Bispecific antibodies targeting SIRPα and PD-L1, or their antigen-binding fragments, and applications

Abstract

Bispecific antibodies or antigen-binding fragments thereof targeting SIRPα and PD-L1 and their applications. The bispecific antibody comprises an SIRPα-binding domain and a PD-L1-binding domain. The SIRPα-binding domain comprises a heavy-chain variable region and a light-chain variable region, and the PD-L1-binding domain comprises a VHH fragment. Further provided are drugs comprising the bispecific antibody or antigen-binding fragment thereof targeting SIRPα and PD-L1, as well as nucleic acid molecules, vectors, host cells transformed with the vectors, and pharmaceutical uses of the antibodies.

Description

Detailed description of the invention

[0001] [Technical field] The present invention relates to the field of biomedical technology, and more specifically to bispecific antibodies or antigen-binding fragments thereof targeting SIRPα and PD-L1, as well as their applications.

[0002] [Background technology] PD-1 (CD279) was first reported in 1992. The human PD-1 coding gene, PDCD1, is located at 2q37.3, has a full length of 2097 bp, and consists of six exons. PD-1 is a membrane protein belonging to the CD28 immunoglobulin superfamily and is mainly expressed on the surface of activated T cells. It is also expressed in small amounts on thymic CD4-CD8-T cells, activated NK cells, and monocytes. PD-1 has two ligands, PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) of the B7 protein family, and PD-L1 and PD-L2 have 40% identical amino acid sequences. The main difference between the two lies in their modes of expression. PD-L1 is constitutively low-expressed in APCs, non-hematopoietic cells (e.g., vascular endothelial cells, pancreatic islet cells), and immune-privileged sites (e.g., placenta, testes, and eyes), while inflammatory cytokines such as type I and type II interferons, TNF-α, and VEGF can induce PD-L1 expression. PD-L2 is expressed only in activated macrophages and dendritic cells. After PD-1 and PD-L1 bind to activated T cells, the ITSM motif of PD-1 undergoes tyrosine phosphorylation, which dephosphorylates downstream protein kinases Syk and PI3K, inhibiting the activation of downstream pathways such as AKT and ERK. Ultimately, this inhibits the transcription and translation of genes and cytokines necessary for T cell activation, thus negatively regulating T cell activity. In tumor cells, the tumor cell microenvironment negatively regulates T cell activity and inhibits the immune response by upregulating PD-L1 expression and binding to PD-1 on the surface of tumor-specific CD8+ T cells. There is growing evidence that tumors utilize PD-1-dependent immunosuppression for immune evasion. High expression of PD-L1 and PD-L2 has been observed in various solid tumors and hematological malignancies. Furthermore, there is a strong correlation between PD-L expression and poor prognosis of tumor cells.

[0003] Phagocytosis of tumor-associated macrophages (TAMs) in the tumor microenvironment is inhibited because the CD47 protein is highly expressed on the surface of almost all tumor cells. It binds to signal regulatory protein α (SIRPα) on the surface of bone marrow cells, sending a "don't eat me" or "self" signal to the body, thereby inhibiting phagocytosis. CD47, also known as an integrin-associated protein (IAP), is a widely expressed transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. CD47 has a molecular weight of 50 kD and its structure includes a large glycosylated N-terminal IgV variable domain, five highly hydrophobic transmembrane domains, and one short C-terminal cytoplasmic tail. The expression of CD47 in various tissues is determined by four alternative splicing configurations of the C-terminal cytoplasmic tail. The corresponding SIRPα, also known as SHPS-1, BIT, or CD172a protein, is a transmembrane protein primarily expressed in myeloid cells, including macrophages, bone marrow dendritic cells, granulocytes, mast cells, and their precursor cells. SIRPα consists of three extracellular Ig-like domains and four intracellular tyrosine residues, which are presumed to be phosphorylation sites. After phosphorylation, SIRPα is activated by binding to the SH2 domain of the SHP-1 / 2 protein, thereby activating downstream signaling pathways. Because the expression of SHP-1 and SHP-2 proteins is tissue-specific, SIRPα is a docking protein that mobilizes and activates downstream protein phosphatases in response to extracellular stimuli. Oldenborg first reported that mature red blood cells (RBCs) protect themselves from the removal of splenic macrophages by binding to SIRPα via CD47.Subsequently, researchers discovered that SIRPα on RBCs can also bind to monocyte SIRPα, inhibiting Fcγ receptor-dependent phagocytosis, which is achieved by dephosphorylating myosin-IIA, a key molecule in phagocytosis. Clinically, high expression of CD47 is observed in various solid tumors and hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), non-Hodgkin lymphoma (NHL), breast cancer, bladder cancer, ovarian cancer, and colon cancer, essentially allowing tumor cells to evade macrophage cellular clearance through the aforementioned regulatory mechanism. CD47 also influences other biological processes through binding to other receptors or signal transduction in its intracellular cytoplasmic domain. The interaction between CD47 and thrombospondin-1 (TSP-1) or vascular endothelial growth factor receptor 2 (VEGFR-2) inhibits angiogenesis, thereby limiting tumor growth.

[0004] Due to the biological function of CD47 itself, it has been determined that CD47 therapeutic antibodies and SIRPα-Fc recombinant protein may carry a risk of causing hematological toxicity or anemia, as reported in CD47 gene knockout NOD mice and mouse models treated with CD47 antibodies. Furthermore, endothelial cell CD47 has been reported to interact with SIRPγ via cell adhesion and promote transendothelial migration of T cells, although SIRPγ is primarily expressed in T cells rather than bone marrow cells. Therefore, using SIRPα antibodies is a more preferable choice for blocking the CD47-SIRPα signaling pathway. Furthermore, the Weissman research group at Stanford University demonstrated that the combination of the screened humanized SIRPα antibody KWAR23 and rituximab effectively inhibits the growth of Burkitt lymphoma in SRG mice (Rag2- / -Il2rγ- / -) with the human SIRPα gene knocked in, but KWAR23 alone does not show any clear therapeutic effect.

[0005] [Overview of the prefecture] [Means for solving the problem] The first object of the present invention is to provide a bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1. The bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 provided by the present invention comprises a SIRPα-binding domain and a PD-L1-binding domain, wherein the SIRPα-binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises VHCDR1, VHCDR2, and VHCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, 4, and 5, respectively, the light chain variable region comprises VLCDR1, VLCDR2, and VLCDR3 whose amino acid sequences are shown in SEQ ID NO: 37, 38, and 9, respectively, and the PD-L1-binding domain comprises a VHH fragment, the VHH fragment comprises CDR1, CDR2, and CDR3 whose amino acid sequences are shown in SEQ ID NO: 63, 64, and 65, respectively.

[0006] Optionally, the sequence of the heavy chain variable region of the SIRPα-binding domain may be as shown in SEQ ID NO:17, or have at least 85% sequence identity thereto, or the sequence of the light chain variable region of the SIRPα-binding domain may be selected from SEQ ID NO:18, or have at least 85% sequence identity thereto.

[0007] Optionally, the sequence of the VHH fragment may be as shown in SEQ ID NO:62, or have at least 85% sequence identity thereto.

[0008] Optionally, the bispecific antibody or its antigen-binding fragment further comprises a heavy chain constant region selected from human IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and a light chain constant region selected from human κ chain, λ chain or a variant thereof.

[0009] Optionally, the heavy chain constant region includes an Fc fragment or a variant thereof, the Fc fragmentary variant being derived from IgG1 and, according to EU counts, containing mutation sites: L234A, L235A, and K338A.

[0010] Optionally, the bispecific antibody or its antigen-binding fragment comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region of the SIRPα-binding domain, a heavy chain constant region, and a VHH fragment, the VHH fragment being fused to the N-terminus of the heavy chain variable region of the SIRPα-binding domain, or the VHH fragment being fused to the C-terminus of the heavy chain constant region, and the second polypeptide chain comprises a light chain variable region of the SIRPα-binding domain and a light chain constant region.

[0011] Optionally, the bispecific antibody or its antigen-binding fragment comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a heavy chain variable region and a heavy chain constant region of the SIRPα-binding domain, and the second polypeptide chain comprises a light chain variable region, a light chain constant region, and a VHH fragment, wherein the VHH fragment is fused to the N-terminus of the light chain variable region of the SIRPα-binding domain.

[0012] Optionally, the bispecific antibody or its antigen-binding fragment has a symmetric structure comprising two first polypeptide chains and two second polypeptide chains.

[0013] Optionally, the bispecific antibody or its antigen-binding fragment further comprises a linking sequence, the linking sequence being (GGGGS)n, where n is an integer from 1 to 4. Optionally, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 66, 26, 69, 84, or 85, or the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 67, 68, 82, or 83.

[0014] Optionally, the amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 66, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 67.

[0015] The second object of the present invention is to provide a drug comprising a bispecific antibody targeting the above-mentioned SIRPα and PD-L1 or an antigen-binding fragment thereof.

[0016] Optionally, the drug further comprises one or more other cancer therapeutic agents. The third object of the present invention is to provide a nucleic acid molecule encoding a bispecific antibody targeting the above-mentioned SIRPα and PD-L1 or an antigen-binding fragment thereof.

[0017] The fourth object of the present invention is to provide a vector comprising the above nucleic acid molecule. The fifth object of the present invention is to provide a host cell transformed with the above vector.

[0018] The sixth object of the present invention is to provide the use of a bispecific antibody targeting the above-mentioned SIRPα and PD-L1 or an antigen-binding fragment thereof in the preparation of a drug for inhibiting or treating a disease, disorder or condition.

[0019] Optionally, the disease, disorder or condition includes cancer, solid tumor, chronic infection, inflammatory disease, multiple sclerosis, autoimmune disease, nervous system disorder, brain injury, nerve injury, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, graft dysfunction or arthritis.

[0020] Optionally, the cancers include anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureteral cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, and skin cancer. The following are selected: prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, T-cell or B-cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, hepatocellular carcinoma, or adenocarcinoma.

[0021] The drug may be used in combination with one or more other drugs, at the discretion of the user. Optionally, the other drugs include rituximab. [Effects of the invention] Compared to the prior art, the present invention has at least the following beneficial effects.

[0022] (1) The bispecific antibodies provided by the present invention can simultaneously target SIRPα and PD-L1, targeting tumor cells while simultaneously mediating the killing of immune cells, and by targeting SIRPα, the risk of hematological toxicity or anemia can be avoided.

[0023] (2) The SIRPα-binding domain sequence of the bispecific antibody of the present invention is novel. (3) The bispecific antibodies provided by the present invention have a unique structure, can target target proteins with high efficiency, and can achieve an efficient tumor-killing effect.

