH1 (VH-CH1), a linker peptide, VHH of the second antigen-binding domain, a linker peptide, and the Fc region of IgG from the N-terminus to the C-terminus; or... The bispecific antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, a second antigen-binding domain VHH, a linker peptide 1, an Fc region of IgG, a linker peptide 2, and a first antigen-binding domain scFv. The scFv comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the first antigen-binding domain, a linker peptide 3, and a light chain variable region of the first antigen-binding domain; or the scFv comprises, from the N-terminus to the C-terminus, a light chain variable region of the first antigen-binding domain, a linker peptide 3, and a heavy chain variable region of the first antigen-binding domain. Wherein, the light chain variable region of the first antigen-binding domain includes LCDR1 as shown in SEQ ID NO: 31, LCDR2 as shown in SEQ ID NO: 32, and LCDR3 as shown in SEQ ID NO: 33; the heavy chain variable region of the first antigen-binding domain includes HCDR1 as shown in SEQ ID NO: 34, HCDR2 as shown in SEQ ID NO: 35, and HCDR3 as shown in SEQ ID NO: 36; and the VHH includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in any one of SEQ ID NO: 22-26, and CDR3 as shown in any one of SEQ ID NO: 27-30.

[0019] In one embodiment, the light chain variable region of the first antigen-binding domain comprises a sequence as shown in SEQ ID NO: 9, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 9, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 9; and / or the heavy chain variable region of the first antigen-binding domain comprises a sequence as shown in SEQ ID NO: 7, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 7; and / or the VHH comprises a sequence as shown in any one of SEQ ID NO: 1-6, or a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO: 1-6, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 1-6.

[0020] In one embodiment, the linker peptides 1-3 independently comprise 4 to 30 amino acids. In one embodiment, the linker peptides 1-3 independently comprise 4 to 24 amino acids selected from G and S. In one embodiment, the linker peptides 1-3 independently comprise (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6.

[0021] In one embodiment, the linker peptides 1-3 are independently selected from the sequences shown in SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 19.

[0022] In one embodiment, the light chain constant region is a κ or λ light chain constant region or a variant thereof. In one embodiment, the light chain constant region is a κ light chain constant region.

[0023] In one embodiment, the heavy chain constant region may comprise an amino acid sequence selected from at least a portion of the hinge region, CH1, CH2, CH3, or combinations thereof, or variants thereof. In one embodiment, the heavy chain constant region or Fc region is the heavy chain constant region or Fc region of IgG (e.g., IgG1, IgG2, IgG3, IgG4), or a variant thereof. In one embodiment, the heavy chain constant region or Fc region is the heavy chain constant region or Fc region of human IgG1, or a variant thereof. In one embodiment, the heavy chain constant region or Fc region is the heavy chain constant region or Fc region of human IgG4, or a variant thereof. In one embodiment, when the C-terminus of the heavy chain constant region or Fc region is linked to a VHH or other antibody fragment that specifically binds to 4-1BB, the lysine (K) at the C-terminus of the heavy chain constant region can be changed to alanine (A).

[0024] In one embodiment, the variant of the heavy chain constant region or Fc region has one or more amino acid modifications, such as substitution, deletion, or insertion. In one embodiment, the variant of the heavy chain constant region or Fc region may have altered (i.e., increased or decreased) antibody-dependent cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In one embodiment, the variant of the heavy chain constant region or Fc region comprises one or more of the mutations N297A, L234A / F234A, L235A, L235E, and G237A (according to Eu numbering). In one embodiment, the variant of the heavy chain constant region or Fc region comprises E345R or S440 Y (according to Eu numbering). In one embodiment, the variant of the heavy chain constant region or Fc region comprises the N297A mutation (according to Eu numbering).

[0025] In one embodiment, the bispecific antibody of the present invention utilizes a "knobs-into-holes" technique (see patent US8216805B2). This technique modifies the interface between different chains of the bispecific antibody of the present invention to promote proper association of the chains. Typically, this technique involves introducing "bumps" ("knobs") at the interface of one chain and corresponding "holes" ("holes") at the interface of the other chain to which it is to pair, allowing the bumps to be placed within the holes. Bumps can be constructed by replacing the amino acid side chains at the interface of the CH3 domain of the heavy chain constant region or Fc region of one chain with larger side chains (e.g., amino acid substitution T366W (according to Eu numbering)). By replacing large amino acid side chains with smaller side chains (e.g., amino acid substitutions of T366S, L368A, and Y407V (according to Eu numbering)), a compensatory hole of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant region or Fc region of the other chain to be paired. In one embodiment, the heavy chain constant region or Fc region of one polypeptide chain contains Y349C, T366S, L368A, and Y407V, and the heavy chain constant region or Fc region of the other polypeptide chain contains S354C and T366W, forming a stable "knob-in-hole" association. In one embodiment, the variant of the heavy chain constant region or Fc region comprises an IgG1 subtype with one or more of the following amino acid mutations: Y349C, S354C, T366W, T366S, L368A, and Y407V (according to Eu numbering).

[0026] In one embodiment, the heavy chain constant region is the heavy chain constant region of IgG1. In one embodiment, the heavy chain constant region of IgG1 has one or more of the following mutations: N297A, Y349C, T366S, L368A, and Y407V mutations (according to Eu numbering). In one embodiment, the heavy chain constant region of IgG1 has N297A, Y349C, T366S, L368A, and Y407V mutations (according to Eu numbering).

[0027] In one embodiment, the Fc region of IgG is the Fc region of IgG1. In one embodiment, the Fc region of IgG1 has one or more of the following mutations: N297A, S354C, and T366W (according to Eu numbering). In one embodiment, the Fc region of IgG1 has the N297A mutation, S354C, and T366W (according to Eu numbering). In one embodiment, the Fc region of IgG1 comprises the sequence shown in SEQ ID NO: 12.

[0028] In one embodiment, the bispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide comprises a light chain with a first antigen-binding domain from the N-terminus to the C-terminus, the second polypeptide comprises a heavy chain with the first antigen-binding domain from the N-terminus to the C-terminus, and the third polypeptide comprises a VHH of the second antigen-binding domain, a linker peptide L1, and an Fc region of IgG1 from the N-terminus to the C-terminus; wherein the light chain of the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 10, the heavy chain comprises the sequence shown in SEQ ID NO: 11, the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6, and the Fc region of IgG1 comprises the sequence shown in SEQ ID NO: 12.

[0029] In one embodiment, the linker peptide L1 comprises 4 to 30 amino acids. In one embodiment, the linker peptide L1 comprises 4 to 24 amino acids selected from G and S. In one embodiment, the linker peptide L1 comprises (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6. In one embodiment, the amino acid sequence of the linker peptide L1 is as shown in SEQ ID NO: 8.

[0030] In one embodiment, the bispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide is an anti-PD-L1 antibody light chain, the second polypeptide is an anti-PD-L1 antibody heavy chain, and the third polypeptide consists of a VHH of anti-4-1BB, a linker peptide L1, and an Fc region of IgG1 from the N-terminus to the C-terminus. The amino acid sequence of the anti-PD-L1 antibody light chain is shown in SEQ ID NO: 10, the amino acid sequence of the anti-PD-L1 antibody heavy chain is shown in SEQ ID NO: 11, the amino acid sequence of the VHH of anti-4-1BB is shown in SEQ ID NO: 3 or 6, the amino acid sequence of the linker peptide L1 is shown in SEQ ID NO: 8, and the amino acid sequence of the Fc region of IgG1 is shown in SEQ ID NO: 12.

[0031] In one embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a light chain of a first antigen-binding domain, a linker peptide L2, and a VHH of a second antigen-binding domain, and the second polypeptide comprises, from the N-terminus to the C-terminus, a heavy chain of the first antigen-binding domain; the light chain comprises the sequence shown in SEQ ID NO: 10, the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6, and the heavy chain comprises the sequence shown in SEQ ID NO: 14 or 18.

[0032] In one embodiment, the linker peptide L2 comprises 4 to 30 amino acids. In one embodiment, the linker peptide L2 comprises 4 to 24 amino acids selected from G and S. In one embodiment, the linker peptide L2 comprises (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6. In one embodiment, the amino acid sequence of the linker peptide L2 is as shown in SEQ ID NO:13.

[0033] In one embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide consists of a light chain of an anti-PD-L1 antibody, a linker peptide L2, and a VHH of an anti-4-1BB antibody from the N-terminus to the C-terminus. The second polypeptide is a heavy chain of the anti-PD-L1 antibody. The amino acid sequence of the light chain is shown in SEQ ID NO: 10. The amino acid sequence of the linker peptide L2 is shown in SEQ ID NO: 13. The amino acid sequence of the VHH is shown in SEQ ID NO: 3 or 6. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 14.

[0034] In one embodiment, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond.

[0035] In one embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a light chain of a first antigen-binding domain from the N-terminus to the C-terminus, and the second polypeptide comprises a heavy chain of the first antigen-binding domain, a linker peptide, and a VHH of the second antigen-binding domain from the N-terminus to the C-terminus, wherein the light chain of the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 10, the heavy chain comprises the sequence shown in SEQ ID NO: 18, and the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6.

[0036] In one embodiment, the linker peptide comprises 4 to 30 amino acids. In one embodiment, the linker peptide comprises 4 to 24 amino acids selected from G and S. In one embodiment, the linker peptide comprises (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6. In one embodiment, the amino acid sequence of the linker peptide is as shown in SEQ ID NO: 16 or 19.

[0037] In one embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises a light chain of an anti-PD-L1 antibody from the N-terminus to the C-terminus. The second polypeptide comprises a heavy chain of an anti-PD-L1 antibody, a linker peptide L3, and a VHH of an anti-4-1BB antibody from the N-terminus to the C-terminus. The amino acid sequence of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 10. The amino acid sequence of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 18. The amino acid sequence of the VHH of the anti-4-1BB antibody is shown in any one of SEQ ID NO: 1-6. The amino acid sequence of the linker peptide L3 is shown in SEQ ID NO: 16.

[0038] In one embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises a light chain of an anti-PD-L1 antibody from the N-terminus to the C-terminus, and the second polypeptide comprises a heavy chain of an anti-PD-L1 antibody, a linker peptide L4, and a VHH of an anti-4-1BB antibody from the N-terminus to the C-terminus. The amino acid sequence of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 10, the amino acid sequence of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 18, the amino acid sequence of the VHH of the anti-4-1BB antibody is shown in SEQ ID NO: 3 or 6, and the amino acid sequence of the linker peptide L4 is shown in SEQ ID NO: 19.