[0024] (4) The bispecific antibodies provided by the present invention can bind to all subtypes of the human SIRPα protein and are beneficial for clinical development. [Brief explanation of the drawing]

[0025] [Figure 1] This is the result of the Binding-ELISA detection. [Figure 2] This is the result of the Binding-ELISA detection. [Figure 3] This is the result of the Binding-ELISA detection. [Figure 4] This is the result of the Binding-ELISA detection. [Figure 5] This is the result of the Binding-ELISA detection. [Figure 6] This is the result of the Binding-ELISA detection. [Figure 7] This is the result of the Blocking-ELISA detection. [Figure 8] This is the result of FACS detection of a SIRPα antibody that binds to human renal cleavage cell adenocarcinoma 786-O cells that naturally express human SIRPa. [Figure 9] This is the ADCP result of an in vitro functional experiment of an anti-SIRPα antibody. [Figure 10] This is the ADCP result of an in vitro functional experiment of an anti-SIRPα antibody. [Figure 11] This is the ADCP result of an in vitro functional experiment of an anti-SIRPα antibody. [Figure 12] The tumor imaging signal values ​​in each group correspond to the tumor growth curve and the D18 imaging signal intensity. [Figure 13] These are the survival curves for each group. [Figure 14] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 15] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 16]This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 17] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 18] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 19] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 20] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 21] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 22] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 23] This figure shows the results of ELISA detection demonstrating the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. [Figure 24] This is an amino acid sequence alignment diagram of known human SIRPα-binding domain alleles. [Figure 25]This is a binding curve diagram between antibody molecules detected by ELISA and human PD-L1 protein. [Figure 26] This is a binding curve diagram between antibody molecules detected by ELISA and human SIRPα V1 protein. [Figure 27] This is a binding curve diagram between antibody molecules detected by ELISA and human SIRPα V2 protein. [Figure 28] This is a coupling curve diagram of antibody molecular blocks PD-L1 and PD-1 detected by ELISA. [Figure 29] This is a coupling curve diagram between the antibody molecule block CD47 detected by ELISA and SIRPα. [Figure 30] This graph shows the results of FACS detection of antibody molecules that synergistically enhance rituxan-dependent ADCP of CD20 antibodies. [Figure 31] This figure shows the results of IL-2 secretion induced by antibody molecules against 48 hours of SEB-stimulated PBMC proliferation. [Figure 32] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 33] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 34] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 35] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 36] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 37] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 38] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 39] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 40] These are protein binding curves for molecules such as Q-1801 detected by ELISA and for human SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 subtypes. [Figure 41] This is a binding curve diagram between molecules such as Q-1801 detected by FACS and human SIRPαV1. [Figure 42] This is a binding curve diagram between molecules such as Q-1801 detected by FACS and human SIRPαV2. [Figure 43] This is a binding curve diagram between molecules such as Q-1801 detected by ELISA and human PD-L1 protein. [Figure 44] This is a binding curve diagram between molecules such as Q-1801 detected by FACS and human PD-L1. [Figure 45] This is a curve diagram showing the ELISA detection of molecules such as Q-1801 that block SIRPα / CD47 binding. [Figure 46] This figure shows the results of ELISA detection of molecules such as Q-1801 that block PD-1 / PD-L1 binding. [Figure 47] This figure shows the results of ELISA detection of molecules such as Q-1801 that block PD-L1 / CD80 binding. [Figure 48] This figure shows the results of Q-1801 synergistically enhancing the CD20 antibody rituxan-dependent ADCP effect. [Figure 49] This figure shows the results of Q-1801 synergistically enhancing the CD20 antibody rituxan-dependent ADCP effect. [Figure 50] This is a diagram showing the results of IL-2 secretion in the supernatant of a 48-hour mixed lymphocyte reaction. [Figure 51] This is a diagram showing the results of IFN-γ secretion in the supernatant of a 120-hour mixed lymphocyte reaction. [Figure 52] This figure shows the results of IL-2 secretion by molecules such as Q-1801 in response to 48 hours of SEB-stimulated PBMC proliferation. [Figure 53] This figure shows the results of IFN-γ secretion by molecules such as Q-1801 in response to 120 hours of SEB-stimulated PBMC proliferation. [Figure 54] This graph shows the tendency for tumor growth after drug administration. [Figure 55] This graph shows the trend in weight change in animals after drug administration. [Figure 56] These are biological imaging images of mice on day 0 after grouping. [Figure 57] These are biological imaging images of mice seven days after grouping. [Figure 58] These are biological imaging images of mice 14 days after grouping. [Figure 59] This graph shows the trend in tumor volume changes after drug administration in the MC38-hPD-L1 colon cancer tumor model. [Figure 60] These are curve diagrams showing tumor growth in individual mice in different groups within the MC38-hPD-L1 colon cancer tumor model. [Figure 61] This is a curve diagram showing the change in body weight of mice in each group after drug administration in the MC38-hPD-L1 colon cancer tumor model. [Figure 62] This graph shows the trend in tumor volume changes after drug administration in a CT26-hPD-L1&hSIRPα colon cancer model. [Figure 63] These are curve diagrams showing tumor volume growth in each mouse after drug administration in the CT26-hPD-L1&hSIRPα colon cancer model. [Figure 64] This is a curve diagram showing the change in mouse body weight in each group after drug administration in the CT26-hPD-L1&hSirpα tumor model. [Figure 65] This graph shows the trend in tumor volume changes when mice whose tumors had regressed after the initial drug administration were reinoculated. [Figure 66] These are curve diagrams showing the tumor volume growth of each mouse after re-inoculation with CT26-hPD-L1 and hSIRPα for colon cancer. [Figure 67] This is a curve diagram showing the changes in body weight of mice in each group after reinoculation with CT26-hPD-L1 and hSIRPα. [Figure 68] This graph shows the changes in tumor volume in different dose groups of noncellular lung cancer HCC827 models administered subcutaneously to NCG mice. [Figure 69] This graph shows the weight changes of mice from different groups. [Modes for carrying out the invention]

[0026] term: An antibody (Ab) is an immunoglobulin molecule (Ig) that contains at least one antigen-binding site and is capable of specifically binding to an antigen.

[0027] An "antigen" is a substance that induces an immune response in the body and specifically binds to an antibody. The binding of an antibody to an antigen is mediated by interactions that form between them, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The binding region between the antigen surface and the antibody is called an "antigen determinant" or "epitope," and generally, each antigen has multiple determinants.

[0028] "Fusion" refers to the linking of components by peptide bonds, either directly or via one or more peptide linkers. Various components of an antibody molecule are linked by "peptide linkers," ensuring correct protein folding and peptide stability. "Peptide linkers" can be selected from amino acid sequences with low immunogenicity. In this specification, "peptide linker" and "linking sequence" have the same meaning. The linking sequence links each component portion of the fusion protein, and in specific implementations, appropriate linking sequences such as (GS)n, (GSGGS (SEQ ID NO: 87))n, (GGGS (SEQ ID NO: 88))n, (GGGGS (SEQ ID NO: 89))n can be selected. n can be selected from 1 to 4 or more.

[0029] The term “antibody” as used in this invention is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) containing at least two different antigen-binding domains. Antibodies further include mouse antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. Antibodies of this invention may originate from any animal, including but not limited to human, non-human primate, mouse, rat, cattle, horse, chicken, camel, and alpaca immunoglobulin molecules. Antibodies may contain additional modifications such as non-natural amino acids, mutations in Fc effector function, and mutations in glycosylation sites. Antibodies further include post-translationally modified antibodies, fusion proteins containing antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to antigen recognition sites, insofar as these antibodies exhibit the desired biological activity.

[0030] The basic structure of conventional antibodies is a Y-shaped monomer consisting of two completely identical heavy chains (H) and two completely identical light chains (L) linked by disulfide bonds. Each chain is composed of 2 to 5 domains (functional regions) with similar sequences but different functions, each containing approximately 110 amino acids. In antibody molecules, the amino acid sequences near the N-terminus of the light and heavy chains change significantly, and the resulting domain is called the variable region (V region), while the region near the C-terminus where the amino acid sequence remains relatively constant is called the constant region (C region).

[0031] The V regions of the heavy and light chains are called VH and VL, respectively. VH and VL each have three amino acid compositions, and their sequence order is highly variable, so they are called hypervariable regions (HVRs). These regions form a spatial structure complementary to the antigen epitope and are also called complementarity determining regions (CDRs). The three CDRs of VH are represented as VHCDR1, VHCDR2, and VHCDR3, respectively, and the three CDRs of VL are represented as VLCDR1, VLCDR2, and VLCDR3, respectively. A total of six CDRs from VH and VL together form the antigen-binding site. The diversity of amino acids in the CDR regions is the molecular basis for antibodies to specifically bind to a large number of different antigens. The amino acid composition and sequence order outside the CDRs of the V regions are relatively less variable and are called framework regions or framework regions (FRs). VH and VL have four framework regions represented by FR1, FR2, FR3, and FR4. VH and VL are each composed of three CDRs and four FRs, and the sequence order from the amino group terminus (N-terminus) to the carboxyl group terminus (C-terminus) is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0032] Human immunoglobulins can be classified into five categories—IgM, IgG, IgA, IgD, and IgE—according to the amino acid sequence of the constant region of the antibody heavy chain. These can be further divided into different subtypes (isotypes); for example, human IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Subtypes of IgM, IgD, and IgE have not been discovered. Based on the light chain amino acid sequence, the light chain can be classified into κ and λ chains. The antibodies of this invention may be any type (e.g., IgM, IgG, IgA, IgD, IgE) or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2).

[0033] The constant regions of the heavy and light chains are called CH and CL, respectively. The heavy chain constant regions of IgG, IgA, and IgD have three domains: CH1, CH2, and CH3, while the heavy chain constant regions of IgM and IgE have four domains: CH1, CH2, CH3, and CH4.

[0034] The area between CH1 and CH2 is a hinge region, rich in proline, making it easily stretchable and bendable. This allows for changes in the distance between the two Y-shaped arms, which is advantageous for simultaneously binding both arms to the antigen epitope.

[0035] An "antigen-binding fragment" refers to Fab fragments, F(ab')2 fragments, Fv fragments, ScFv fragments, etc., that possess antigen-binding activity. A "Fab fragment" (fragment of antigen binding, Fab) refers to an antibody fragment consisting of VL, VH, CL, and CH1 domains that bind to a single antigen epitope (monovalent). Those skilled in the art know that papain hydrolyzes IgG to form two identical Fab segments and one Fc segment, and that pepsin hydrolyzes IgG to form one F(ab')2 segment and several polypeptide fragments (pFc'). When the F(ab')2 inter-helicopter disulfide bond is cleaved, two Fab' fragments can be formed, the latter of which can be enzymatically cleaved into Fv fragments. The Fv fragment contains the heavy chain variable region and light chain variable region of the antibody, but does not contain the constant region. A single-chain variable fragment scFv (single-chain antibody fragment), or single-chain antibody, is formed by linking the heavy chain variable region and light chain variable region of the antibody via a linker.

[0036] In 1993, the Hamers Laboratory discovered that, in addition to conventional quadruple antibodies, camel serum also contains a large number of molecules similar to immunoglobulin G (IgG). These molecules are called heavy-chain antibodies (HCAbs), and while they naturally lack the constant CH1 region of the light and heavy chains of conventional antibodies, they still possess a strong binding ability to antigens. The Hamers Laboratory analyzed and identified the structure and sequence of heavy-chain antibodies in camel serum, finding that the antigen-binding region of heavy-chain antibodies consists solely of a variable region fragment functionally equivalent to the antigen-binding fragment (Fab) of conventional antibodies. Therefore, the antigen-recognition region fragment of heavy-chain antibodies is called VHH (variable domain of the heavy-chain antibody, VHH), and based on this, nanobodies containing only the VHH domain are developed. Nanobodies are also called single-domain antibodies (sdAbs).

[0037] Nanobodies are readily modified to form multivalent forms. Because of their small molecular weight, nanobodies are encoded by a single gene, making genetic manipulation easy. Furthermore, multiple nanobodies can be polymerized through short linkage sequences, and they can be linked and combined with conventional antibody Fab fragments, Fv fragments, ScFv fragments, etc., to form multivalent or multispecific antibody structures. Bivalent or multivalent antibodies can recognize the same epitope but have higher affinity than monovalent antigens. Bispecific or multispecific antibodies can bind to different targets or different binding regions on the same target, and have stronger antigen recognition ability than monovalent antibodies.

[0038] Nanobodies can readily form novel fusion molecules with other structures (e.g., BSA, IgG-Fc, etc.). In these novel fusion molecules, the nanobodies bind directionally to their target antigens, and the fused portion exerts its corresponding function. Therefore, they can be used in combination with other drugs or as a tool for diagnostic and experimental research in various fields. Nanobody screening can be divided into steps such as alpaca immunization, lymphocyte extraction, nanobody library construction, phage library construction, specific phage screening, E. coli expression, and antibody purification.

[0039] The terms "Fc," "Fc segment," or "Fc fragment" refer to a crystallizable fragment that does not possess antigen-binding activity and is the interaction site between the antibody and the effector molecule or cell surface Fc receptor (FcR). An Fc fragment contains the constant region polypeptide of an antibody, excluding the heavy chain constant region CH1. Fc fragments bind to cells that have the corresponding Fc receptor on their surface and produce various biological effects. In the ADCC effect (antibody-dependent cell-mediated cytotoxicity), the Fab segment of the antibody binds to the antigenic epitope of virus-infected cells or tumor cells, and its Fc segment binds to the FcR on the surface of the killer cell (NK cell, macrophage, etc.), directly killing the target cell via the killer cell. ADCP is antibody-dependent cellular phagocytosis, and its mechanism involves the activation of the FcγR mechanism on the surface of macrophages by target cells acting on an antibody, inducing phagocytosis, which leads to the target cells' internal migration and subsequent acidification and degradation by phagosomes. Under certain circumstances, the removal of antibody Fc function may be beneficial. These circumstances include the use of (1) receptor agonists that induce cellular signaling, (2) receptor antagonists that block receptor and ligand binding to inhibit signaling, or (3) antibodies as drug vectors to deliver drugs to target cells expressing the corresponding antigen. If Fc function is maintained, antibody drugs may inadvertently damage cells expressing the corresponding receptor, or antibody-coupling drugs may inadvertently damage important immune cells when they are off-target.