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

[0040] In one embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a light chain of a first antigen-binding domain from the N-terminus to the C-terminus, and the second polypeptide comprises VH-CH1 of the first antigen-binding domain, a linker peptide L3, VHH of the second antigen-binding domain, a linker peptide L1, and an Fc region of IgG4 from the N-terminus to the C-terminus; wherein the light chain of the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 10, the VH-CH1 comprises the sequence shown in SEQ ID NO: 20, the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6, and the Fc region of IgG4 comprises the sequence shown in SEQ ID NO: 15 or 21.

[0041] In one embodiment, the linker peptide L1 or L3 independently comprises 4 to 30 amino acids. In one embodiment, the linker peptide L1 or L3 independently comprises 4 to 24 amino acids selected from G and S. In one embodiment, the linker peptide L1 or L3 independently comprises (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6. In one embodiment, the amino acid sequence of the linker peptide L1 is as shown in SEQ ID NO: 8. In one embodiment, the amino acid sequence of the linker peptide L3 is as shown in SEQ ID NO: 16.

[0042] In one embodiment, the bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide is a light chain of an anti-PD-L1 antibody, and the second polypeptide consists of a VH-CH1 domain of a first antigen-binding domain, a linker peptide L3, a VHH domain of a second antigen-binding domain, a linker peptide L1, and an Fc region of IgG4 from the N-terminus to the C-terminus. The amino acid sequence of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 10, the amino acid sequence of the VH-CH1 is shown in SEQ ID NO: 20, the amino acid sequence of the linker peptide L3 is shown in SEQ ID NO: 16, the amino acid sequence of the VHH of the anti-4-1BB antibody is shown in SEQ ID NO: 3 or 6, the amino acid sequence of the linker peptide L1 is shown in SEQ ID NO: 8, and the amino acid sequence of the Fc region of IgG4 is shown in SEQ ID NO: 21.

[0043] In one embodiment, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond.

[0044] In one embodiment, the bispecific antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, a second antigen-binding domain VHH, a linker peptide L1, an Fc region of IgG4, a linker peptide L3, and a first antigen-binding domain scFv; wherein, the VHH comprises a sequence as shown in any one of SEQ ID NO: 1-6, the amino acid sequence of the Fc region of IgG4 is shown in SEQ ID NO: 15, and the scFv of the first antigen-binding domain comprises a sequence as shown in SEQ ID NO: 17.

[0045] In one embodiment, the linker peptides L1 and / or L3 comprise 4 to 30 amino acids. In one embodiment, the linker peptides L1 and / or L3 comprise 4 to 24 amino acids selected from G and S. In one embodiment, the linker peptides L1 and / or L3 comprise (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6. In one embodiment, the amino acid sequence of the linker peptide L1 is as shown in SEQ ID NO: 8. In one embodiment, the amino acid sequence of the linker peptide L3 is as shown in SEQ ID NO: 16.

[0046] In one embodiment, the bispecific antibody comprises a polypeptide consisting of a VHH for anti-4-1BB, a linker peptide L1, an Fc region of IgG4, a linker peptide L3, and an scFv for anti-PD-L1 from the N-terminus to the C-terminus, wherein the VHH comprises the sequence shown in SEQ ID NO: 3 or 6, the amino acid sequence of the linker peptide L1 is shown in SEQ ID NO: 8, the amino acid sequence of the Fc region of IgG4 is shown in SEQ ID NO: 15, the amino acid sequence of the linker peptide L3 is shown in SEQ ID NO: 16, and the amino acid sequence of the scFv for anti-PD-L1 is shown in SEQ ID NO: 17.

[0047] In one embodiment, the bispecific antibody comprises two identical polypeptides linked by a disulfide bond.

[0048] In a second aspect, the present invention provides a biomaterial, which is: 1) A nucleic acid molecule encoding the bispecific antibody or a portion thereof of the present invention; 2) An expression vector comprising the nucleic acid molecule of the present invention; 3) Host cell containing the nucleic acid molecule or expression vector of the present invention.

[0049] In a third aspect, a method for preparing the bispecific antibody of the present invention is provided, comprising: 1) Chemical synthesis method: Based on the amino acid sequence of the bispecific antibody provided in this invention, it is prepared by chemical synthesis; 2) Biosynthesis method: Cultivate the host cells provided by the present invention to express the bispecific antibodies of the present invention.

[0050] In one embodiment, the method further includes isolating antibodies from the obtained culture and purifying the antibodies.

[0051] In a fourth aspect, a pharmaceutical composition is provided comprising the bispecific antibody of the present invention. In one embodiment, a pharmaceutically acceptable excipient, such as a pharmaceutically acceptable excipient, diluent, or carrier, is also included.

[0052] In a fifth aspect, the invention provides the use of the bispecific antibody, biomaterial, or pharmaceutical composition in the preparation of a medicament. In one embodiment, the medicament is used to treat or prevent tumors or viral infections. In one embodiment, the tumors include 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, bile duct cancer, thyroid cancer, hepatocellular carcinoma, mesothelioma, etc., and the viral infections include hepatitis C, hepatitis B, etc.

[0053] In a sixth aspect, the invention provides the use of the bispecific antibody, biomaterial, or pharmaceutical composition in the treatment or prevention of tumors or viral infections. In one embodiment, the tumors include 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, bile duct cancer, thyroid cancer, hepatocellular carcinoma, mesothelioma, etc., and the viral infections include hepatitis C, hepatitis B, etc.

[0054] In a seventh aspect, a method for treating or preventing tumors or viral infections is provided, comprising administering an effective amount of the bispecific antibody or biomaterial or pharmaceutical composition of the present invention to an individual in need. In one embodiment, 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, bile duct cancer, thyroid cancer, hepatocellular carcinoma, mesothelioma, etc., and the viral infection includes hepatitis C, hepatitis B, etc.

[0055] The bispecific antibody of this invention has an IgG-like structure, wherein the anti-PD-L1 antibody is in Fab form or a conventional IgG antibody, and 4-1BB is a VHH single-domain antibody, employing various forms such as Fab-VHH-Fc or IgG-VHH. Fc represents the sequence of natural IgG (e.g., IgG1 or IgG4), preserving the natural antibody structure and weakening binding to FcγR and C1q, thus reducing ADCC and CDC effects. Simultaneously, the PD-L1 binding domain in this structure remains unchanged compared to the parent antibody, preserving the activity of the anti-PD-L1 antibody. The bispecific antibody of this invention exhibits a PD-L1 affinity at least 10 times, and potentially 100 times, 150 times, or 200 times, greater than that of 4-1BB, thereby achieving a balance between activity and safety. Attached Figure Description

[0056] Figure 1 Results of the binding experiment between the 4-1BB-VHH-Fc fusion protein and cells expressing 4-1BB; Figure 2 and Figure 3 Competition experiment for binding of 4-1BB-VHH-Fc fusion protein to 4-1BB ligand; Figure 4A Comparison of activation of the 293T-4-1BB reporter system by different fusion proteins in the presence of cross-linked antibodies, wherein the cross-linked antibody was anti-human IgG (Fc specific). Figure 4B Comparison of the activation of reporter systems after different fusion proteins were immobilized and cross-linked in 96-well plates; Figure 5A Schematic diagrams of the structures of different bispecific antibodies (BsAb); Figure 5B Different bispecific antibodies were identified by PAGE electrophoresis, with 105 being the INBRX-105 control. Figure 6A PD-L1-4-1BB BsAb-mode 7 cell binding assay; Figure 6B and 6C Detection of PD-L1 activity in different BsAbs; Figure 7 A~G: 4-1BB activity detection of BsAbs with different structures in mode 7; Figure 8A ~C: 4-1BB activity detection of BsAbs with different structures in modes 1, 3, and 4; Figure 9 A~G: Detection of 4-1BB activity of BsAbs with different structures at positions 2, 5, and 7 of sequences 6 and 15 of VHH; Figure 10 A~B and Figure 11 : Co-culture experiment of human PBMCs with cells expressing or not expressing PD-L1; Figure 12A ~H: In vitro cytokine release assay; in the figure, aCD3 represents anti-CD3 antibody, Ure represents ursulcimbrine antibody, and medium represents medium control. Figure 13A Efficacy assay of different BsAbs with structures 2, 5, and 7 in sequences 6 and 15 in Balb / c tumor-bearing mice; comparison of tumor inhibition rates of different BsAbs in Balb / c-hCD137-CT26.WT tumor-bearing mouse model; where Ctrl represents hIgG4. Figure 13B Efficacy assay of different BsAbs with structures 2, 5, and 7 of sequence 15 in C57BL / 6 tumor-bearing mice; comparison of tumor inhibition rates of different BsAbs in C57BL / 6-hCD137-B16F10-hPD-L1 tumor-bearing mouse models. Figure 13C Tumor-bearing mice rechallenge experiment. Detailed Implementation

[0057] the term Unless otherwise stated, each of the following terms shall have the meaning described below.

[0058] definition The term “a” refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

[0059] As used herein, the terms “comprising” or “including” mean that antibodies, compositions, or methods include the listed elements, such as components or steps, but do not exclude others. “Substantially composed of” means that antibodies, compositions, or methods exclude other elements that fundamentally affect the characterization of the composition, but do not exclude elements that do not substantially affect the antibody, composition, or method. “Composed of” means excluding elements not specifically listed.

[0060] An "antibody" is a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody ("immunoglobulin, IgG") and any antigen-binding fragment thereof, or a single chain thereof, such as an antigen-binding domain. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Examples of antibodies, including but not limited to, include the heavy chain, the light chain, its ligand-binding region, complementarity-determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), framework region (FR), or any portion thereof, or at least a portion of the binding protein. The CDR region includes the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3). 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 (e.g., γ1-γ4). The properties of this chain determine the "type" of immunoglobulin, namely IgG, IgM, IgA, IgD, or IgE, and subclasses (isotypes), such as IgG which can have subclasses such as IgG1, IgG2, IgG3, and IgG4. All types of immunoglobulins are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type, whose two heavy chains and two light chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region.

[0061] Those skilled in the art will understand that the CDR region of an antibody is responsible for the antibody's binding specificity to the antigen. Given the known sequences of the antibody's heavy and light chain variable regions, several methods exist for determining the antibody's CDR region, including the Kabat, IMGT, Chothia, and AbM numbering systems. However, the application of each definition of the CDR for an antibody or its variants will be within the scope of the terminology defined and used herein. Given the amino acid sequence of the antibody's variable region, those skilled in the art can generally determine which residues are contained in a particular CDR without relying on any experimental data outside of the sequence itself.