[0040] Fc variants or mutation combinations are not limited to the following forms (according to EU counts):

[0041] [Table 1] TIFF0007853730000002.tif244170TIFF0007853730000003.tif79170

[0042] Currently, mouse-derived antibodies are a major source of antibody drugs. Because mouse-derived antibodies are immunogenic, they are typically humanized. The following examples provide mouse-derived antibodies, chimeric antibodies, and humanized antibodies. A "chimeric antibody" is an antibody formed by fusing the variable region of a mouse-derived antibody with the constant region of a human antibody, and can reduce the immune response induced by mouse-derived antibodies. The constant region of the human antibody is a heavy chain constant region selected from human IgG1, IgG2, IgG3, IgG4 or their variants, and a light chain constant region selected from human κ, λ chains or their variants. A "humanized antibody" is an antibody obtained by transplanting the CDR sequence of a mouse-derived antibody into the variable region framework of a human antibody, and can overcome the strong reaction caused by chimeric antibodies that hold a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available reference literature. To avoid a decrease in activity due to reduced immunogenicity, activity can be maintained by performing minimal retrograde or reverse mutations on the human antibody variable region framework sequence.

[0043] Theoretically, increasing antibody affinity improves antibody specificity and efficacy, leading to reduced drug dosage and mitigated toxic side effects. Actual research has shown that, particularly in the treatment of solid tumors, an increase in affinity and an increase in antibody titer are not always linearly related; however, in many cases, this linear relationship is clearly present. The humanized antibodies of this invention also include humanized antibodies that have undergone affinity maturation of CDRs by phage display. The theoretical basis for in vitro antibody affinity maturation is to mimic the process of in vivo antibody affinity. By constructing a random mutation library and mimicking the high-frequency mutations in B cells within the body, high-affinity antibodies can be screened.

[0044] The drugs provided by the present invention may contain a "therapeutic dose" of an antibody or antigen-binding fragment. "Therapeutic dose" refers to the amount of a therapeutic agent that effectively prevents or alleviates a specific disease, and may vary based on various factors, including the patient's disease state, age, and weight, as well as the drug's ability to produce the desired effect on different patients.

[0045] "Sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides, to the extent that the two polynucleotides or two polypeptides share the same base or amino acid. In this invention, "having at least 85% sequence identity" refers to at least 85%, 90%, 95%, 97%, or 99% identity.

[0046] In this specification, amino acid substitutions are named by a single-letter amino acid code, followed by the amino acid position, and then the single-letter amino acid code of the substitution. For example, L234A refers to the substitution of the L amino acid at position 234 with A.

[0047] The gene encoding SIRPα is a polymorphic gene, and 10 SIRPα variants are known in the human population. Katsuto Takenaka et al. discovered 10 different SIRPα IgV-coding alleles by sequencing the IgV-coding SIRPα domain of 37 unrelated normal Caucasians, Africans, Chinese, and Japanese individuals from the Human HapMap Genome Project (Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells, NATURE IMMUNOLOGY VOLUME 8 NUMBER 12 DECEMBER 2007). The 10 SIRPα variants are the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 subtypes. Although SIRPalpha is highly polymorphic, amino acid sequence alignments of known human SIRPalpha alleles by ChiaChiM.Ho et al. revealed two unique sequences at the binding interface between SIRPα and CD47, corresponding to allele types V1(a2d1) and V2(a1d1), respectively. ("Velcro" Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis, JOURNAL OF BIOLOGICAL CHEMISTRY, VOLUME 290·NUMBER 20·MAY 15, 2015).

[0048] As shown in Figure 24, amino acid sequence alignment of known human SIRPα-binding domain alleles revealed only two variants at the CD47 contact interface: a1d1 and a2d1. The first line of text in Figure 24 is the amino acid sequence of the most important human SIRPα allele V1 (a2d1), and the second line of text in Figure 24 is the amino acid sequence of the most important human SIRPα allele V2 (a1d1). Black boxes indicate CD47-interacting residues, and shading indicates residues different from the V1 sequence. Janet Sim et al. identified two SIRPα variants v1 and v2 using SIRPα sequences from 2535 individuals and Sanger sequencing of 510 samples, representing three gene groups: homozygous v1 / v1, homozygous v2 / v2, and heterozygous v1 / v2. The distribution and frequency of SIRPα v1 and v2 allele clusters are determined in various populations and unrelated subpopulations. Here, the distribution of v1 / v2 heterozygotes in the five superpopulations of Europe (EUR), the United States (AMR), East Asia (EAS), Africa (AFR), and South Asia (SAS) is similar, with a distribution range of 42.0% to 47.2%. In the East Asian population, the number of v2 / v2 cases is significantly higher than that of v1 / v1 cases, with incidences of 42.3% and 13.3%, respectively. In Africans, Europeans, Americans, and South Asians, the number of v1 / v1 cases is higher than that of v2 / v2 cases, with incidence ranges of 30.3% to 49.1% and 8.9% to 24.2%, respectively (see MABS, 2019, VOL.11, NO.6, 1036¨C1052, https: / / doi.org / 10.1080 / 19420862.2019.1624123 for references). Aduro Biotech also studied that the prevalence of v2 / v2 homozygosity in the East Asian population is 41.3%, and the prevalence of v1 / v1 homozygosity is 34.6%, thus demonstrating that 41.3% of the East Asian population is V2 / V2 homozygous (see Voets et al. Journal for ImmunoTherapy of Cancer (2019) 7:340 for reference).

[0049] Based on the results of SIRPα polymorphism analysis, anti-SIRPα antibodies can simultaneously bind to SIRPα v1 and SIRPa v2 genes, which is important for accelerating clinical development.

[0050] The technical solutions of the present invention will be described in detail below, along with specific embodiments. In the following embodiments, experimental methods that do not specify particular conditions are based on conventional conditions, conditions recommended by the raw material or product manufacturer, or biotechnology textbooks such as molecular cloning, laboratory manuals, Cold Spring Harbor Laboratory, modern molecular biology methods, and cell biology. Reagents whose specific source is not indicated are conventional reagents purchased through commercial channels.

[0051] Tables 1 and 2 below show the molecules and cell lines used in this study.

[0052] [Table 2]

[0053] [Table 3]

[0054] Acquisition of anti-SIRPα antibodies Example 1: Acquisition of anti-SIRPα mouse antibody (1) Mouse immunization: Anti-human SIRPα monoclonal antibodies are produced by immunizing mice. The experiment uses Balb / c white female mice, 6 weeks old. Rearing environment: SPF level. After purchasing the mice, they are reared in a laboratory environment for one week, with a 12 / 12 hour light-dark cycle, temperature 20-25°C, and humidity 40-60%. After immunizing the Balb / c mice, they are first immunized with recombinant protein QP009(SIRPα) 50 μg / mice using complete Freund's adjuvant (CFA). Two weeks later, they are then mutually immunized weekly with QP009(SIRPα) + complete Freund's adjuvant (IFA) or QP009(SIRPα) + aluminum salt Alum+CpG ODN 1826 at 25 μg / mice.

[0055] QP009(SIRPα) has the following amino acid sequence (SEQ ID NO:1).

[0056] [ka]

[0057] (2) Cell fusion: Mice with high serum antibody titers are selected for splenocyte fusion. 72 hours prior to fusion, the selected mice are immunized by intraperitoneal injection of Dash. Hybridoma cells are obtained by fusion of splenic lymphocytes and myeloma Sp2 / 0 cells using an optimized PEG-mediated fusion step. The fused hybridoma cells are resuspended in HAT complete medium (IMDM medium containing 20% ​​FBS, 1× HAT, and 1× OPI), dispensed into 96-well cell culture plates (1×10⁵ cells / 150 μl / well), and cultured at 37°C and 5% CO₂. Five days post-fusion, 50 μl / well of IMDM medium with 20% FBS (containing 2× HAT and 1× OPI) is added, and the cells are cultured at 37°C and 5% CO₂. On days 7-8 of fusion, all fluids are replaced according to the cell growth density. The culture medium is HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT and 1×OPI), 250 μl / well, and cultured at 37°C and 5% CO2.

[0058] (3) Screening of hybridoma cells: Depending on the cell growth density, ELISA detection is performed 10 to 14 days after fusion to screen for anti-SIRPα antibodies in the hybridoma supernatant. Supernatant is collected from the hybridoma fusion wells, and preliminary screening of the entire 96-well plate is performed by ELISA. Anti-SIRPα antibodies in the detected supernatant can block the binding of SIRPα / CD47, which is a hole in the preliminary screening. Supernatant is collected from the preliminary screening positive wells, and binding to QP009 (SIRPα) is detected by ELISA. Clones that are positive for binding to SIRPα and blocking of SIRPα / CD47 binding are selected, i.e., anti-SIRPα antibody positive clone wells. The positive clones are enlarged and transferred to a 24 / 6-well plate in a timely manner, and the cell culture supernatant is detected again by ELISA. Clone wells that are positive for binding to SIRPα and blocking of SIRPα / CD47 binding are detected, i.e., anti-SIRPα antibody positive clone wells. Positive clones are diluted to the limit 2-3 times to obtain single-cell clones, and the positive single-cell line is cryopreserved to obtain single-cell clone 71C10.

[0059] (4) Hybridoma monoclonal antibody sequencing to obtain antibody sequences: Hybridoma-positive monoclonal cell line 71C10 was obtained, mRNA was extracted, the mRNA was reverse transcribed into cDNA, the cDNA was used as a template for PCR amplification, PCR-positive clones were selected and sent for sequencing, and the variable region sequences of the light and heavy chains of the monoclonal antibody were obtained through sequence analysis. The number and position of CDR amino acid residues conform to known Kabat numbering rules.

[0060] The heavy chain variable region sequence of 71C10 is SEQ ID NO:2, and is specifically as follows:

[0061] [ka]

[0062] Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The bolded and underlined parts are VHCDR1 (SEQ ID NO:3), VHCDR2 (SEQ ID NO:4), and VHCDR3 (SEQ ID NO:5), respectively.

[0063] The light chain variable region sequence of 71C10 is SEQ ID NO:6, and is specifically as follows:

[0064] [ka]

[0065] Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The bolded and underlined parts are VLCDR1 (SEQ ID NO:7), VLCDR2 (SEQ ID NO:8), and VLCDR3 (SEQ ID NO:9), respectively.

[0066] Example 2: Anti-SIRPα Chimeric Antibody SPR Detection Affinity (1) A chimeric antibody molecule is obtained by fusing the mouse-derived variable region sequence of monoclonal cell line 71C10 with a human constant region gene. The antibody light chain uses the kappa light chain constant region CL. Simultaneously, various antigen sequences are designed for performance testing of the antibody molecule. The molecular cloning designs of the antigen and chimeric antibody are shown in Tables 3 and 4.

[0067] [Table 4] TIFF0007853730000010.tif64170

[0068] Note: Antibodies with protein numbers QP026027 and QP026249 were used as control antibodies. Both use the variable region sequence of the known anti-SIRPα antibody KWAR23, with the difference being in the constant region. QP163164 and QP163245 both use the variable region of the monoclonal cell line 71C10, with the difference being in the constant region. The sequences shown in the above sequence numbers represent the heavy chain and light chain sequences of each antibody molecule, respectively.

[0069] pQD is the vector name for a combination of a signal peptide and a constant region gene (CH1-FC / CL) fragment, where pQDH is used for ligation and expression of the heavy chain variable region and contains the signal peptide and the constant region gene (CH1-FC) fragment, and pQDK is used for ligation and expression of the light chain variable region and contains the signal peptide and the constant region gene (CL) fragment. "H" represents the heavy chain and "L" represents the light chain. "(IgG4)" indicates that the heavy chain adopts the constant region of human IgG4. If "(IgG4)" is not marked, the constant region of human IgG1 is used by default. 180122VH represents the heavy chain variable region derived from monoclonal cell line 71C10, and 180122VL represents the light chain variable region derived from monoclonal cell line 71C10.