[0062] The "heavy chain constant region" includes at least one of the following: CH1 domain, hinge domain (e.g., upper, middle, and / or lower hinge regions), CH2 domain, CH3 domain, or variants or fragments. 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 may include a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4.

[0063] The "light chain constant region" consists of a single domain CL. In some implementations, the light chain constant region may be derived from either the κ constant region domain or the λ constant region domain.

[0064] The antibodies disclosed in this invention can be derived from any animal, including but not limited to fish, birds, and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse, or chicken-derived antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

[0065] The term "bispecific antibody" refers to an antibody (including the antibody or its antigen-binding fragment, such as single-chain antibodies and nanobodies) that can specifically bind to two different antigens or two different antigenic epitopes of the same antigen. Based on the integrity of the IgG molecule, it can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Based on the number of antigen-binding regions, it can be divided into bivalent, trivalent, tetravalent, or more valent bispecific antibodies. Based on whether the structure is symmetrical, it can be divided into symmetrical and asymmetrical bispecific antibodies. Fragment-type bispecific antibodies, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. They have lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively larger molecular weight. The Fc fragment helps in antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life. Bispecific antibody structural models include 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, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery, volume 18, pages 585–608 (2019); Chen S1 et al., J...) Immunol Res. 2019 Feb11;2019:4516041).

[0066] VHH, or "single-domain antibody" or "sdAb," refers to an antigen-binding fragment containing only a single antibody variable domain. A standalone sdAb can bind to an antigen. In some cases, sdAbs are engineered from camel-derived HcAbs, whose heavy chain variable domain is referred to herein as "VHH" (heavy chain variable domain of a heavy chain antibody). Some VHHs are also called nanobodies. A VHH has the following structure from the N-terminus to the C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0067] As used herein, the twenty common amino acids and their abbreviations follow conventional usage. Stereoisomers of the twenty common amino acids (e.g., D-amino acids), non-natural amino acids (such as α- and α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be components applicable to the polypeptides disclosed herein. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide representation used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxyl-terminal direction, consistent with standard usage and convention. 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 (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), valine (Val, V), etc.

[0068] "Identity or sequence identity" of a polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that, when sequence alignment is performed, that percentage of bases (or amino acids) are identical in the two compared sequences. This alignment and identity percentage or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignment is preferably performed using default parameters. One alignment procedure is BLAST using default parameters, such as BLASTN and BLASTP, which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides with the same or similar biological activities.

[0069] Minor variations in the amino acid sequence of antibody or immunoglobulin molecules are covered within this disclosure, provided that the amino acid sequence identity is maintained at least 90%, such as at least 92%, 95%, 98%, or 99%. In some embodiments, the variation is a conserved amino acid substitution. A conserved amino acid substitution is a substitution that occurs within the relevant amino acid family in its side chain. Genetically encoded amino acids are broadly classified into the following categories: (1) acidic amino acids, such as aspartate and glutamate; (2) basic amino acids, such as lysine, arginine, and histidine; (3) nonpolar amino acids, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, such as glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine from the aliphatic-hydroxy family; (ii) asparagine and glutamine from the amide family; (iii) alanine, valine, leucine, and isoleucine from the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine from the aromatic family. In some embodiments, the conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. For example, it is reasonable to predict that the substitution of leucine with isoleucine or valine alone, the substitution of aspartic acid with glutamate, the substitution of threonine with serine, or the substitution of one amino acid with a structurally related amino acid, will not have a significant impact on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the binding site. Whether an amino acid change results in a functional peptide can be readily determined by measuring the specific activity of the polypeptide derivative. This measurement is described in detail herein. Fragments or analogues of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art.

[0070] In some embodiments, amino acid substitution has the following effects: (1) reducing sensitivity to proteolytic activity, (2) reducing sensitivity to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity, or (5) imparting or improving other physicochemical or functional properties to such analogs. Analogs may include various mutant proteins with sequences different from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conserved amino acid substitutions) may be made in the naturally occurring sequence (preferably in the polypeptide portion outside the domains forming intermolecular contacts). Conserved amino acid substitutions should not significantly alter the structural characteristics of the parental sequence (e.g., the substituted amino acid should not tend to disrupt the helical structure present in the parental sequence or disrupt other types of secondary structures characterizing the parental sequence).

[0071] The term "polypeptide" is intended to encompass both the singular and plural forms of "polypeptide" 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 imply a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and "polypeptide" can be used in place of any of the foregoing terms or interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide 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. Polypeptides can be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence; they can be produced in any manner, including chemical synthesis.

[0072] The antibody and antigen-binding fragments disclosed in this invention include modified derivatives, i.e., modified by covalently linking any type of molecule to the antibody or antigen-binding fragment, wherein the covalent link does not prevent the antibody or antigen-binding fragment from binding to the epitope. The antibody or antigen-binding fragment can be glycosylated, acetylated, polyethylene glycol-modified, phosphorylated, amidated, derivatized by known protecting / blocking groups, cleaved by proteolytic hydrolysis, linked to cellular ligands or other proteins, etc. Any of the numerous chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin.

[0073] In some implementations, the antibody or antigen-binding fragment may be conjugated to a therapeutic agent, drug precursor, peptide, protein, enzyme, virus, lipid, biological response modifier, oligonucleotide (e.g., siRNA), or PEG.

[0074] As used herein, the terms “specific binding” or “immune response” refer to a non-covalent interaction between an immunoglobulin molecule and one or more antigenic determinants of its target antigen. The strength or affinity of an immunobinding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD represents a larger affinity. The immunobinding properties of a selected peptide can be quantified using methods well known in the art. One such method measures the rates of formation and dissociation of antigen-binding sites / antigen complexes, where those rates depend on the concentration of the complex coupler, the affinity of the interaction, and other geometric parameters that equally affect the rate. Both the “binding rate constant” (kon) and the “dissociation rate constant” (koff) can be determined by calculating the concentration and the actual association and dissociation rates (see Malmqvist, M., Nature 361:186-87 (1993)). The koff / kon ratio eliminates parameters that are not related to affinity and is equal to the equilibrium dissociation constant KD (see Davies et al. (1990) Annual Rev Biochem 59:439-473). Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR) assays, flow cytometry binding assays, or similar assays known to those skilled in the art.

[0075] The term "isolated" as used in this invention for cells, nucleic acids, peptides, antibodies, etc., such as "isolated" DNA, RNA, and peptides, refers to molecules isolated from one or more other components such as DNA or RNA, respectively, in the cellular natural environment. The term "isolated" as used in this invention also refers to nucleic acids or peptides that, when produced by recombinant DNA technology, are substantially free of cellular material, viral material, or cell culture medium, or chemical precursors or other chemicals used in chemical synthesis. Furthermore, "isolated nucleic acids" is intended to include nucleic acid fragments that are not naturally present and will not exist in their natural state. The term "isolated" is also used in this invention to refer to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides are intended to include purified and recombinant peptides. Isolated peptides, etc., are typically prepared by at least one purification step. In one or more embodiments, the purity of the isolated nucleic acids, peptides, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including extreme values) between any two of these values, or any value therein.

[0076] When the term “encoding” is applied to polynucleotides, it refers to a polynucleotide called the “encoding” polypeptide that, in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce the polypeptide and / or fragments thereof.

[0077] "Approximately" refers to a typical error range for the corresponding value that is readily known to those skilled in the art. In some embodiments, "approximately" as used herein refers to the described value and its range of ±10%, ±5%, or ±1%.

[0078] "EC50" stands for half-maximal concentration, which refers to the concentration that produces a 50% maximum effect.

[0079] "Treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing, mitigating, improving, or stopping adverse physiological changes or disorders, such as disease progression, including but not limited to the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, mitigation, reduction, or disappearance of the disease state (whether partial or complete), and prolongation of expected survival without treatment. Patients requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those needing prevention of the condition or disorder, as well as those who can or are expected to benefit from the application of the antibody or pharmaceutical composition disclosed in this invention for detection, diagnostic procedures, and / or treatment.

[0080] The term "tumor" refers to or is intended to describe a physiological state in mammals characterized by uncontrolled cell growth, including both benign and malignant tumors such as cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, or leukemia. More specific examples of such cancers include, but are not limited to, colorectal 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 carcinoma, nasopharyngeal carcinoma, laryngeal cancer, lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, glioblastoma, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, rectal cancer, prostate cancer, vulvar cancer, testicular cancer, squamous cell carcinoma, small cell lung cancer, cervical cancer, bladder cancer, retinoblastoma, neuroblastoma, mesothelioma, oral epithelioid carcinoma, choriocarcinoma, and head and neck cancer.

[0081] The effective dosage and treatment regimen for a specific patient will depend on various factors, including the specific antibody, antigen-binding fragment or derivative used, the patient's age and weight, general health condition, sex and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors will be determined by a healthcare professional, including those skilled in the art. The dosage used can be determined using pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the antibody of the present invention is administered to the patient at a dose ranging from 0.01 mg / kg to 100 mg / kg of patient body weight per administration. In some embodiments, it is administered once weekly or monthly.

[0082] The term "patient" refers to any mammal requiring diagnosis, prognosis, or treatment, including but not limited to humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc. In some implementations, the patient is a human patient.

[0083] The term "pharmaceuticalally acceptable" refers to a substance approved by a government regulatory agency or listed in a recognized pharmacopoeia for use in animals, and especially in humans. Furthermore, "pharmaceuticalally acceptable excipients" generally refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0084] The term "excipient" refers to a diluent, adjuvant, excipient, or carrier that can be administered to the patient along with the active ingredient. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline, glucose, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid, chelating agents, and tonic agents such as dextran are also foreseeable. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions will contain a clinically effective dose of an antibody or antigen-binding fragment or fusion protein, preferably in a purified form, along with an appropriate amount of excipients to provide a formulation suitable for administration.

[0085] In some embodiments, pharmaceutical compositions can be formulated for intravenous injection into the human body. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The compositions may also contain solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site. For injection, they can be diluted to the required concentration with sterile pharmaceutical-grade water or physiological saline. In some embodiments, pharmaceutical compositions can be formulated for subcutaneous injection. The formulation can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0086] The antibody or antigen-binding fragment or fusion protein of the present invention may be in neutral or salt form. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0087] Antibody preparation Various methods for preparing antibodies are known in this field, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, and phage display library methods.