[0070] For example, "pQDH-KWAR23-H" means that the control sequence KWAR23 is fused to the pQDH vector, and pQDH contains a signal peptide and a constant region gene (CH1-FC) fragment, using the constant region of human IgG1. "pQDH-180122VH" means that the heavy chain variable region sequence 180122VH is fused to the pQDH vector, using the constant region of human IgG1. Specifically, the sequences shown in the above sequence numbers are as follows:

[0071] [ka] Here, the double-underlined portion represents the stationary region array.

[0072] [ka] Here, the single-underlined portion is the signal peptide, and the double-underlined portion is the heavy chain constant region sequence.

[0073] [ka] Here, the single-underlined portion is the signal peptide, and the double-underlined portion is the heavy chain constant region sequence.

[0074] [ka] Here, the double-underlined portion represents the stationary region array.

[0075] [ka] Here, the double-underlined portion represents the stationary region array.

[0076] [ka] Here, the single-underlined portion is the signal peptide, and the double-underlined portion is the heavy chain constant region sequence.

[0077] [Table 5] TIFF0007853730000018.tif35170

[0078] Note: QP098 is the cynomolgus monkey SIRPα sequence (uniprot database sequence number I7G9Z7), QP100 is the cynomolgus monkey SIRPα sequence (uniprot database sequence number G7PGS8), QP271 is the rhesus monkey SIRPα sequence, obtained by the inventors by sequencing monkey PBMCs, and QP273 is the cynomolgus monkey SIRPα sequence, obtained by the inventors by sequencing monkey PBMCs.

[0079] (2) Expression and purification of antigens and chimeric antibodies The culture density of 293E cells is (0.2~3) × 10⁻⁶. 6 The cells were maintained at a concentration of / ml and cultured in maintenance medium (GIBCO Freestyle 293 expression medium). One day before transfection, the cells to be transfected were centrifuged, the medium was changed, and the cell density was increased to (0.5~0.8) × 10⁻¹⁶. 6 Adjust to / ml. On the same day as transfection, the density of 293E cells should be (1~1.5) × 10 6 The amount is / ml. Prepare the plasmid and transfection reagent PEI. The amount of plasmid required for transfection is 100 μg / 100 ml of cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI uniformly and let stand for 15 minutes, not exceeding 20 minutes. Slowly add the plasmid and PEI mixture to 293E cells and culture in a shaker at 8% CO2, 120 rpm, 37°C. On day 5 of transfection, centrifuge at 4700 rpm for 20 minutes using a horizontal centrifuge and collect the cell supernatant.

[0080] Protein A affinity chromatography purification: Pass the equilibrium solution through the column at least 3 CV, ensuring the actual volume is 20 ml, and confirm that the pH and conductivity of the solution effluent from the final instrument match the equilibrium solution, with a flow rate of 1 ml / min. After centrifugation, pass the culture supernatant through the column and load 40 ml of sample, with a flow rate of 0.33 ml / min. Pass the eluent through the column, and when UV280 rises to 15 mAU, begin collecting the elution peak (PAC-EP). When UV280 drops to 15 mAU, stop collecting, with a flow rate of 1 ml / min. After sample collection is complete, adjust the PAC-EP to neutral with pH adjusting solution.

[0081] (3) Surface plasmon resonance (SPR) detection affinity The affinity of the anti-SIRPα chimeric antibody QP163164 to human SIRPα type 1 (protein number QP094) and human SIRPα type 2 (protein number QP096) was measured using Biacore T200(GE). Tables 5 and 6 show the detection results for QP163164 and QP026027. The results show that the SIRPα chimeric antibody QP163164 binds to human SIRPα type 1 with an affinity KD of 5.27E-10M and to human SIRPα type 2 with an affinity KD of 6.78E-10M. The binding affinity to human SIRPα type 1 and human SIRPα type 2 is significantly superior compared to the control antibody KWAR23 (QP026027).

[0082] [Table 6]

[0083] The affinity between the chimeric antibody and cynomolgus monkey SIRPα, as measured by biacore, is shown in the following table.

[0084] [Table 7]

[0085] Example 3: Humanization using anti-SIRPα hybridoma monoclonal antibody By aligning the IMGT Human Antibody Heavy and Light Chain Variable Region Germline Gene Database and MOE software, heavy and light chain variable region germline genes with high homology to QP163164 are selected as templates. The CDRs of mouse-derived antibodies are then transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Several important amino acid residues are selected as combinations of reverse mutations. Here, the amino acid residues are determined and annotated by the Kabat numbering system. In the following examples, the heavy chain FR region sequence is derived from the combined sequence of human germline heavy chains IGHV1-18 and IGHJ2*01, which includes the FR1, FR2, and FR3 regions of human germline heavy chain IGHV1-18 and the FR4 region of IGHJ2*01. The light chain FR region sequence is derived from a combination sequence of human germline light chain IGKV4-1 and IGKJ2*01, which includes the FR1, FR2, and FR3 regions of human germline light chain IGKV4-1 and the FR4 region of IGKJ2*01.

[0086] (1) Cloning of humanized anti-SIRPα antibody molecules Primer PCR is designed to construct the VH / VK gene fragments for each humanized antibody, and homologous recombination is performed with the expression vector pQD (containing the signal peptide and constant region gene (CH1-FC / CL) fragment) to construct the full-length antibody expression vector VH-CH1-FC-pQD / VK-CL-pQD.

[0087] Using the online software DNAWorks (v3.2.2) (http: / / helixweb.nih.gov / dnaworks / ), multiple primers are designed to synthesize VH / VK containing the gene fragments necessary for recombination: 5'-30bp signal peptide + VH / VK + 30bp CH1 / CL-3'. Following the instructions for use of TaKaRa STAR GXL DNA polymerase primers, the above-designed primers are used for two-step PCR amplification to obtain VH / VK containing the gene fragments necessary for recombination. For the construction and enzymatic cleavage of the expression vector pQD, several specialized restriction enzymes such as BsmBI, which have different recognition sequences and restriction sites, are used to design and construct the expression vector pQD. BsmBI is used to enzymatically cleave the vector, cleave the gel, recover, and store it. Construction of heavy chain expression vector pQD-VH-CH1-FC and light chain expression vector pQD-VL-CL: Mix the heavy chain variable region VH gene fragment and the BsmBI enzyme-cleaved vector pQD (containing the signal peptide and heavy chain constant region (CH1-FC) fragment) in a 3:1 ratio, and mix the light chain variable region VL gene fragment and the BsmBI enzyme-cleaved vector pQD (containing the signal peptide and light chain constant region (CL) fragment) in a 3:1 ratio. Transfer each mixture to DH5a competent cells, heat shock in an ice bath at 0°C for 30 minutes and then at 42°C for 90 seconds, add 5 times the volume of LB medium, incubate at 37°C for 45 minutes, spread onto an LB-Amp plate, culture overnight at 37°C, select a single clone and sequence it to obtain the desired clone.

[0088] The following table provides specific information regarding the humanization design of QP163164. The protein expression number is QP256253. In this table, the antibody light chain uses the kappa light chain constant region CL, and the antibody heavy chain uses the human IgG4 constant region (see Example 2 for the specific sequence of the constant region). The humanization design of the light chain and heavy chain variable region sequences is not limited to the sequences shown in the following table.

[0089] [Table 8]

[0090] Note: The light chain variable region of QP256253 is encoded by plasmid number QD253. The specific sequence of the light chain variable region sequence, SEQ ID NO:16, is as follows:

[0091] [ka]

[0092] The heavy chain variable region of QP256253 is encoded by plasmid number QD256. The specific sequence of the heavy chain variable region sequence, SEQ ID NO:17, is as follows:

[0093] [ka]

[0094] (2) Expression of humanized anti-SIRPα antibody The culture density of 293E cells is (0.2~3) × 10⁻⁶. 6 The cells are maintained at a concentration of / ml and cultured in maintenance medium (GIBCO Freestyle 293 expression medium). One day before transfection, the cells to be transfected are centrifuged, the medium is changed, and the cell density is increased to (0.5~0.8) × 10⁻¹⁶. 6 Adjust to / ml. On the day of transfection, the density of 293E cells should be (1~1.5) × 10 6 The amount of plasmid and transfection reagent PEI required for transfection is 100 μg / 100 ml of cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI uniformly and let stand for 15 minutes, not exceeding 20 minutes. Slowly add the plasmid and PEI mixture to 293E cells, culture in a shaker at 8% CO2, 120 rpm, 37°C, and on the 5th day after transfection, centrifuge at 4700 rpm for 20 minutes using a horizontal centrifuge and collect the cell supernatant.

[0095] (3) Purification of humanized anti-SIRPα antibody protein Protein A affinity chromatography purification: Pass the equilibrium solution through the column at least 3 CV, the actual volume being 20 ml, ensuring that the pH and conductivity of the solution effluent from the final instrument match the equilibrium solution, with a flow rate of 1 ml / min. After centrifugation, pass the culture supernatant through the column and load 40 ml of sample, with a flow rate of 0.33 ml / min. Pass the equilibrium solution through the column at least 3 CV, the actual volume being 20 ml, ensuring that the pH and conductivity of the solution effluent from the final instrument match the equilibrium solution, with a flow rate of 0.33 ml / min. Pass the eluent through the column, increasing the UV280 to 15 mAU to begin collecting the elution peak (PAC-EP), and decreasing the UV280 to 15 mAU to stop collection, with a flow rate of 1 ml / min. After sample collection is complete, adjust the PAC-EP to neutral with pH adjusting solution.

[0096] (4) Identification of humanized SIRPα antibody activity (Binding-ELISA) Binding-ELISA experimental method: QP094 (SIRPαV1-flag-his), QP096 (SIRPαV2-Flag-his), and QP100 (cynoSIRPα-flag-his) were coated at 0.5 μg / ml and 50 μl / well respectively, and left overnight at 4°C. Washed three times with PBS, incubated in 200 μl / well of 3% BSA / PBS at room temperature for 2 hours, washed three times with PBST, added different concentrations of antibody, incubated at room temperature for 1 hour, washed three times with PBST, washed three times with PBS, incubated with secondary antibody HRP-anti-Fab 1:2500 dilution, incubated at room temperature for 1 hour, washed three times with PBST, washed three times with PBS, displayed on TMB, stopped with 2MH2SO4, and read at 450 nm.

[0097] (5) Identification of SPR affinity of humanized SIRPα antibody As shown in Table 8 below, the affinity of the humanized antibody to human SIRPα type 1, human SIRPα type 2, and cynomolgus monkey SIRPα was measured using biacore. The results show that the anti-SIRPα humanized antibody QP256253 binds to human SIRPα type 1 with an affinity KD of 3.36E-10M and to human SIRPα type 2 with an affinity KD of 3.19E-10M.

[0098] [Table 9]

[0099] Example 4: Affinity maturation of anti-SIRPα antibody QP163164 (1) Construction of humanized phagemide vectors Humanized QP256253 is constructed in a phagemide vector using the scFv mode (VH-3 GGGGS-VL) as the wild-type sequence (i.e., the original sequence or start sequence, or a mutant sequence obtained by affinity maturation screening). VH, the (GGGGS)3 linker, and VL are spliced ​​using over-lap PCR and ligated into the phagemide vector using NcoI and NotI restriction sites.

[0100] (2) Construction of a phage display library Using the constructed wild-type scFv as a template, codon-based primers are used during the primer synthesis process. The mutant region codons consist of 50% wild-type codons and 50% NNK (reverse primers are MNN), and mutations are introduced into all CDR regions to construct a mutant library. PCR fragments are enzymatically cleaved with NcoI and NotI, ligated into phagemide vectors, and finally electrotransformed into E. coli TG1. Each codon-based primer is independently used to construct a library.

[0101] (3) Panning of the library After the library is rescued and phage granules are packaged for panning, liquid-phase panning is performed using biotinylated QP098 (cynoSIRPα(ECD)) antigen and streptavidin magnetic beads, and the antigen concentration is decreased in each round of screening compared to the previous round. After 3 rounds of panning, 250 clones are selected for phage ELISA to detect binding activity, and positive clones are sequenced. The sequenced clones are aligned and analyzed, and after removing redundant sequences, the non-redundant sequences are converted to full-length IG (heavy chain constant region: CH1-CH2-CH3 of hIgG4 selected, light chain constant region: κ light chain CL selected) and expressed in mammalian cells. Full-length IG protein is obtained after affinity purification. The specific sequences are shown in the table below. In this table, the antibody light chain uses the kappa light chain constant region CL, and the antibody heavy chain uses the human IgG4 constant region (see Example 2 for specific sequences of the constant regions).

[0102] [Table 10] TIFF0007853730000026.tif36170

[0103] Note: The naming convention for protein numbers is a combination of the heavy chain plasmid number and the light chain plasmid number. For example, in an antibody molecule with protein number QP256279, the heavy chain plasmid number is QD256 and the light chain plasmid number is QD279. The sequences shown in the sequence numbers in the table are sequences of the heavy chain variable region or light chain variable region of different antibodies. The specific sequences of the light chain variable region are as follows:

[0104] [ka]

[0105] [ka]

[0106] [ka]

[0107] [ka]

[0108] [ka]

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] The bolded and underlined parts above are VLCDR1, VLCDR2, and VLCDR3 of each antibody molecule, respectively, and a specific comparison with the wild-type sequence QP256253 is as follows.

[0113] [Table 11] TIFF0007853730000036.tif186170

[0114] Note: " / " indicates that the sequence is the same as QP256253, while blackbody and bold text indicate amino acids that are different from QD253. (4) ELISA detection Binding-ELISA experimental method: Coat with 0.5 μg / ml of QP094 (SIRPαV1-flag-his), QP096 (SIRPαV2-Flag-his), QP098 (cynoSIRPα-flag-his), and QP100 (cynoSIRPα-flag-his), 50 μl / well, respectively, at 4°C overnight. Wash three times with PBS, incubate in 200 μl / well with 3% BSA / PBS at room temperature for 2 hours, wash three times with PBST, add different concentrations of antibody, incubate at room temperature for 1 hour, wash three times with PBST, wash three times with PBS, incubate with secondary antibody HRP-anti-Fab 1:2500 dilution, incubate at room temperature for 1 hour, wash three times with PBST, wash three times with PBS, display on TMB, stop with 2MH2SO4, and read at 450 nm. EC50 values ​​are as shown in the following table. The following table further shows the detection results for the humanized antibody QP256253, the chimeric antibody QP163245, and the control antibody QP026249. The results are shown in Figures 1 to 6.

[0115] [Table 12] TIFF0007853730000038.tif48170

[0116] Blocking-ELISA experimental method: Coated with QP001, 2 μg / ml, overnight at 4°C, washed three times with PBS, blocked with 250 μl / well of 5% milk, incubated with a 1:1 mixture of Biotin-QP002 0.05 μg / ml + Abs 50 μg / ml, incubated at 25°C for 1 hour, and then treated with HRP-streptavidin (1:5000). The results are shown in Figure 7.

[0117] (5) Detection of affinity by surface plasmon resonance (SPR) The affinity of anti-SIRPα antibodies to human SIRPα type V1, human SIRPα type V2, and cynomolgus monkey SIRPα was measured using biacore, and some of the results are shown in Table 12. As can be seen from Table 12, anti-SIRPα antibodies QP2561589, QP2561586, QP2561581, QP256279, and QP2561770 all bind to human SIRPα type V1 and human SIRPα type V2. At the same time, QP2561589, QP2561586, QP256279, QP2561770, and QP256253 all bind to different cynomolgus monkey and rhesus monkey SIRPα proteins.

[0118] [Table 13] TIFF0007853730000040.tif30170

[0119] As can be seen from the table above, the affinity of the mature antibodies QP2561589, QP2561586, and QP256279 proteins for human SIRPα V1 and SIRPα V2 is more than 50 times higher than the affinity of the control antibody KWAR23 (QP026249).

[0120] Example 5: FACS detection of anti-SIRPα antibodies that bind to human renal clear cell adenocarcinoma 786-O cells that naturally express human SIRPα. Experimental steps: Collect 2E5 / well of 786-O cells, wash once with PBS, centrifuge at 300g for 3 minutes, and discard the supernatant. Blocking: Resuspend 2E5 / well in 2% FBS, inoculate 200 μl / well into a 96-well U-bottom plate, and incubate in an ice bath for 1 hour. Centrifuge at 300g for 3 minutes and discard the supernatant. Antibody incubation: Add 10 μg / ml of antibody diluted 1:3 to 100 μl / well and incubate in an ice bath for 1 hour. Centrifuge and discard the supernatant. Add 200 μl / well of pre-cooled PBS, centrifuge at 300g for 5 minutes, discard the supernatant, and repeat twice. Secondary antibody: Add 50 μl / well of PE-anti-human FC (1:200) and incubate in an ice bath for 0.5 hours. Centrifuge and discard the supernatant. Pre-cooled PBS was centrifuged at 300g for 5 minutes in 200 μl / wells, the supernatant was discarded, and the process was repeated three times. The average fluorescence value was read by FACS. The results are shown in Figure 8, and all SIRPα antibodies QP163245, QP256253, QP256279, QP2561586, and QP2561589 bound to human renal cleavage cell adenocarcinoma 786-O cells that naturally express human SIRPα, and their binding affinity was superior to that of the control antibody QP026249 (KWAR23).

[0121] Example 6: In vitro functional experiment of anti-SIRPα antibody ADCP (1) Anti-SIRPα antibodies were created for different IgG subtypes, and the molecular cloning design was as follows:

[0122] [Table 14] TIFF0007853730000042.tif237170TIFF0007853730000043.tif93170

[0123] Note: The protein numbering convention is a combination of the heavy chain plasmid number and the light chain plasmid number. The sequence shown in the heavy chain sequence number is the heavy chain sequence of a different subtype of antibody. The sequence shown in the light chain sequence number is the light chain or light chain variable region sequence of a different subtype of antibody. (L234A, L235A, K338A) refers to a mutation in the FC segment that removes FCγR function (EU count L234A / L235A / K338A).

[0124] Here, the specific sequence of the heavy chain (SEQ ID NO: 26) of QP32700279 is as follows: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWIAMIDPSDSETHYNQIFKDRATLTTDTSTSTAYMELRSLRSDDTAVYYCAMDYGSLYAMDYWGRG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISAAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0125] The sequence of the light chain variable region of QP32700279 is as shown in SEQ ID NO:18. (2) In vitro functional experiment of anti-SIRPα antibody ADCP Macrophage preparation: Resuscitate PBMCs and use the EasySep kit. TMMonocytes were isolated using a human monocyte isolation kit (Stemcell-19359), human recombinant M-CSF (final concentration 50 ng / mL) was added, mixed thoroughly and uniformly, and the cells were cultured at 37 °C for 6 days to induce macrophages. The cells were collected, counted and stored. Raji cells were labeled with CFSE. Raji was resuspended to 6 2×10 5 cells / ml, then 50 μl / well (1×10 5 / well) was added to a 96-well plate containing macrophages. Antibody dilution: Rituximab was diluted to 80 μg / ml in complete medium and serially diluted in nine gradients. Anti-SIRPα was diluted to 20 μg / ml in complete medium. Antibody mixing: In the Combination group, the two diluted antibodies were mixed 1:1. In the Rituximab group, an equal volume of medium was mixed. 50 μl / well was added to the 96-well plate of pre-seeded cells and cultured at 37 °C for 2 hours. FACS detection: Phagocytosis was measured by gating live CFSE+ / CD14+ cells.

[0126] The affinity matured molecule and control antibody were used in combination with rituximab in the ADCP assay. The experimental results showed that in the combination of SIRPα antibody and rituximab, compared with rituximab alone, the EC50 was decreased, indicating that the synergistic effect of ADCP was enhanced in the original article. The results are as shown in Figures 9, 10, and 11.

[0127] Example 7: Evaluate the inhibitory effect of anti-SIRPα antibody on Raji-Luc tumor growth in the B-NDG-hSIRPΑ mouse model QP32700279 To investigate the killing effect of anti-SIRPα antibodies against tumors, B-NDG-hSIRPα was intravenously inoculated into a Raji-Luc tumor model, and the inhibitory effects of SIRPα antibodies and rituximab on tumor growth were evaluated. Raji-Luc cells were cultured in RPMI1640 culture medium containing 10% fetal bovine serum. Raji-Luc cells resuspended in PBS were cultured in 5 × 10⁶ cells. 5 The drug is inoculated into the tail vein of B-NDG-hSIPRa mice at a concentration of 0.2 mL per cell and a volume of 0.2 mL per mouse. On days 0 and 3 after inoculation, tumor imaging signal values ​​are measured using a small animal imager, and the average imaging signal intensity is approximately 1 × 10⁻⁶. 6 If the P / S level is reached, the animals will be selected to an appropriate group according to their tumor imaging signal value and body weight, and will be equally distributed among four experimental groups, with eight animals in each group. Administration will begin on the day of group assignment, and the specific administration plan is shown in Table 14 below.

[0128] [Table 15]

[0129] Note: a: The dose volume is calculated according to 10 μL / g based on the body weight of the experimental animal.

[0130] b: Q3D refers to administration once every 3 days, and Q2W refers to administration once every 2 weeks. Group assignments and the day of administration were counted as D0. At D18, the tumor growth curves and D18 imaging signal intensity data, which reflect the tumor imaging signal values ​​for each group, are shown in Figure 12 and Table 15.

[0131] [Table 16]

[0132] The tumor growth curve results showed that rituximab, QP32700279, and the QP32700279 and rituximab combination therapy groups all significantly inhibited Raji-Luc tumor growth, with tumor growth inhibition rates (TGI) of 58.6%, 46.4%, and 84.5%, respectively, indicating that the combination therapy groups exhibited stronger antitumor activity than the single-drug groups.

[0133] Based on the model characteristics, the mice exhibited abnormal movement or paralysis in the later stages of the experiment. At this point, the mice were euthanized, and their survival curves were recorded. The survival curves for each group were as shown in Figure 13 until all mice in group G1 died (D25).

[0134] For survival analysis, the Kaplan-Meier method was used, and for comparisons between groups, the Log-rank test was used, with p<0.05 considered statistically significant. Compared to the control group, QP32700279 and the combination therapy group (QP32700279 + rituximab) all significantly extended the survival time of Raji-Luc tumor-bearing mice (p=0.0445*, p<0.001**), but the rituximab group did not effectively extend the survival time of tumor-bearing mice (p=0.23). These test results suggest that the combination therapy of QP32700279 and QP32700279 + rituximab effectively inhibits tumor growth in Raji-Luc tumor-bearing mice, thereby improving mouse survival rates.

[0135] Example 8: ELISA detection of anti-SIRPα antibodies that bind to all subtypes of human SIRPα According to the SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 sequences reported in the literature ("Velcro" Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein (SIRPα) Antagonists That Enhance Antibody-dependent Cellular Phagocytosis, JOURNAL OF BIOLOGICAL CHEMISTRY, VOLUME 290·NUMBER 20·MAY 15, 2015), the above SIRPα C-terminus was fused to mouse IgG2a subtype Fc (mouse IgG2a) by gene synthesis, constructed in a eukaryotic expression vector pQD, and the supernatant was purified on day 5 of transient transfection with 293E, and each of the SIRPα sequences was obtained. V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10 fusion Fc (mouse IgG2a) protein was obtained, and further ELISA was performed to detect binding of the SIRPα antibody to all subtypes of SIRPα. The sequences are shown below.

[0136] > SIRPα V1 (SEQ ID NO: 51) GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS > SIRPαV2 (SEQ ID NO:52) GVAGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPDTEFKSGAGTELSVRAKPS > SIRPα V3(SEQ ID NO:53) GVAGEELQVIQPDKSVSVAAGESAILLCTVTSLIVPGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRAKPS > SIRPα V4(SEQ ID NO:54) GVAGEEGLQVIQPDKSVSVAAGESAILHCTATSLIVPGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS > SIRPα V5(SEQ ID NO:55) GVAGEEELQVIQPDKFVLVAAGETATLRCTATSLIVPGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS > SIRPα V6(SEQ ID NO:56) GVAGEELQVIQPDKSVLVAAGETATLRCTATSLIVPGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFPIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS > SIRPα V7(SEQ ID NO:57) GVAGEELQVIQPDKSVSVAAGESAILHCTVTSLIVPGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRGKPS > SIRPα V8(SEQ ID NO:58) GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRAKPS > SIRPα V9 (SEQ ID NO: 59) GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPS >SIRPα V10 (SEQ ID NO:60) RVAGEEELQVIQPDKSVSVAAGESAILHCTVTSLIPVGPIQWFRGAGPARELIYNQKEGHFPRVTTVSESTKRENMDFSISISNITPADAGTYYCVKFRKGSPD-TEFKSGAGTELSVRAKPS >FC (Mouse IgG2a) (SEQ ID NO: 61) EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAP IERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0137] SIRPα antibody to be detected: The SIRPα antibody QP256279 is stably expressed in CHOS cells, and the number of the stably expressed CHOS protein is CHO71.