[0088] Antibodies can be prepared using conventional recombinant DNA techniques. Vectors and cell lines for antibody production 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, FM Wurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which, including supplementary information, are incorporated herein by reference.

[0089] In some embodiments, antibody-encoding DNA can be designed and synthesized according to the antibody amino acid sequence described herein using conventional methods, placed into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in a culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody-encoding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters of long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as actin promoters. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, 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.

[0090] In some implementations, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the aforementioned genes. After culturing in a selective medium, successfully transfected cells grow in large numbers, producing the desired target protein. The resulting antibody can be separated or purified using conventional techniques, such as protein A-agarose gel chromatography, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0091] Table 1: Sequence information involved in this application:

[0092] Table 2: CDR1-3 sequence information of VHH against 4-1BB

[0093] The technical solution of the present invention will be further illustrated below through specific embodiments. These specific embodiments do not represent a limitation on the scope of protection of the present invention, and any non-essential modifications or adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0094] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0095] The DNA sequence of the antibody or its fragment involved in this invention can be set according to its amino acid sequence and codon optimized according to the selected cells, which is a conventional technique in the field.

[0096] The cross-linked antibody or antibody cross-linking used in the following examples indicates that anti-human IgG1 (Fc specific) (goat antibody, Sigma-Aldrich, catalog number I2136) is cross-linked with the fusion protein of the present invention. The working principle is that one anti-human IgG1 can bind to at least two 4-1BB-VHH-Fc fusion proteins, producing an aggregation effect, thereby activating the activity of the fusion protein.

[0097] Example 1: Preparation of 4-1BB-VHH-Fc fusion protein Six fusion proteins were designed and expressed, each consisting of a single polypeptide chain from the N-terminus to the C-terminus with the following structure: VHH domain of anti-4-1BB single-domain antibody (sdAb) (sequences shown in SEQ ID NO: 1~6 respectively) - linker peptide (linker peptide L1, sequence shown in SEQ ID NO: 8) - Fc domain of IgG1 (sequence shown in SEQ ID NO: 38), structurally represented as: VHH-Fc. Specific nomenclature and corresponding sequences are shown in Table 3.

[0098] Table 3: Structure and corresponding sequence of the 4-1BB-VHH-Fc fusion protein

[0099] The positive control antibodies were urelumab and utomilumab.

[0100] Plasmids expressing the above six fusion proteins and two positive control antibodies were prepared and transiently expressed in 293F cells. The proteins were purified using protein A. Sequencing analysis showed that the sequences of the eight proteins were consistent with the design.

[0101] Example 2: Determination of the binding of the 4-1BB-VHH-Fc fusion protein to the target The ability of fusion proteins to bind 4-1BB can be determined using surface plasmon resonance (e.g., Biacore), fluorescence-assisted cell sorting (FACS), or a combination thereof, and the analysis can be performed on activated T cells.

[0102] The binding kinetics of different fusion proteins to 4-1BB were determined using the BIACORE T200 (GE Healthcare) surface plasmon resonance (SPR) biosensor. Different concentrations of the 4-1BB-VHH-Fc fusion protein were prepared by serially diluting 3-fold with BIAcore's HEPES buffer, starting at 100 nM, for a total of seven gradients. Each sample was immobilized on the sensor chip using the Fc capture method. The antigen 4-1BB (Sinochem, catalog number 10041-H08H) was used as the analyte. The dissociation (kd) and binding (ka) rate constants were obtained using the T200 evaluation software (Biacore, GE Healthcare). The apparent equilibrium dissociation constant (K) was also determined. D ) by K d For K a The ratio was calculated. The results are shown in Table 4. Each fusion protein exhibited binding kinetics to 4-1BB comparable to the positive control antibody.

[0103] Table 4: Affinity test results of 4-1BB-VHH-Fc fusion protein

[0104] Example 3: FACS analysis of the binding of the 4-1BB-VHH-Fc fusion protein to cells expressing 4-1BB The binding of the 4-1BB-VHH-Fc fusion protein to HEK-293T (293T-4-1BB) cells overexpressing 4-1BB was evaluated using a fluorescence-activated cell sorting (FACS) assay. Different fusion protein samples (11 concentrations, starting at 100 nM and serially diluted 2-fold) were prepared as the primary antibodies for FACS analysis. 293T-4-1BB cells were detached from adherent culture flasks and mixed with different concentrations of the fusion protein samples (all in 96-well plates). Urelimumab and utorumab were used as positive controls against 4-1BB antibodies. The mixture was equilibrated at 4°C for 60 min and washed three times with PBS buffer. Then, phycoerythrin (PE)-conjugated goat anti-human IgG Fc antibody (Invitrogen) was added as a secondary antibody, and the cells were equilibrated at 4°C in the dark for 30 min. The cells were washed again with PBS buffer and analyzed by flow cytometry. Nonlinear regression was used to analyze the data using GraphPad PRISM 8 (GraphPad Software, San Diego, CA). The results are as follows: Figure 1As shown, FACS binding assays revealed that all fusion proteins could bind to 4-1BB cells.

[0105] The preparation method of HEK-293T (293T-4-1BB) cells is as follows: HEK-293T cells were transfected with a luciferase reporter gene vector containing an NF-κB promoter, and stable cell lines were obtained by selection with hygromycin resistance (100 μg / ml). Then, the cells were transfected with a vector expressing h4-1BB, and selected with puromycin (0.3 μg / ml) to obtain a stable 4-1BB / NF-κB reporter gene-HEK-293T cell line. The amino acid sequence of h4-1BB is shown in SEQ ID NO: 39.

[0106] Example 4: FACS analysis of the inhibition of 4-1BB-VHH-Fc fusion protein binding to 4-1BB and its ligand 4-1BBL. To evaluate the inhibitory effect of the fusion protein on the binding of 4-1BB and its ligand 4-1BBL (competitive assay), the 4-1BB-VHH-Fc fusion protein was prepared (12 concentrations, starting at 100 nM and serially diluted 2-fold). 293T cells expressing human 4-1BB (i.e., HEK-293T (293T-4-1BB), preparation method as described in Example 3) were dissociated from adherent culture flasks and mixed with different concentrations of each fusion protein and 1 nM of biotinylated h4-1BBL protein (Sinochem, catalog number 15693-H42H-B). Functionally similar utolumab and urerutumab were used as positive controls against 4-1BB antibodies. The mixture was equilibrated at 4°C for 60 min and washed three times with PBS buffer. Then, PE-streptoacidin secondary antibody (Invitrogen) was added to the mixture and equilibrated at 4°C in the dark for 30 min. Subsequently, the cells were washed with PBS buffer and analyzed by flow cytometry. Nonlinear regression was used to analyze the data using GraphPad PRISM 8 (GraphPad Software, San Diego, CA). Competitive assays demonstrated that urorumab effectively inhibited 4-1BB / 4-1BBL interactions at low concentrations (0.1–10 nM), while different fusion protein samples and urorumab at high concentrations (10–100 nM) failed to inhibit 4-1BB / 4-1BBL interactions. Figure 2 and Figure 3 The binding data indicate that neither the 4-1BB-VHH-Fc fusion protein nor urinumab blocks the binding of 4-1BB and 4-1BBL.

[0107] Example 5: In vitro functional assay of 4-1BB-VHH-Fc fusion protein A variety of bioassays can be used to investigate the activation of the 4-1BB pathway by the 4-1BB-VHH-Fc sample, which monitor T cell proliferation, IFN-γ release, IL-2 secretion, or reporter gene expression driven by signal transduction in the 4-1BB pathway.

[0108] Six different 4-1BB-VHH-Fc fusion proteins were selected for in vitro bioactivity assessment. The bioactivity of anti-4-1BB agonist antibodies was characterized using 293T-4-1BB cells in 4-1BB cell-based assays, as follows: Figure 4A As shown. The specific experiments are as follows: Different fusion proteins were mixed with anti-human IgG1 (Fc specific) (goat antibody, Sigma-Aldrich, catalog number I2136) at a 1:1 ratio, or without anti-human IgG1 (Fc specific). 293T-4-1BB cells were mixed with serially diluted fusion protein samples (starting at 500 nM, 3-fold serial dilutions, resulting in 10 concentrations) and cultured in a cell culture incubator. After approximately 6 hours of stimulation, Bio-Lite... TM The Luciferase Assay System used luciferase reagent (Novazia) to add to cells to measure NF-κB activity. Data were analyzed using nonlinear regression with GraphPad PRISM 8 (GraphPad Software, San Diego, CA), and EC50 was calculated. 50 Value. Anti-human IgG (Fc specific) (goat anti-, polyclonal antibody) can specifically bind to the Fc of at least two 4-1BB-VHH-Fc fusion proteins, producing an aggregation effect of the fusion protein, thereby activating the 4-1BB activation activity of the fusion protein. For example... Figure 4A The reporter gene assay showed that each fusion protein could effectively activate NF-κB signaling in the presence of anti-human IgG (Fc specific), but did not activate NF-κB signaling when present alone. In contrast, the control antibody urinumab could effectively activate NF-κB signaling on its own. This indicates that the fusion proteins of the present invention require cross-linking to be activated and have higher safety.

[0109] Another scenario also reflects the effective activation of NF-κB signaling by different fusion proteins under cross-linking conditions. In this case, fusion protein samples were prepared separately (starting at 100 nM, serially diluted 2-fold, with 10 concentrations). These samples were coated overnight at 4°C in an ELISA plate, and then 293T-4-1BB cells were added. After stimulation for approximately 6 hours, Bio-Lite... TMThe Luciferase Assay System used luciferase reagent (Novazia) to add to cells to measure NF-κB activity. Data were analyzed using nonlinear regression with GraphPad PRISM 8 (GraphPad Software, San Diego, CA), and EC50 was calculated. 50 value. Figure 4B Reporter gene assays showed that all constructs cross-linked on the plate effectively activated NF-κB signaling.

[0110] Example 6: Construction, expression and characterization of PD-L1 / 4-1BB bispecific antibody This embodiment describes the construction and expression of an exemplary PD-L1 / 4-1BB bispecific antibody (BsAb) vector. Six bispecific structures were designed and expressed, and schematic diagrams of the six structures are shown below. Figure 5A As shown, each contains the following polypeptide chains.

[0111] Mode 1 structure: It contains a first polypeptide, a second polypeptide, and a third polypeptide, wherein the first polypeptide is the light chain of the anti-PD-L1 antibody; the second polypeptide is the heavy chain of the anti-PD-L1 antibody (N297A-knob); and the third polypeptide contains from the N-terminus to the C-terminus: VHH of anti-4-1BB, linker peptide L1, and Fc region of IgG1 (N297A-hole).

[0112] Representative bispecific antibodies are 1-6 and 1-15; their sequence composition is shown in Table 5-1.

[0113] Table 5-1: Sequence composition of Mode 1 bispecific antibodies

[0114] Mode 2 structure: contains two identical first polypeptides and two identical second polypeptides, wherein the first polypeptide contains, from the N-terminus to the C-terminus: an anti-PD-L1 antibody light chain, a linker peptide L2, and an anti-4-1BB VHH; the second polypeptide is the heavy chain of an IgG4 type anti-PD-L1 antibody.

[0115] Representative bispecific antibodies are 2-6 and 2-15, and their sequence composition is shown in Table 5-2.

[0116] Table 5-2: Sequence composition of mode 2 bispecific antibodies

[0117] Mode 3 structure: contains two identical first peptides, which from the N-terminus to the C-terminus contain: VHH for anti-4-1BB, linker peptide L1, Fc region of IgG4, linker peptide L3, and ScFv for anti-PD-L1.

[0118] Representative bispecific antibodies are 3-6 and 3-15, and their sequence composition is shown in Table 5-3.

[0119] Table 5-3: Sequence composition of mode 3 bispecific antibodies

[0120] Mode 4 structure: contains two identical first polypeptides and two identical second polypeptides, wherein the first polypeptide is the light chain of the anti-PD-L1 antibody; the second polypeptide contains, from the N-terminus to the C-terminus: the heavy chain of the anti-PD-L1 antibody, the linker peptide L4, and the VHH of the anti-4-1BB.

[0121] Representative bispecific antibodies are 4-6 and 4-15, and their sequence composition is shown in Table 5-4.

[0122] Table 5-4: Sequence composition of mode 4 bispecific antibodies

[0123] Pattern 5 structure: contains two identical first polypeptides and two identical second polypeptides, wherein the first polypeptide is the light chain of the anti-PD-L1 antibody; the second polypeptide contains from the N-terminus to the C-terminus: VH-CH1 of the anti-PD-L1 antibody, linker peptide L3, VHH of the anti-4-1BB antibody, linker peptide L1, and the Fc region of IgG4.

[0124] Representative bispecific antibodies are 5-6 and 5-15, and their constituent sequences are shown in Table 5-5.

[0125] Table 5-5: Sequence composition of mode 5 bispecific antibodies

[0126] Pattern 7 structure: contains two identical first polypeptides and two identical second polypeptides, wherein the first polypeptide is the light chain of the anti-PD-L1 antibody; the second polypeptide contains, from the N-terminus to the C-terminus: the heavy chain of the anti-PD-L1 antibody, the linker peptide L3, and the VHH of the anti-4-1BB antibody.

[0127] Representative bispecific antibodies are 7-1, 7-2, 7-6, 7-8, 7-13, and 7-15, and their sequence composition is shown in Table 5-6.

[0128] Table 5-6: Sequence composition of mode 7 bispecific antibodies

[0129] Plasmids expressing the aforementioned bispecific antibodies were prepared and transiently expressed in 293F cells, then purified using protein A. Figure 5BAs shown, under both reducing and non-reducing conditions, the composition and purity of purified BsAb were analyzed by SDS-PAGE. The size of the polypeptide chain and the full-length molecule were consistent with the molecular weight calculated based on the amino acid sequence.

[0130] Example 7: Assay of BsAb binding to target antigen in Mode 7 structure The binding kinetics of different BsAbs to 4-1BB were determined using the BIACORE T200 (GE Healthcare) surface plasmon resonance (SPR) biosensor. Different concentrations of BsAb samples were prepared by serial 3-fold dilutions, starting at 100 nM. Each sample was immobilized on the sensor chip using Fc capture. The antigen 4-1BB was used as the analyte. The dissociation (kd) and binding (ka) rate constants were obtained using the T200 evaluation software. The apparent equilibrium dissociation constant (Kd) was also determined. D ) by K d For K a The ratio was calculated. As shown in Table 6-1, different BsAbs exhibited comparable binding kinetics to 4-1BB. The BsAbs of this invention have a lower affinity for 4-1BB than positive antibodies, making the BsAbs dependent on the binding of PD-L1 antibodies, thereby generating cross-linking activation and ensuring safety.

[0131] Table 6-1: Affinity test results of BsAb in Model 7 structure and control

[0132] Similarly, the surface plasmon resonance (SPR) biosensor BIACORE T200 (GE Healthcare) was used to determine the binding kinetics of different BsAbs to PD-L1. Different concentrations of BsAb samples were prepared by serial 2-fold dilutions, starting at 100 nM. Each sample was immobilized on the sensor chip using Fc capture. The antigen PD-L1 was used as the analyte. The dissociation (kd) and binding (ka) rate constants were obtained using the T200 evaluation software. The apparent equilibrium dissociation constant (Kd) was also determined. D ) by K d For K a The ratio was calculated. As shown in Table 6-2, different BsAbs exhibited comparable binding kinetics to PD-L1.

[0133] PD-L1 antigen preparation: An 8×HIS tag was added to the C-terminus of the human PD-L1 extracellular domain (ECD) protein to construct a recombinant PD-L1 protein. The ECD region of human PD-L1 was synthesized and subcloned into a mammalian expression vector (e.g., pcDNA3.1(+) expression vector). After transient transfection of HEK293F cells, His-tagged PD-L1 antigen was obtained through nickel-based immobilized metal affinity chromatography purification (GE Healthcare). The amino acid sequence of the human PD-L1 extracellular domain (ECD) is as follows: MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGA DYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO: 40).

[0134] Table 6-2: Affinity test of BsAb in Mode 7 structure

[0135] Example 8: Cell binding determination and activity detection of BsAb protein with pattern 7 structure The binding of different BsAb proteins of pattern 7 structure to HEK-293T cells overexpressing 4-1BB (293T-4-1BB) was evaluated using a fluorescence-activated cell sorting (FACS) assay. Different BsAb samples (starting from 25 nM, serially diluted 2-fold, for a total of 12 concentrations) were prepared as the primary antibodies for FACS analysis. 293T-4-1BB cells were detached from adherent culture flasks and mixed with different concentrations of the samples (all in 96-well plates). Urejuvenumab was used as a positive control against 4-1BB. The mixture was equilibrated at 4°C for 60 min and washed three times with PBS buffer. Then, phycoerythrin (PE)-conjugated goat anti-human IgG FC antibody (Invitrogen) was added as a secondary antibody, and the cells were equilibrated at 4°C in the dark for 30 min. Cells were washed again with PBS buffer and analyzed by flow cytometry. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) using nonlinear regression. Figure 6AAs shown in the figure, FACS binding assays demonstrated that ursulcimbrolizumab binds to EC4-1BB on the cell surface. 50 The strongest binding ability is found in 4-1BB, but the plateau is relatively low. The binding ability of 7-15 is the weakest, while the binding ability of other BsAbs is comparable, indicating that these bispecific antibodies can bind to 4-1BB very well.

[0136] Bioactivity of anti-PD-L1 neutralizing antibodies in PD-1 / PD-L1 cell-based assays using PD-1 / NFAT reporter gene-Jurkat cells. Briefly, CHO-K1 cells (Promega, CS187108) stably expressed with human PD-L1 and engineered T-cell receptor (TCR) activators were utilized. PD-1 / NFAT reporter gene-Jurkat cells (Promega, CS187102) were pre-incubated for 30 min with serially diluted BsAb (starting concentration 30 nM, 2-fold dilution, 10 consecutive gradients) and then co-cultured with engineered CHO-K1 cells (Promega, CS187108). After approximately 6 hours of stimulation, Bio-Lite... TM The Luciferase Assay System used luciferase reagent (Novazia) to add to cells to measure NFAT activity. Data were analyzed using nonlinear regression with GraphPad PRISM 8 (GraphPad Software, San Diego, CA), and EC50 was calculated. 50 Value and shown in Figure 6B and 6C In the study, reporter gene assays showed that 1-6, 1-15, 3-6, and 3-15 exhibited decreased activity compared to PD-L1 monoclonal antibodies, while the other BsAbs showed similar ability to effectively activate NFAT signaling as the PD-L1 monoclonal antibody L1 (sequence from L1-R2-4-71 antibody, patent publication number: CN114057877A) and the control antibody INBRX-105 (sequence from US 2020 / 0199243 A1).

[0137] Example 9: In vitro bioactivity assay of BsAb with Mode 7 structure Characterization of the bioactivity of BsAb as an anti-4-1BB agonist antibody using 293T-4-1BB cells in a 4-1BB cell-based assay, as follows: Figure 7As shown. In this case, different BsAb and control antibody INBRX-105 samples (100 nM starting concentration, 3-fold serial dilution, with 10 concentrations) were prepared. 293T-4-1BB cells were mixed with the serially diluted samples and then co-cultured with engineered CHO-K1 cells overexpressing PD-L1 (CHO-PD-L1) (Promega, CS187108) or CHO-K1 cells in a cell culture incubator (37°C, 5% CO2). After approximately 6 hours of stimulation, Bio-Lite... TM The Luciferase Assay System used luciferase reagent (Novazia) to add to cells to measure NF-κB activity. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) with nonlinear regression, and EC50 was calculated. 50 Value. Reporter gene assay results are as follows: Figure 7 As shown in Tables A-G and 7-1 and 7-2, all BsAbs effectively activated NF-κB signaling in the presence of CHO-PD-L1 cells. All BsAbs showed comparable ECG activity to the control antibody INBRX-105. 50 The activation of CHO-K1 cells was not significant at low concentrations (below 10 nM), while activation was significant at high concentrations (10-100 nM), but still much lower than that of CHO-PD-L1 cells. Furthermore, the EC50 activity of different BsAbs was significantly lower. 50 Both are much larger than the control antibody, indicating that the safety is far superior to INBRX-105.