[0138] Molecular cloning was constructed according to the sequence provided by patent WO2017178653, and OSE's anti-SIRPα antibody 18D5 was expressed and purified as an experimental control. Simultaneously, QP026249 is, as mentioned above, Forty Seven's anti-SIRPα antibody KWAR23, and is referred to here as KWAR23.

[0139] Experimental steps for ELISA detection of SIRPα antibodies that bind to SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10: SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10, 1 μg / ml, 60 μl / well, coated overnight at 4°C, washed twice with PBST, blocked with 200 μl / well of 5% nonfat milk (Sangon), incubated at room temperature for 1 hour, washed twice with PBST, antibody 10 μg / ml, 5-fold dilution, 10 gradients, incubated with 60 μl / well, incubated at room temperature for 1 hour, washed five times with PBST, Secondary antibody incubation: Anti-hFab1: 10000, 60 μl / well, incubated at room temperature for 1 hour, washed five times with PBST, Stain development: Equilibrate TMB at room temperature 1 hour prior, stain with 100 μl / well for 10 minutes, stop with 2M H2SO450 ul / well, read at 450 nm with a microplate reader.

[0140] The experimental results are shown in Figures 14 to 23. The SIRPα antibody CHO71 of the present invention binds to all subtypes of SIRPα V1 / V2 / V3 / V4 / V5 / V6 / V7 / V8 / V9 / V10. OSE's SIRPα antibody 18D5 does not bind to SIRPα V2 / V3 / V7 / V8 / V10.

[0141] Construction and detection of bispecific antibodies targeting SIRPα and PD-L1 Based on the above results, a bispecific antibody targeting SIRPα and PD-L1 is constructed using the sequence of an anti-SIRPα antibody with protein number QP256279. The SIRPα-binding domain of the bispecific antibody includes a heavy chain variable region and a light chain variable region, with the sequence of the heavy chain variable region selected from QD256 and the sequence of the light chain variable region selected from QD279. By combining this with the sequence of a PD-L1 nanobody initially obtained by the applicant (Title of Invention: Anti-PD-L1 Nanobody and its Uses, Patent Publication No.: CN112574309A, Application No.: 202011309419.7), a bispecific antibody targeting SIRPα and PD-L1 is constructed, with the plasmid number of the selected PD-L1 nanobody being QD509, obtained by immunizing and humanizing alpacas. In this study, QD509 can represent the VHH fragment of anti-PD-L1, and can also represent a fusion protein of the VHH fragment and FC. The amino acid sequence of the VHH fragment is as shown in SEQ ID NO:62, and specifically as follows:

[0142] [ka] Here, the sequence order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, with the bolded and underlined parts being CDR1, CDR2, and CDR3, respectively, and the amino acid sequences are as shown in SEQ ID NO: 63, 64, and 65, respectively.

[0143] Example 9: Clone Design Anti-SIRPα / PD-L1 bispecific antibody molecules were designed, and the anti-PD-L1 nanobody QP509 VHH was fused to the C-terminus (e.g., QD3282 in Table 16) or N-terminus (e.g., QD626 in Table 16) of the SIRPα antibody heavy chain, or to the N-terminus (e.g., QD623 in Table 16) of the SIRPα antibody light chain via G4S linkage sequences with different repeat counts. Primers were designed according to the sequences, the full-length versions of each bispecific antibody gene designed were constructed by PCR, homologous recombination was performed with expression vectors pQD, each constructed expression vector pQD was plasmid-numbered as shown in Table 16, and the resulting antibody molecules were protein-numbered. In Table 16, the amino acid sequences of QD623, QD624, and QD625 are fundamentally similar, differing only in the number of G4S repeats. Similarly, the amino acid sequences of QD626, QD627, and QD628 are fundamentally similar, with the difference being the number of G4S repeats. The bispecific antibody sequences and protein expression numbers are as follows:

[0144] [Table 17] TIFF0007853730000048.tif210170

[0145] In Table 16, the specific sequences of SEQ ID NO:66 and SEQ ID NO:67 that constitute QP32820279 are as follows:

[0146] >QD3282(SEQIDNO:66) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWINWVRQAPGQGLEWIAMIDPSDSETHYNQIFKDRATLTTDTSTSTAYMELRSLRSDDTAVYYCAMDYGSLYAMDYWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISAAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTLDDYAIGWFRQAPGKGREGVSCISKSGETTFFVESVKDRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATGSWCTVGSMSRQFYRQFFHSWGQGTLVTVSS*。

[0147] >QD279(SEQIDNO:67) DIVLTQSPDSLAVSLGERATINCRASQSVRSSGYNWIFWYQQKPGQPPKLLIYLASNRDSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0148] Anti-SIRPα monoclonal antibodies and anti-PD-L1 nanobodies were designed to fuse with FC molecules and used as controls. The anti-SIRPα monoclonal antibody protein number is QP32700279, and the anti-PD-L1 nanobodies fused FC molecule numbers are QP509 and QP3447, as specifically shown in Table 17.

[0149] [Table 18]

[0150] Furthermore, based on existing literature, we will design additional anti-SIRPα and anti-PD-L1 antibodies to be used as controls. Specifically, these are shown in Table 18. Here, QP026249 is an analog of the SIRPα monoclonal antibody KWAR23 from 47. QP250251 is an analog of the anti-SIRPα monoclonal antibody 18D5 from OSE Immunotherapeutics. QP37503751 is an analog of the anti-SIRPα monoclonal antibody 1H9 from 47. QP11801181 is a Tecentriq analog, which is the anti-PD-L1 monoclonal antibody atezolizumab from Roche.

[0151] [Table 19] TIFF0007853730000051.tif31170

[0152] Based on existing literature, the SIRPα antigen was designed for experimental use and is shown in Table 19.

[0153] [Table 20] TIFF0007853730000053.tif117170

[0154] Note: QP098 is the cynomolgus monkey SIRPα sequence (uniprot database sequence number I7G9Z7), and QP271 is the rhesus monkey SIRPα sequence, obtained by the inventors by sequencing monkey PBMCs. QP532~QP538 represent sequences encoding the SIRPα molecule, and the mFC sequence is as shown in SEQ ID NO:86.

[0155] Example 10: Protein expression and purification The protein of Example 9 was expressed and purified referring to the protein expression and purification method of Example 2. Each antibody was further purified by SEC, and the results showed good transient expression yield of the anti-SIRPα / PD-L1 bispecific antibody, good SEC purity, and stable physical and chemical properties.

[0156] Example 11: SPR detection of anti-SIRPα / anti-PD-L1 bispecific antibody that binds to SIRPα / PD-L1 affinity (1) In this study, the affinity between the antibody molecule and the antigen SIRPα is detected via Biacore8K. As mentioned above, QP026249 is an analog of the SIRPα monoclonal antibody KWAR23 from 47 companies. See the table below for the results.

[0157] [Table 21]

[0158] The results demonstrate that all of the different forms of biantibody molecules designed in this invention bind to the SIRPαV1 recombinant protein with high affinity. (2) Next, the affinity between the antibody molecule and the antigen PD-L1 is detected via Biacore 8K. QP11801181 is an analog of Roche's PD-L1 monoclonal antibody Tecentriq. QP509 is the N-terminus where the PD-L1 nanobody VHH is fused to the FC, and QP3447 is the C-terminus where the PD-L1 nanobody is fused to the FC. See the following table for the results.

[0159]

Table 22

[0160] The results show that different forms of bispecific antibody molecules designed in this invention bind to human PD-L1 recombinant protein with high affinity, where the C-terminal affinity of the PD-L1 nanobody VHH fused to FC is slightly higher than that in the form fused to the N-terminal.

[0161] Example 12: ELISA Detection of Anti-SIRPα / Anti-PD-L1 Bispecific Antibody Binding to PD-L1 and SIRPα Proteins (1) ELISA Detection of the Binding between Antibody and PD-L1: Coating plate: Anti-his, 1 μg / ml in PBS, 60 μl / well, overnight at 4°C, wash twice with PBST. Block: 5% non-fat milk (non-fat milk, Sangon), 200 μl / well, incubate at 25°C, 120 rpm for 1 hour. Antigen QP003 (PDL1-his), 1 μg / ml, 60 μl / well, incubate at 25°C, 120 rpm for 1 hour, wash 5 times with PBST. Dilute the primary antibody 5-fold starting from 13.3 nM, with seven gradients and a final 10-fold dilution, 60 μl / well, incubate at 25°C, 120 rpm for 1 hour, wash 5 times with PBST. Secondary antibody anti-hFc1:5000, 60 μl / well, incubate at 25°C, 120 rpm for 1 hour, wash 5 times with PBST. Color development: Equilibrate TMB at room temperature 1 hour in advance, develop color for 3 minutes, Stop: Stop color development with 1 M H2SO4. The results are as shown in Figure 25.

[0162] (2) ELISA Detection of Antibody Molecules Binding to SIRPα: Coating plates: Coat QP093 (SIRPαV1) and QP095 (SIRPaV2) respectively with 1 μg / mlinPBS, 60 μl / well, overnight at 4°C, and wash twice with PBST. Blocking: Incubate 200 μl / well in 5% non-fat milk (Sangon) at 25°C, 120 rpm, for 1 hour. Incubate 66.7 nM primary antibody in a 5-fold dilution, seven gradients, and the final 50-fold dilution in 60 μl / well at 25°C, 120 rpm, for 1 hour, and wash five times with PBST. Secondary antibody incubation: Incubate anti-hFab (glycerol-free) 1:10000 in 60 μl / well at 25°C, 120 rpm, for 1 hour, and wash five times with PBST. Stain development: Equilibrate TMB to room temperature 1 hour prior, allow stain development for 3 minutes, and stop: Stop stain development with 1 MH2SO4. The results are shown in Figures 26 and 27.

[0163] Example 13: ELISA detection of antibody molecules that block the binding of human PD-L1 to PD-1 protein. The substrates were coated with 2 μg / ml of protein QP1138 (PD1-FC) in 50 μl / well and incubated overnight at 4°C. Washed three times with PBS. Blocking: 250 μl / well of 3% BSA, incubated at room temperature for 1 hour. Prepared 2 μg / ml of PD-L1-mouse FC and antibodies of different concentrations, mixed uniformly in equal volumes, and incubated at room temperature for 1 hour. Washed three times with PBST and three times with PBS. Secondary antibody incubation: 50 μl / well of HRP-mouse IgG (1:5000), washed six times with PBST and three times with PBS. Stain development: 100 μl / well of TMB, allowed to develop for 10 minutes. Stopped with 50 μl / well of 2MH2SO4. The results are shown in Figure 28, and the biantibody molecule can block the binding of human PD-L1 to the PD-1 protein.

[0164] Example 14: ELISA detection of antibody molecules that block the binding of human CD47 to SIRPα protein. Blocking-ELISA experimental method: Coat with CD47-FC (QP001) 2 μg / ml overnight at 4°C, wash three times with PBS, block with 250 μl / well of 5% milk, incubate with a 1:1 mixture of biotin-SIRPα-FC (QP002) 0.05 μg / ml + Abs 50 μg / ml, and incubate with HRP-streptavidin (1:5000) at 25°C for 1 hour. The results are shown in Figure 29.

[0165] Example 15: Synergistic enhancement of CD20 antibody rituxan-dependent ADCP effect Antibody-dependent macrophage-mediated phagocytosis (ADCP) refers to the binding of an antibody's Fab segment to an antigen epitope on tumor cells, and its Fc segment to FcγR on the macrophage surface, thereby phagocytosing target cells via the macrophage. Simultaneously, SIRPα expressed on macrophages binds to CD47 expressed on tumor cells, forming an inhibitory signal. We will further investigate how ADCP blocks the binding of a biantibody molecule between macrophage SIRPα and Raji cell CD47, thereby synergistically enhancing the biological activity of human Burkitt lymphoma cells (Raji) through CD20 antibody-rituxan-dependent macrophages.