[0138] Table 7-1: Comparison of the activities of BsAbs with Mode 7 structure in the CHO-PD-L1 / 293T-41BB reporter system

[0139] Table 7-2: Comparison of the activities of BsAbs with Mode 7 structure in the CHO-K1 / 293T-41BB reporter system

[0140] Example 10: In vitro functional assay of BsAb in modes 1, 3, and 4 The bioactivity of BsAbs against 4-1BB agonist antibodies was characterized using 293T-4-1BB cells in a 4-1BB cell-based assay, as shown in Figure 8. Different BsAb samples were prepared (3-6, 3-15, INBRX-105: starting at 10 nM, 3-fold dilution; 1-6, 1-15: starting at 30 nM, 2-fold dilution; 4-6, 4-15: starting at 20 nM, 3-fold dilution; each with 10 concentrations). 293T-4-1BB cells were mixed with the serially diluted samples and then co-cultured with engineered CHO-K1 cells overexpressing PD-L1 (CHO-PD-L1) (Promega, CS187108) or CHO-K1 cells. After approximately 6 hours of stimulation, Bio-Lite... TM The Luciferase Assay System used luciferase reagent (Novazia) to add to cells to measure NF-κB activity. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) with nonlinear regression, and EC50 was calculated. 50 value. Figure 8A The ~C and Table 8 reporter gene assays show that: Table 8: Comparison of the activities of different BsAbs in the CHO-PD-L1 / 293T-41BB reporter system

[0141] The BsAb with the Mode 1 structure activated NF-κB signaling weakly in the presence of CHO-PD-L1 cells compared to the control antibody. Low concentrations (below 10 nM) of BsAb in the presence of CHO-PD-L1 cells did not show significant activation, while high concentrations (10-100 nM) showed significant activation, but the activation intensity was much lower than that in the presence of CHO-PD-L1 cells, indicating that its safety was far superior to that of INBRX-105.

[0142] The BsAb with the mode 3 structure activated NF-κB signaling better than the control antibody in the presence of CHO-PD-L1 cells; activation was not obvious in the presence of low concentration (below 1 nM) of CHO-K1 cells, but was obvious in the presence of high concentration (1-100 nM).

[0143] The BsAb with the mode 4 structure activated NF-κB signaling more effectively than the control antibody in the presence of CHO-PD-L1 cells; activation was not significant in the presence of low concentrations (below 1 nM) of CHO-K1 cells, but was significant in the presence of high concentrations (1-100 nM).

[0144] Example 11: In vitro functional assay of 4-1BB in different BsAbs with structures of modes 2, 5, and 7 The 293T-4-1BB cells used in the in vitro functional assays of the above embodiments were polyclonal cell populations. To further improve the stability of activity detection, monoclonal cell lines of 293T-4-1BB were screened, and these monoclonal cell lines were used in all the following embodiments. The screening of monoclonal cell lines was carried out using a flow cytometry instrument to plate monoclonal cells, ensuring that there was only one cell in each well. The obtained monoclonal cells were used in the experiments in Example 10 using an INBRX-105, and finally, monoclonal cells with low background and large window size were selected.

[0145] Characterization of the bioactivity of anti-4-1BB agonist antibodies using 293T-4-1BB cells in 4-1BB cell-based assays, as follows: Figure 9 As shown in A~G. The specific procedure is as follows: Different BsAb samples were prepared (30 nM starting concentration, 3-fold serial dilution, resulting in 10 concentrations). 293T-4-1BB monoclonal cells were mixed with the serially diluted BsAb samples, and then co-cultured with engineered CHO-K1 cells overexpressing PD-L1 (CHO-PD-L1) (Promega, CS187108) or CHO-K1 cells in a cell culture incubator. After approximately 6 hours of stimulation, Bio-Lite... TM The Luciferase Assay System used luciferase reagent (Novazia) added to cells to measure NF-κB activity. Data were analyzed using GraphPad PRISM 8 (GraphPad Software, San Diego, CA) with nonlinear regression, and EC50 was calculated. 50 value. Figure 9 The A~G and reporter gene assays in Table 9 show that: Table 9: Comparison of the activities of different BsAbs in the CHO-PD-L1 / 293T-4-1BB reporter system

[0146] The BsAb with the mode 2 structure activated NF-κB signaling in the presence of CHO-PD-L1 cells, which was comparable to that of the control antibody. In the presence of CHO-K1 cells, low concentrations (below 10 nM) showed no significant activation, while high concentrations (greater than 10 nM) showed slight activation, which was much lower than the activation intensity of the control antibody, indicating that its safety was far superior to that of INBRX-105.

[0147] The BsAb with the pattern 5 structure activated NF-κB signaling in the presence of CHO-PD-L1 cells, which was comparable to that of the control antibody; however, the activation in the presence of CHO-K1 cells was not significant and was much lower than that of the control antibody, indicating that its safety was far superior to that of INBRX-105.

[0148] The BsAb with the pattern 7 structure activated NF-κB signaling in the presence of CHO-PD-L1 cells in a manner comparable to the control antibody. In the presence of CHO-K1 cells, activation was not significant at low concentrations (below 10 nM) and only slightly at high concentrations (greater than 10 nM), which was much lower than the activation intensity of the control antibody, indicating that its safety was far superior to that of INBRX-105.

[0149] Example 12: Detection of activation of human peripheral blood mononuclear lymphocytes with PD-L1 / 4-1BB bispecific antibody By identifying target cells expressing PD-L1 (such as dendritic cells or tumor cells; in this example, PD-L1-overexpressing 293F cells (293F-PD-L1 cells, prepared as follows: 293F cells were transfected with a plasmid containing hPD-L1 (pCDH-CMV-MCS-EF1-Hygro lentiviral plasmid), and selected under pressure with hygromycin (200 μg / ml) to finally obtain hPD-L1-containing 293F cells) and the secretion levels of IL-2 and IFN-γ in human peripheral blood mononuclear lymphocytes (PBMCs), the activation of the PD-L1-dependent 4-1BB pathway by 7-15BsAb was investigated. Human PBMCs were isolated from blood using sample density separation medium (Dakow, China). One night before, each well was coated with 200 ng / μl of anti-CD3 antibody (Tongli Haiyuan). The anti-CD3 antibody was removed the next day, and 1×10⁻⁶ oz / μl of anti-CD3 antibody was added to each well. 4 293F-PD-L1 cells (50 μg / well), 1×10 5 PBMC cells (50 μg / well) were used. 7–15 BsAb were added to each well at different concentrations (starting from 33.3 nM, 3-fold dilution, 10 gradients) to a final working volume of 200 μl. Wells without antibody were used as background controls. Antibody L1 was used as a PD-L1 monospecific antibody control, and uroselumab was used as a positive control. After incubation at 37°C, 5% CO2 for 72 hours, 100 μl of culture medium was removed from each test well for IL-2 and IFN-γ measurements (Xinbosheng Biotechnology, China). Figure 10 As shown in A and 10B, 7-15 BsAb exhibited superior activation potential compared to ursulcimbrine, while PD-L1 monoclonal antibody showed no significant activation effect under the same conditions.

[0150] Similar to the activity reporter system in the previous examples, the effect of different PD-L1 / 4-1BB bispecific antibodies on PBMC activation in the absence of PD-L1 expression cells was also tested to determine the safety of different bispecific antibodies. Human PBMCs were isolated from blood using sample density separation buffer (Dakow, China). 200 ng / μl of anti-CD3 antibody (Tonglihaiyuan) was coated into each well overnight. The anti-CD3 antibody was removed the next day, and 1×10⁻⁶ ng / μl of anti-CD3 antibody was added to each well.5 PBMCs were cultured, and BsAb was added to each well at different concentrations (starting from 33.3 nM, 3-fold dilution, 10 gradients) to a final working volume of 200 μl. Wells without antibody were used as background controls. Antibody L1 was used as a PD-L1 monospecific antibody control, and ursulcimbrine antibody was used as a positive control. After incubation at 37°C, 5% CO2 for 72 hours, 100 μl of culture medium was removed from each well for IL-2 and IFN-γ measurements (Xinbosheng Biotechnology, China). Figure 11 As shown, different PD-L1 / 4-1BB bispecific antibodies have no activation potential in the absence of PD-L1-positive cells, while uroselumab has a significant activation effect under the same conditions. This indicates that the activation of 4-1BB by bispecific antibodies is specific to PD-L1 expression; they can only activate immune cells to release cytokines in the presence of PD-L1 (such as in the presence of a tumor), but cannot be activated in the absence of PD-L1 in peripheral blood, making them safer. In contrast, uroselumab activation does not require PD-L1 and has no conditional limitations; it can be activated even in peripheral blood, which could lead to side effects.

[0151] The full-length hPD-L1 sequence is: MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPV TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET (SEQ IDNO: 41) Example 13: Cytokine Release Assay of PD-L1 / 4-1BB Bispecific Antibody Cytokine release syndrome (CRS) is a group of clinical syndromes caused by excessive activation of immune cells and rapid release of pro-inflammatory factors, and is a common adverse reaction to immunomodulatory drugs. For immunomodulatory drugs, a complete non-clinical evaluation requires integrating all available in vivo and in vitro experimental data. In addition to routine in vivo toxicity studies in animals, in vitro cytokine release assays should also be performed. Using human whole blood or human peripheral blood mononuclear cells to evaluate cell activation and cytokine release in vitro can, to some extent, compensate for the limitations of animal models in fully mimicking the human immune stimulation process due to species differences. When in vivo test results are negative, positive in vitro test results can indicate potential clinical safety risks. It is recommended to design appropriate in vitro cytokine release assays based on the cytokine release mechanism. Both liquid-phase and solid-phase incubation systems should generally be considered.

[0152] Based on the above considerations, this embodiment used liquid-phase and solid-phase incubation systems to evaluate the cytokine release of different BsAbs. The dry-packing method, i.e., the solid-phase incubation system, involved coating the test sample at a volume of 25 μg / mL in 40 μL, drying it overnight in a clean bench at speed 2, and then adding 1×10⁻⁶ ppm to each well. 5 Two PBMC cells (Red Biotech, Guangzhou) from different donors, each 200 μL in volume, were used. A wet pack method, i.e., a liquid-phase incubation system, was employed. The sample concentration was 25 μg / mL, 100 μL, with 1 × 10⁻⁶ cells added to each well. 5 PBMCs were incubated for three days. Anti-CD3 antibody, uroselumab (Ure), and INBRX-105 were used as control antibodies; TGN1412 (cd28 agonist antibody, sequence derived from patent US8709414B2) was used as a positive control antibody; IgG4 (HG4K, Sinocare) was used as a negative control; and RPMI 1640 medium was used as a systemic background control. After three days of incubation, the supernatant was collected to detect IL-2 and IFN-γ (Xinbosheng Biotechnology, China). Figure 12A As shown in Figure H, TGN1412 significantly activated PBMCs in both liquid and solid-phase incubation systems, exhibiting significantly higher IL-2 and IFN-γ release levels than other test samples. Anti-CD3 antibody, ursulcimbrine antibody, and INBRX-105, used as control antibodies, also activated PBMCs to varying degrees. However, the 2-15, 5-15, and 7-15 constructs failed to activate PBMCs in either case. These cytokine release experiments demonstrate that, regardless of whether fixed in solution or solid states on the plate, the different BsAbs in PD-L1 / 4-1BB exhibit good safety profiles and do not lead to excessive cytokine release. In contrast, the anti-CD3 antibody and TGN1412, used as positive controls, resulted in excessive cytokine release, raising serious safety concerns.

[0153] Example 14: In vivo antitumor efficacy of PD-L1 / 4-1BB bispecific antibody This embodiment describes in vivo experiments demonstrating the blocking effect of the PD-L1 bispecific antibody on PD-L1 and the activation effect of 4-1BB. Antitumor efficacy was evaluated in a tumor model developed using human 4-1BB knock-in mice. Since the bioactively similar anti-PD-L1 monoclonal antibody L1 also binds to mouse PD-L1, the humanization of 4-1BB in mice enabled direct in vivo evaluation of the efficacy of BsAbs in a mouse tumor xenograft model.

[0154] The mouse tumor-bearing model was established by implanting tumor cells into Balb / c-hCD137KI mice. The mouse colon adenocarcinoma cell line CT26-WT was used in this assay. CT26-WT (5 × 10⁻⁶ cells) was used in this assay. 5 (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) A Balb / c-hCD137-CT26.WT tumor-bearing mouse model was obtained by subcutaneous injection into 8-week-old Balb / c-hCD137KI mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). Tumor size was measured using calipers, and tumor volume was calculated using a modified elliptic formula: length × (width). 2 / 2. On day 13 post-inoculation, the average tumor volume reached 101.55 mm. 3 Around [time period missing], 64 mice were randomly divided into 8 groups of 8 mice each, based on tumor volume. Human control IgG4 (Essential), anti-PD-L1 antibody (antibody L1), and BsAbs with different structures were administered to the mice via intraperitoneal injection. Day 0 was defined as the day of grouping, and medication was initiated on Day 0, administered twice a week. Antibody L1 was administered at 5 mg / kg, and the others at 6 mg / kg. Tumor volume was measured twice. The efficacy of different BsAbs was evaluated by assessing tumor size inhibition. Figure 13A As shown in Table 10, this model is exceptionally sensitive to PD-L1 monoclonal antibodies, with antibody L1 exhibiting very significant efficacy. Different test samples all showed relatively significant tumor-inhibiting effects. Among them, 2-15, 5-15, and 7-15 BsAbs showed similar effects to antibody L1, while 2-6, 5-6, and 7-6 showed slightly weaker effects.

[0155] Table 10: Comparison of tumor inhibition rates of different antibodies in Balb / c-hCD137-CT26.WT tumor-bearing mouse model

[0156] In another mouse tumor-bearing model, tumor cells were implanted into C57BL / 6-hCD137KI mice. The mouse melanoma cell line B16F10, which stably expresses human PD-L1, was used in this assay. B16F10-hPD-L1 (1×10⁻⁶) was used. 6 Tumors were subcutaneously injected into 8-week-old C57BL / 6-hCD137KI mice (Shanghai Southern Model Biotechnology Co., Ltd.). Tumor size was measured using calipers, and tumor volume was calculated using a modified elliptic formula: length × (width). 2 / 2. On day 13 post-inoculation, the average tumor volume reached 56 mm. 3 Around 10:00 AM, 48 mice were randomly divided into 6 groups of 8 mice each, based on tumor volume. Mice were administered PBS control, anti-PD-L1 antibody (antibody L1), uroselumab, and BsAbs with different structures via intraperitoneal injection (ip). Day 0 was defined as the day of grouping, and medication was initiated on Day 0, twice a week. The dosage of uroselumab and antibody L1 was 15 mg / kg, and 20 mg / kg for 2-15 / 5-15 / 7-15 weeks. Tumor volume was measured twice. The efficacy of different BsAbs was evaluated by assessing tumor size inhibition. Figure 13B As shown in Table 11, the model was insensitive to PD-L1 monoclonal antibodies, with no significant efficacy against L1 antibody. Urerucizumab showed a more significant effect, and all tested products exhibited significant tumor-inhibiting effects. Among them, 2-15, 5-15 BsAb, and urrerucizumab showed similar effects, while 7-15 showed the best effect. Notably, one mouse in each of the urrerucizumab, 2-15, 5-15, and 7-15 groups achieved complete tumor remission and remained relapse-free during the observation period.

[0157] Table 11: Comparison of tumor inhibition rates of different antibodies in Balb / c-hCD137-CT26.WT tumor-bearing mouse model

[0158] Mice whose tumors (B16F0-hPD-L1) had completely regressed were observed for 3 months without further drug administration, and the tumors remained in a state of complete regression. On D91, the 91st day after the start of the experiment, tumors were injected into the other side of the above four mice to conduct a tumor-bearing mouse rechallenge experiment. At the same time, three mice in the control group were also injected with tumors (C57BL / 6-hCD137 KI mice that had not been injected with tumor cells, B16F10-hPD-L1 (1×10)). 6 (Subcutaneous injection), continue to observe. Figure 13CIn mice in groups 2-15, 5-15, and 7-15, tumor growth was completely inhibited. However, the tumor volume of the control group (3 mice) and the uroselumab group increased rapidly. This indicates that the BsAb can promote immune memory function in mice, enabling it to kill tumors even when the same tumor cells are present.

Claims

1. A bispecific antibody comprising a first antigen-binding domain targeting PD-L1 and a second antigen-binding domain targeting 4-1BB, in, The first antigen-binding domain targeting PD-L1 includes LCDR1 as shown in SEQ ID NO: 31, LCDR2 as shown in SEQ ID NO: 32, LCDR3 as shown in SEQ ID NO: 33, HCDR1 as shown in SEQ ID NO: 34, HCDR2 as shown in SEQ ID NO: 35, and HCDR3 as shown in SEQ ID NO: 36; the second antigen-binding domain targeting 4-1BB includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in any one of SEQ ID NO: 22-26, and CDR3 as shown in any one of SEQ ID NO: 27-30. Alternatively, 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 the sequence shown in SEQ ID NO: 7, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 7; and / or the light chain variable region comprises the sequence shown in SEQ ID NO: 9, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 9, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 9; and / or the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6, or a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO: 1-6, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 1-6.

2. The bispecific antibody according to claim 1, wherein, The first antigen-binding domain and / or the second antigen-binding domain further include a light chain constant region and / or a heavy chain constant region; optionally, the first antigen-binding domain and / or the second antigen-binding domain further include an Fc region; further optionally, the VHH is linked to the heavy chain variable region, light chain variable region, light chain constant region, heavy chain constant region or Fc region via a linker peptide.

3. A bispecific antibody targeting PD-L1 and 4-1BB, wherein, The bispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide includes a light chain variable region and a light chain constant region of a first antigen-binding domain from the N-terminus to the C-terminus. The second polypeptide includes a heavy chain variable region and a heavy chain constant region of the first antigen-binding domain from the N-terminus to the C-terminus. The third polypeptide includes the VHH region of the second antigen-binding domain, a linker peptide, and the Fc region of IgG from the N-terminus to the C-terminus. Alternatively... The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide, from its N-terminus to its C-terminus, comprises a light chain variable region, a light chain constant region, a linker peptide, and a VHH region of the second antigen-binding domain. The second polypeptide, from its N-terminus to its C-terminus, comprises a heavy chain variable region and a heavy chain constant region of the first antigen-binding domain. Alternatively... The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide includes a light chain variable region and a light chain constant region of a first antigen-binding domain from the N-terminus to the C-terminus. The second polypeptide includes a heavy chain variable region, a heavy chain constant region, a linker peptide, and a VHH of the second antigen-binding domain from the N-terminus to the C-terminus. Alternatively... The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide includes, from the N-terminus to the C-terminus, a light chain variable region and a light chain constant region of a first antigen-binding domain. The second polypeptide includes, from the N-terminus to the C-terminus, a heavy chain variable region of the first antigen-binding domain and CH1 (VH-CH1), linker peptide 1, VHH of the second antigen-binding domain, linker peptide 2, and the Fc region of IgG; or... The bispecific antibody comprises a polypeptide comprising, from the N-terminus to the C-terminus, a second antigen-binding domain VHH, a linker peptide 1, an Fc region of IgG, a linker peptide 2, and a first antigen-binding domain scFv. The scFv comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the first antigen-binding domain, a linker peptide 3, and a light chain variable region of the first antigen-binding domain; or the scFv comprises, from the N-terminus to the C-terminus, a light chain variable region of the first antigen-binding domain, a linker peptide 3, and a heavy chain variable region of the first antigen-binding domain. Wherein, the light chain variable region of the first antigen-binding domain includes LCDR1 as shown in SEQ ID NO: 31, LCDR2 as shown in SEQ ID NO: 32, and LCDR3 as shown in SEQ ID NO: 33; the heavy chain variable region of the first antigen-binding domain includes HCDR1 as shown in SEQ ID NO: 34, HCDR2 as shown in SEQ ID NO: 35, and HCDR3 as shown in SEQ ID NO: 36; and the VHH includes CDR1 as shown in SEQ ID NO: 37, CDR2 as shown in any one of SEQ ID NO: 22-26, and CDR3 as shown in any one of SEQ ID NO: 27-30. The linker peptide, linker peptide 1-3 independently contains 4 to 30 amino acids; or, the linker peptide 1-3 independently contains 4 to 24 amino acids selected from G and S; or, the linker peptide 1-3 independently contains (GGGGS)x, where x is 1, 2, 3, 4, 5 or 6. And / or, the light chain variable region of the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 9, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 9, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 9; and / or the heavy chain variable region of the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 7, or a sequence having at least 80% identity with the sequence shown in SEQ ID NO: 7, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO: 7; and / or the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6, or a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO: 1-6, or a sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO: 1-6.