[0166] Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of healthy individuals, and macrophage differentiation was induced by adding 50 ng / mL of human recombinant M-CSF. Raji cells were labeled with green fluorescent CFSE, and Raji cells and macrophages were inoculated into 96-well plates in a 2:1 ratio. Various concentrations of CD20 antibody rituxan were added, either alone or in combination with a SIRPα antibody molecule. After incubation at 37°C for 2 hours, the reaction was stopped, and APC anti-human CD11b antibody was incubated. The results were read via FACS, and the percentage of APC / FITC double-positive cells at each antibody concentration represented the percentage of macrophages undergoing phagocytosis.

[0167] The results are shown in Figure 30. The results indicate that the combination of the antibody of the present invention with rituximab resulted in a lower EC50 than rituximab alone, and that the ADCP synergistic effect was significantly enhanced.

[0168] Example 16: Biological activity of a biantibody molecule that stimulates human PBMC proliferation in vitro Human peripheral blood mononuclear cells (PBMCs) are composed mainly of various white blood cells, including monocytes, B cells, T cells, NK cells, dendritic cells, and macrophages. In vitro, PBMCs are stimulated by adding the superantigen SEB, leading to the activation and proliferation of lymphocytes through the presentation and activation of APC cells, which then generate various cytokines. The PD-L1 antibody promotes T cell proliferation and the release of cytokines such as IL-2 by blocking the PD-1 / PD-L1 binding immunosuppressive signal. The amount of IL-2 released is detected by ELISA to further study the biological activity of the antibody molecule in in vitro PBMC proliferation experiments. PBMC cells are inoculated into a 96-well plate, different concentrations of SEB are prepared and added to the PBMC cell wells, then the antibody molecule of the present invention and other control antibodies are added, gently mixed, and cultured for 2 days. The amount of IL-2 secreted in the cell culture supernatant is detected by ELISA. The results show that QP32700624 and QP32820279 significantly enhance the activation and proliferation of SEB-stimulated PBMCs in in vitro proliferation experiments, and can enhance IL-2 production. This is comparable to the control antibody Tecentriq. The results are shown in Figure 31.

[0169] Example 17: Production and purification of Q-1801 protein The sequence of the anti-SIRPα / anti-PD-L1 bispecific antibody QP32820279 was transferred to a pCHO vector. The pCHO vector is a laboratory-modified vector containing GS as a screening marker and can be used to screen stably transfected CHO cells. CHO cells were stably transfected, pressurized and screened with GS to obtain a cell line highly expressing the anti-SIRPα / anti-PD-L1 bispecific antibody for protein synthesis. This cell line was purified to obtain the target molecule, number CHO44 (named Q-1801), and the sequence number of the CHO44 protein is the same as QP32820279 (amino acid sequence numbers are SEQIDNO:66, SEQIDNO:67). Simultaneously, the SIRPα monoclonal antibody QP32700279 was also expressed and purified in CHO cells, number CHO71, and used as a single-component control for SIRPα. The PD-L1 nanobody VHH is fused to the C-terminus of FC, with protein number QP3447, and is used as a single-component PD-L1 control.

[0170] Example 18: Q-1801 that binds to human and monkey SIRPα (1) SPR The gene encoding human SIRPα is a polymorphic gene, and 10 variants have been identified in the human population. Polymorphisms in human SIRPα cause changes in surface-exposed amino acids but do not affect binding to CD47. The most common protein variants are SIRPαV1 and V2 (accession numbers NP_542970(P78324) and CAA71403). In this study, Biacore8K was used to detect the affinity between molecules such as Q-1801 and the antigen SIRPα. The results for the binding affinity between Q-1801, QP026249 and human SIRPαV1, and the binding affinity between Q-1801 and human SIRPαV2, cynomolgus monkey SIRPα, and rhesus monkey SIRPα are shown in Table 22. The results show that Q-1801 binds to human SIRPαV1 with high affinity, with a KD value of 6.01E-11(M). The KD value for binding to SIRPαV2 is 1.03E-10(M), the KD value for binding to cynomolgus monkey SIRPα is 2.14E-09(M), and the KD value for binding to rhesus monkey SIRPα is 5.22E-10(M). The affinity of Q-1801 for binding to human SIRPαV1 is significantly higher than that of the KWAR23 analog (QP026249), with an affinity KD value of 4.69E-09M for the KWAR23 analog to human SIRPαV1. The affinity of Q-1801 for binding to human SIRPαV2 is also significantly higher than that of the KWAR23 analog (QP026249), with an affinity KD value of 1.75E-08(M) for the KWAR23 analog to human SIRPαV2.

[0171] [Table 23]

[0172] (2) ELISA Based on existing literature reports on SIRPα gene polymorphisms in different races, the ratio of SIRPαV1 / V2 differs among races, with the SIRPαV2 gene reaching 42.3% in East Asians. ELISA is used to detect the binding of molecules such as Q-1801 to different subtypes of human SIRPα and to mouse SIRPα.

[0173] The inventors constructed nine previously reported SIRPα genotypes V1-V9. Human SIRPαV1 recombinant protein (QP093), human SIRPαV2 recombinant protein (QP095), human SIRPαV3 recombinant protein (QP532), human SIRPαV4 recombinant protein (QP533), human SIRPαV5 recombinant protein (QP534), human SIRPαV6 recombinant protein (QP535), human SIRPαV7 recombinant protein (QP536), human SIRPαV8 recombinant protein (QP537), and human SIRPαV9 recombinant protein (QP538) were coated onto ELISA plates, and test molecules such as gradient-diluted Q-1801 were added and detected with an HRP-labeled anti-human Fc secondary antibody. The results are shown in Figures 32 to 40. Q-1801 binds to human SIRPαV1 with an EC50 of 0.1768 nM, to human SIRPαV2 with an EC50 of 0.2101 nM, to human SIRPαV3 with an EC50 of 0.1543 nM, to human SIRPαV4 with an EC50 of 0.1631 nM, to human SIRPαV5 with an EC50 of 0.1667 nM, to human SIRPαV6 with an EC50 of 0.2721 nM, to human SIRPαV7 with an EC50 of 0.2182 nM, to human SIRPαV8 with an EC50 of 0.4176 nM, and to human SIRPαV9 with an EC50 of 0.3991 nM. The 18D5 analog does not bind to human SIRPαV2, human SIRPαV3, human SIRPαV7, or human SIRPαV8.

[0174] In summary, Q-1801 has high affinity for all SIRPα genotypes, but QP250251 (18D5 analog) does not bind to SIRPαV2 / V3 / V7 / V8 / V10, and QP026249 (KWAR23 analog) has weaker binding to SIRPαV2 / V3 / V7 / V8 / V10.

[0175] (3) FACS According to existing literature reports, the U-937 cell line is a human histiocytic lymphoma cell that expresses endogenous SIRPαV1, the THP-1 cell line is a human monocytic leukemia cell that expresses endogenous SIRPαV2, and the binding of Q-1801 to human SIRPαV1 and human SIRPαV2 proteins is tested by FACS measurement. Incubate U-937 cells and THP-1 cells with molecules such as Q-1801 at different concentrations respectively, detect the fluorescence values by FACS, plot the fitting curves, and compare the EC50 values.

[0176] The results are as shown in Figure 41. The EC50 of Q-1801 binding to U-937 (human SIRPαV1) is 0.1057 nM, the CHO71 binding EC50 is 0.07598 nM, the QP026249 (KWAR23 analog) binding EC50 is 0.21 nM, the QP37503751 (1H9 analog) binding EC50 is 0.1583 nM, and the QP250251 (18D5 analog) binding EC50 is 0.5811 nM. The affinity of Q-1801 binding to human SIRPαV1 is comparable to that of the monoclonal antibody CHO71.

[0177] The results are as shown in Figure 42. The EC50 of Q-1801 binding to THP-1 (human SIRPαV2) is 0.1037 nM, the CHO71 binding EC50 is 0.0743 nM, the QP026249 (KWAR23 analog) binding EC50 is 0.2606 nM, the QP37503751 (1H9 analog) binding EC50 is 0.2103 nM, and QP250251 (18D5 analog) does not bind. The affinity of Q-1801 binding to human SIRPαV2 is comparable to that of the monoclonal antibody CHO71.

[0178] In summary, Q-1801 binds to human SIRPαV1 and human SIRPαV2 with high affinity. QP250251 (18D5 analog) does not bind to human SIRPαV2.

[0179] Example 19: Q-1801 that binds to human PD-L1 and cynomolgus PD-L1 (1) SPR Biacore8K was used to detect the affinity between molecules such as Q-1801 and the antigen PD-L1. The affinity results for Q-1801, QP3447, QP11801181 (Tecentriq analog) and human PD-L1, as well as the affinity results for Q-1801 and rhesus monkey PD-L1, are shown in Table 23.

[0180] [Table 24]

[0181] The results show that Q-1801 has a high affinity for binding to human PD-L1 with a KD value of 4.31E-10(M), and a KD value for binding to rhesus monkey PD-L1 of 4.99E-10(M). The KD value for QP3447 that binds to human PD-L1 is 4.79E-10(M). The KD value for the tecentriq analog that binds to human PD-L1 is 1.39E-09(M).

[0182] In summary, both Q-1801 and QP3447 bind to human PD-L1 with high affinity, and their affinities are comparable. The affinity of Q-1801 to human PD-L1 is higher than that of the PD-L1-positive antibody Tecentriq analog.

[0183] (2) ELISA ELISA is used to detect the binding of molecules such as Q-1801 to human PD-L1.

[0184] Anti-HIS antibodies were coated onto ELISA plates, different species of PD-L1 proteins were added, and after incubation, molecules such as Q-1801 were added in gradient dilutions. HRP-labeled anti-human Fc secondary antibodies were detected. The results are shown in Figure 43. Q-1801 bound to human PD-L1 protein with an EC50 value of 0.1218 nM. QP3447 bound to human PD-L1 protein with an EC50 value of 0.08847 nM. Tecentriq bound to human PD-L1 protein with an EC50 value of 0.09194 nM.

[0185] (3) FACS According to existing literature reports, the HCC827 cell line is a human lung cancer cell line expressing endogenous PD-L1, and the binding of Q-1801 to HCC827 cells that naturally express human PD-L1 protein was tested by FACS. Different concentrations of molecules such as Q-1801 were incubated with HCC827 cells, fluorescence values ​​were detected by FACS, fitting curves were plotted, and EC50s were compared. The results are shown in Figure 44, where the EC50 for Q-1801 binding to human PD-L1 was 0.1416 nM, the EC50 for QP3447 binding was 0.1188 nM, and the EC50 for Tecentriq binding was 0.1089 nM.

[0186] In summary, Q-1801, the PD-L1 monoclonal antibody QP3447, and Tecentriq all bind to HCC827 cells that naturally express human PD-L1, and their binding affinities are equivalent.

[0187] Example 20: Q-1801 blocks the binding of SIRPα to CD47 CD47 protein overexpressed on the surface of tumor cells binds to SIRPα expressed on the surface of macrophages, evading phagocytosis by macrophages. The Q-1801 molecule may block the binding of CD47 to SIRPα, causing the "don't eat me" signal to disappear and promoting macrophage attack on tumors. Competitive ELISA is used to detect the ability of molecules such as Q-1801 to block the binding of SIRPα to CD47. Human CD47 protein is coated onto an ELISA plate, SIRPα-mouse Fc protein is added, and after incubation, molecules such as Q-1801 are added in gradient dilution, and an HRP-labeled anti-mouse IgG secondary antibody is detected. The results are shown in Figure 45. The IC50 of Q-1801, which blocks the binding of SIRPα to CD47, is 0.8149 nM. The IC50 of CHO71, which blocks the binding of SIRPα to CD47, is 0.7074 nM. The IC50 of QP026249 (KWAR23 analog), which blocks the binding of SIRPα to CD47, is 3.12 nM. The IC50 of QP37503751 (1H9 analog), which blocks the binding of SIRPα to CD47, is 2.277 nM. The IC50 of QP250251 (18D5 analog), which blocks the binding of SIRPα to CD47, is 32.98 nM.

[0188] In summary, the ability of Q-1801 to block the binding of SIRPα to CD47 is comparable to that of the SIRPα monoclonal antibody CHO71, and significantly more potent than that of QP026249 (KWAR23 analog), QP37503751 (1H9 analog), and QP250251 (18D5 analog).

[0189] Example 21: Q-1801 blocking PD-L1 / PD-1 and PD-L1 / CD80 binding PD-L1 has two ligands: PD-1 and CD80. The C-terminus of the Q-1801 molecule is an anti-PD-L1 nanobody that not only blocks the binding of PD-L1 to PD-1, but also blocks the binding of CD80 to PD-L1.