4. A bispecific antibody targeting PD-L1 and 4-1BB, wherein, The bispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide comprises a light chain with a first antigen-binding domain from its N-terminus to its C-terminus. The second polypeptide comprises a heavy chain with the first antigen-binding domain from its N-terminus to its C-terminus. The third polypeptide comprises a VHH of the second antigen-binding domain, a linker peptide L1, and an Fc region of IgG1 from its N-terminus to its C-terminus. The light chain with the first antigen-binding domain comprises the sequence shown in SEQ ID NO:

10. The heavy chain comprises the sequence shown in SEQ ID NO:

11. The VHH comprises the sequence shown in any one of SEQ ID NO: 1-6. The Fc region of IgG1 comprises the sequence shown in SEQ ID NO:

12. The linker peptide L1 comprises 4 to 30 amino acids. Optionally, the linker peptide L1 comprises 4 to 24 amino acids selected from G and S. Alternatively, the linker peptide L1 comprises (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6. Alternatively, the amino acid sequence of the linker peptide L1 is as shown in SEQ ID NO:

8. The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises, from its N-terminus to its C-terminus, a light chain with a first antigen-binding domain, a linker peptide L2, and a VHH with a second antigen-binding domain. The second polypeptide comprises, from its N-terminus to its C-terminus, a heavy chain with a first antigen-binding domain. The light chain comprises the sequence shown in SEQ ID NO:

10. The VHH comprises the sequence shown in any one of SEQ ID NO: 1-6. The heavy chain comprises the sequence shown in SEQ ID NO: 14 or 18. The linker peptide L2 comprises 4 to 30 amino acids, or 4 to 24 amino acids selected from G and S, or (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6, or the amino acid sequence of the linker peptide L2 is shown in SEQ ID NO:

13. Alternatively, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond. The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises a light chain with a first antigen-binding domain from its N-terminus to its C-terminus. The second polypeptide comprises a heavy chain with the first antigen-binding domain, a linker peptide, and a VHH with the second antigen-binding domain from its N-terminus to its C-terminus. The light chain with the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 10, the heavy chain comprises the sequence shown in SEQ ID NO: 18, and the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6. The linker peptide comprises 4 to 30 amino acids, or 4 to 24 amino acids selected from G and S, or (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6, or the amino acid sequence of the linker peptide is shown in SEQ ID NO: 16 or 19. Alternatively, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond. The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises a light chain with a first antigen-binding domain from the N-terminus to the C-terminus. The second polypeptide comprises, from the N-terminus to the C-terminus, a VH-CH1 of the first antigen-binding domain, a linker peptide L3, a VHH of the second antigen-binding domain, a linker peptide L1, and an Fc region of IgG4. The light chain with the first antigen-binding domain comprises the sequence shown in SEQ ID NO: 10; the VH-CH1 comprises the sequence shown in SEQ ID NO: 20; the VHH comprises the sequence shown in any one of SEQ ID NO: 1-6; and the Fc region of IgG4 comprises the sequence shown in SEQ ID NO: 15 or 21. The linker peptide L1 or L3 independently comprises 4 to 30 amino acids; or, the linker peptide L1 or L3 independently comprises 4 to 24 amino acids selected from G and S; or, the linker peptide L1 or L3 independently comprises (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6; or, the amino acid sequence of the linker peptide L1 is as shown in SEQ ID NO: As shown in Figure 8, the amino acid sequence of the linker peptide L3 is as shown in SEQ ID NO:16; or, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond; or, The bispecific antibody comprises a polypeptide comprising, from its N-terminus to its C-terminus, a second antigen-binding domain (VHH), a linker peptide L1, an Fc region of IgG4, a linker peptide L3, and a first antigen-binding domain (scFv); wherein the VHH comprises a sequence as shown in any one of SEQ ID NO: 1-6, the amino acid sequence of the Fc region of IgG4 is as shown in SEQ ID NO: 15, and the scFv of the first antigen-binding domain comprises a sequence as shown in SEQ ID NO: 17; the linker peptides L1 and / or L3 comprise 4 to 30 amino acids; or, the linker peptides L1 and / or L3 comprise 4 to 24 amino acids selected from G and S; or, the linker peptides L1 and / or L3 comprise (GGGGS)x, where x is 1, 2, 3, 4, 5, or 6; or, the amino acid sequence of the linker peptide L1 is as shown in SEQ ID NO: 8, and the amino acid sequence of the linker peptide L3 is as shown in SEQ ID NO:

8. As shown in Figure 16; the two polypeptides are linked by a disulfide bond; or, the bispecific antibody comprises two identical polypeptides linked by a disulfide bond.

5. A bispecific antibody targeting PD-L1 and 4-1BB, wherein, The bispecific antibody comprises a first polypeptide, a second polypeptide, and a third polypeptide. The first polypeptide is an anti-PD-L1 antibody light chain, the second polypeptide is an anti-PD-L1 antibody heavy chain, and the third polypeptide consists of a VHH of anti-4-1BB, a linker peptide L1, and the Fc region of IgG1 from the N-terminus to the C-terminus. The amino acid sequence of the anti-PD-L1 antibody light chain is shown in SEQ ID NO: 10, the amino acid sequence of the anti-PD-L1 antibody heavy chain is shown in SEQ ID NO: 11, the amino acid sequence of the VHH of anti-4-1BB is shown in SEQ ID NO: 3 or 6, the amino acid sequence of the linker peptide L1 is shown in SEQ ID NO: 8, and the amino acid sequence of the Fc region of IgG1 is shown in SEQ ID NO: 12; or... The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide, from its N-terminus to its C-terminus, consists of a light chain of an anti-PD-L1 antibody, a linker peptide L2, and a VHH of an anti-4-1BB antibody. The second polypeptide is the heavy chain of the anti-PD-L1 antibody. The amino acid sequence of the light chain is shown in SEQ ID NO: 10, the amino acid sequence of the linker peptide L2 is shown in SEQ ID NO: 13, the amino acid sequence of the VHH is shown in SEQ ID NO: 3 or 6, and the amino acid sequence of the heavy chain is shown in SEQ ID NO:

14. Alternatively, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond. The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises a light chain of an anti-PD-L1 antibody from its N-terminus to its C-terminus. The second polypeptide comprises a heavy chain of an anti-PD-L1 antibody, a linker peptide L3, and a VHH of an anti-4-1BB antibody from its N-terminus to its C-terminus. The amino acid sequence of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO:

10. The amino acid sequence of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO:

18. The amino acid sequence of the VHH of the anti-4-1BB antibody is shown in any one of SEQ ID NO: 1-6. The amino acid sequence of the linker peptide L3 is shown in SEQ ID NO:

16. Alternatively, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond. The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide comprises a light chain of an anti-PD-L1 antibody from its N-terminus to its C-terminus. The second polypeptide comprises a heavy chain of an anti-PD-L1 antibody, a linker peptide L4, and a VHH of an anti-4-1BB antibody from its N-terminus to its C-terminus. The amino acid sequence of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO:

10. The amino acid sequence of the heavy chain of the anti-PD-L1 antibody is shown in SEQ ID NO:

18. The amino acid sequence of the VHH of the anti-4-1BB antibody is shown in SEQ ID NO: 3 or 6. The amino acid sequence of the linker peptide L4 is shown in SEQ ID NO:

19. Alternatively, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond. The bispecific antibody comprises a first polypeptide and a second polypeptide. The first polypeptide is the light chain of an anti-PD-L1 antibody. The second polypeptide, from its N-terminus to its C-terminus, consists of a first antigen-binding domain (VH-CH1), a linker peptide (L3), a second antigen-binding domain (VHH), a linker peptide (L1), and the Fc region of IgG4. The amino acid sequence of the light chain of the anti-PD-L1 antibody is shown in SEQ ID NO: 10; the amino acid sequence of VH-CH1 is shown in SEQ ID NO: 20; the amino acid sequence of the linker peptide (L3) is shown in SEQ ID NO: 16; the amino acid sequence of the anti-4-1BB VHH is shown in SEQ ID NO: 3 or 6; the amino acid sequence of the linker peptide (L1) is shown in SEQ ID NO: 8; and the amino acid sequence of the Fc region of IgG4 is shown in SEQ ID NO:

21. Alternatively, the bispecific antibody comprises two identical first polypeptides and two identical second polypeptides, wherein one first polypeptide and one second polypeptide are linked by a disulfide bond, and the two second polypeptides are linked by a disulfide bond. The bispecific antibody comprises a polypeptide consisting of, from N-terminus to C-terminus, a VHH for anti-4-1BB, a linker peptide L1, an Fc region of IgG4, a linker peptide L3, and an scFv for anti-PD-L1. The VHH comprises the sequence shown in SEQ ID NO: 3 or 6, the amino acid sequence of the linker peptide L1 is shown in SEQ ID NO: 8, the amino acid sequence of the Fc region of IgG4 is shown in SEQ ID NO: 15, the amino acid sequence of the linker peptide L3 is shown in SEQ ID NO: 16, and the amino acid sequence of the anti-PD-L1 scFv is shown in SEQ ID NO:

17. Alternatively, the bispecific antibody comprises two identical polypeptides linked by a disulfide bond.

6. A biomaterial, comprising: 1) A nucleic acid molecule encoding the bispecific antibody or a portion thereof as described in any one of claims 1 to 5; 2) An expression vector comprising the nucleic acid molecule described in 1); 3) Host cell, comprising the nucleic acid molecule described in 1) or the expression vector described in 2).

7. The method for preparing the bispecific antibody according to any one of claims 1 to 5 is as follows: 1) Chemical synthesis method: Based on the amino acid sequence of the bispecific antibody, it is prepared through chemical synthesis; 2) Biosynthesis method: culturing the host cells of claim 6 to express bispecific antibodies; optionally, further comprising isolating antibodies from the obtained culture; and purifying the antibodies.

8. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 5; optionally, further comprising a pharmaceutically acceptable excipient; optionally, the pharmaceutically acceptable excipient is a pharmaceutically acceptable excipient, diluent, or carrier.

9. The use of the bispecific antibody according to any one of claims 1 to 5, the biomaterial according to claim 6, or the pharmaceutical composition according to claim 8 in the preparation of a medicament.

10. The application according to claim 9, wherein, The drug is used to treat or prevent tumors or viral infections, such as melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colon cancer, prostate cancer, stomach cancer, kidney cancer, bladder cancer, pancreatic cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, bile duct cancer, thyroid cancer, hepatocellular carcinoma, and mesothelioma, and the viral infections include hepatitis C and hepatitis B.

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