[0190] Competitive ELISA was used to detect molecules such as Q-1801 that block the binding of PD-1 to PD-L1. Human PD-1 protein was coated onto an ELISA plate, PD-L1-mouse Fc protein was added, and after incubation, molecules such as Q-1801 were added using gradient dilution, and an HRP-labeled anti-mouse IgG secondary antibody was detected. The results are shown in Figure 46. The IC50 of Q-1801, which blocks PD-L1 and PD-1, was 0.9043 nM. The IC50 of QP3447, which blocks PD-L1 and PD-1, was 0.9511 nM. The IC50 of Tecentriq, which blocks PD-L1 and PD-1, was 2.422 nM.

[0191] Competitive ELISA was used to detect the ability of molecules such as Q-1801 to block the binding of CD80 to PD-L1. Human CD80 protein was coated onto an ELISA plate, PD-L1-mouseFc protein was added, and after incubation, test molecules such as Q-1801 were added using gradient dilution. HRP-labeled anti-mouse IgG secondary antibodies were detected, and the results are shown in Figure 47. The IC50 of Q-1801, which blocks PD-L1 and CD80, was 0.7415 nM, the IC50 of QP3447, which blocks PD-L1 and CD80, was 0.746 nM, and the IC50 of Tecentriq, which blocks PD-L1 and CD80, was 1.683 nM.

[0192] In summary, Q-1801 not only blocks PD-L1 / PD-1 binding but also PD-L1 / CD80 binding, and its ability to block both PD-L1 / PD-1 and PD-L1 / CD80 binding is superior to that of Tecentriq.

[0193] Example 22: Q-1801 synergistically enhances the CD20 antibody rituxan-dependent ADCP effect. Antibody-dependent macrophage-mediated phagocytosis (ADCP) refers to the Fab segment of an antibody that binds to an antigen epitope on tumor cells. Its Fc segment binds to FcγR on the surface of macrophages, phagocytosing target cells via macrophages. However, SIRPα expressed on macrophages binds to CD47 expressed on tumor cells, forming an inhibitory signal. Raji cells are human Burkitt's lymphoma cells that endogenously express CD47 / CD20. We will further investigate how ADCP blocks the binding of Q-1801 between macrophage SIRPα and Raji cell CD47, thereby synergistically enhancing the biological activity of human Burkitt's lymphoma cells (Raji) through CD20 antibody-rituxan-dependent macrophages.

[0194] Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) from two different donors, and differentiation into macrophages was induced by adding 50 ng / mL of human recombinant M-CSF. Raji cells were labeled with green fluorescent CFSE, and Raji cells and macrophages were inoculated into 96-well plates in a 2:1 ratio. Different concentrations of CD20 antibody rituxan were added either alone or in combination with a SIRPα antibody molecule such as Q-1801. After incubation at 37°C for 2 hours, the reaction was stopped, and APC anti-human CD11b antibody was incubated. The percentage of APC / FITC double-positive cells at each antibody concentration was obtained via FACS reading, i.e., the percentage of macrophages that underwent phagocytosis.

[0195] The results are shown in Figure 48. With donor P121031405C, rituximab caused macrophages to phagocytose Raji cells in a concentration-dependent manner, with a maximum phagocytosis rate of approximately 26.57% and an EC50 value of approximately 0.02115 μg / mL. Q-1801 had a stronger synergistic effect on phagocytosis of human Burkitt lymphoma Raji cells in conjunction with rituximab-dependent macrophages, with a maximum phagocytosis rate increasing from 26.57% to 32.38% and an EC50 value of approximately 0.01188 μg / mL. The single-component control CHO71 was comparable to Q-1801, with a maximum phagocytosis rate increasing from 26.57% to 32.14% and an EC50 value of approximately 0.01302 μg / mL. The maximum phagocytosis rate of the positive control QP026249 (KWAR23 analog) increased from 26.57% to 30.09%, and its EC50 value was approximately 0.01765 μg / mL. The maximum phagocytosis rate of the positive control QP37503751 (1H9 analog) was approximately 28.07%, and its EC50 value was approximately 0.01485 μg / mL.

[0196] The results are shown in Figure 49. With donor P121070501C, rituximab caused macrophages to phagocytose Raji cells in a concentration-dependent manner, with a maximum phagocytosis rate of approximately 31.32% and an EC50 value of approximately 0.0578 μg / mL. The combined use of Q-1801 and rituximab showed that Q-1801 significantly increased rituximab-dependent macrophage phagocytosis of Raji cells, with a maximum phagocytosis rate of approximately 44.17% and an EC50 value of approximately 0.02733 μg / mL. The maximum phagocytosis rate for single-component control CHO71 was approximately 43.66%, and the EC50 value was approximately 0.02784 μg / mL. The maximum phagocytosis rate for positive control QP026249 (KWAR23 analog) was approximately 40.71%, and the EC50 value was approximately 0.04938 μg / mL. The maximum phagocytosis rate of the positive control QP37503751 (1H9 analog) was approximately 37.73%, and its EC50 value was approximately 0.03626 μg / mL.

[0197] In summary, the combination of Q-1801 and rituximab significantly increases phagocytosis of Raji cells by rituximab-dependent macrophages. Example 23: Q-1801 that can stimulate T cell proliferation in mixed lymphocyte reaction Mixed lymphocyte reaction refers to a mixed co-culture of human T cells and allogeneic dendritic cells. Lymphocytes are activated and proliferate in response to stimulation from allogeneic antigens, generating a wide variety of cytokines. PD-L1 antibodies block the PD-1 / PD-L1 binding immunosuppressive signal in an antibody concentration-dependent manner, stimulating T cell proliferation and releasing cytokines such as IL-2 / IFN-γ. We will detect IL-2 / IFN-γ release using ELISA and further investigate the biological activity of Q-1801 in stimulating T cell proliferation in vitro in mixed lymphocyte reactions.

[0198] Monocytes were isolated from PBMCs, rhGM-CSF and rhIL-4 were added to induce DCs (inducible dendritic cells), and CD4+ T cells were isolated from another donor PBMC. DC cells and T cells were mixed in a 1:10 ratio, different concentrations of antibodies were added, and the mixture was cultured for 2-5 days. The expression of IL-2 and IFN-γ in the culture supernatant was detected. The results showed that Q-1801, the single-component control QP3447, and the control antibody Tecentriq all stimulated T cell proliferation and enhanced the production of IL-2 and IFN-γ in the mixed lymphocyte reaction (MLR). (See Figures 50 and 51).

[0199] In summary, Q-1801 stimulates T cell proliferation in the mixed lymphocyte reaction (MLR), enhances the production of IL-2 and IFN-γ, and IFN-γ secretion is superior to that of Tecentriq.

[0200] Example 24: Q-1801 stimulates the biological activity of in vitro proliferation of human PBMCs in vitro. Human peripheral blood mononuclear cells (PBMCs) are composed mainly of various white blood cells, including monocytes, B cells, T cells, NK cells, dendritic cells, and macrophages. In vitro stimulation of PBMCs with the superantigen SEB leads to the presentation and activation of APC cells, which in turn activate and proliferate lymphocytes, generating a wide variety of cytokines. PD-L1 antibodies enhance T cell proliferation and the release of cytokines such as IL-2 / IFN-γ by blocking the PD-1 / PD-L1 binding immunosuppressive signal. IL-2 / IFN-γ release is detected by ELISA to further study the biological activity of Q-1801 in in vitro PBMC proliferation experiments. PBMC cells are inoculated into 96-well plates, different concentrations of SEB are prepared and added to the PBMC cell wells, Q-1801 and other control antibodies are added, gently mixed uniformly, and cultured for 2-5 days. ELISA was used to detect IL-2 secretion in the cell culture supernatant, and ELISA was also used to detect IFN-γ secretion in the cell culture supernatant. The results show that Q-1801 significantly enhances the activation and proliferation of SEB-stimulated PBMCs and enhances the production of IL-2 and IFN-γ in in vitro proliferation experiments of SEB-stimulated PBMCs. The single-component control QP3447 significantly enhances the activation and proliferation of SEB-stimulated PBMCs and enhances the production of IL-2 and IFN-γ in in vitro proliferation experiments of SEB-stimulated PBMCs. The control antibody Tecentriq significantly enhances the activation and proliferation of SEB-stimulated PBMCs and enhances the production of IL-2 and IFN-γ in in vitro proliferation experiments of SEB-stimulated PBMCs. The results are shown in Figures 52 and 53.

[0201] In summary, in in vitro proliferation experiments of SEB-stimulated PBMCs, Q-1801 significantly enhanced T cell activation and proliferation, and increased the production of IL-2 and IFN-γ, with activity comparable to that of Tecentriq.

[0202] Example 25: Inhibitory effect of Q-1801 on Raji-Luc tumor growth in a B-NDG-hSIRPα mouse model B-luc-GFPRaji cells resuspended in PBS, 1 × 10⁶ 5Female B-NDG-hSIRPa mice were inoculated via the tail vein at a concentration of 0.2 mL per cell and a volume of 0.2 mL per mouse. On day 0 after inoculation, tumor inoculation status was observed using a small animal imager, and on day 4 after inoculation, tumor cell growth status was measured using a small animal imager. Tumor-bearing mice with excessively strong / weak biological imaging signals were eliminated, and 70 mice with moderate tumor imaging signals were selected and randomly assigned to seven groups of 10 mice each. The average imaging signal for each group was approximately 2.92E+06 p / sec. The day of group assignment was designated as day D0, and administration was started on the day of group assignment according to the experimental design, with a dose volume of 10 μL / g. Imaging and drug administration were scheduled on the same day, with an interval of more than 4 hours between imaging and drug administration. Detailed administration methods, doses, and routes are shown in Table 24 below.

[0203] [Table 25]

[0204] After initiating drug administration, the mice's condition is carefully observed daily, and imaging is performed twice a week using a small animal imager to obtain imaging signal diagrams and signal intensity. After the last drug administration, the experimental animals' body weight and tumor growth (detected and recorded by the small animal imager) are continuously monitored for three days, after which the mice are euthanized.

[0205] The following analysis method was used to perform the data analysis, where TGI (%) = (1 - TR / CR) × 100%, where TR and CR are the relative tumor imaging signal sizes (R) of the treatment group and the control group at a specific time point, respectively, and R = Vt / V0 (where V0 is the mean imaging signal at the time of group division, and Vt is the mean imaging signal at each measurement after treatment).

[0206] The tumor inhibition rate (TGI) was calculated based on the imaging signal intensity, and the results are shown in Table 25 and Figure 54. Figure 54 is a graph showing the trend of tumor growth after drug administration. The body weight of the mice in each group and the tumor imaging signal intensity of individual mice in each group after drug administration are shown in Figures 55 to 58. Figure 55 is a graph showing the trend of changes in animal body weight after drug administration. Figure 56 is a biological imaging image of the mice on day 0 after grouping. Figure 57 is a biological imaging image of the mice on day 7 after grouping. Figure 58 is a biological imaging image of the mice on day 14 after grouping.

[0207] [Table 26] TIFF0007853730000060.tif64170

[0208] Note: a: Mean ± standard error, b: Grouping and statistical comparison of tumor imaging signal intensity between the treatment group and the solvent control group on day 14 of administration, t-test. (**P<0.01, ****P<0.0001).

[0209] Experimental results: After grouping and 14 days after administration, compared to the PBS control group, the experimental groups, including 1H9, showed significant inhibition of tumor imaging signal intensity growth with CHO71, CHO44, and rituximab. The groups treated with CHO71 and CHO44 in combination with rituximab, respectively, exhibited a more pronounced inhibitory effect on tumor imaging signal intensity growth than the monotherapy groups (P<0.0001 and P<0.0001). Mouse body weight did not decrease significantly during the administration process, indicating that the antibody molecules did not cause any apparent toxic side effects in the mice.

[0210] Example 26: In vitro inhibition of growth in an MC38-hPD-L1 tumor model in C57BL / 6-hPD-L1 mice. Experimental Objective: To evaluate the inhibitory activity of an anti-human SIRPα antibody against tumor growth in an in vivo drug efficacy study of an MC38-hPD-L1 tumor model in C57BL / 6-hPD-L1 mice.

[0211] Logarithmically growing mouse colon cancer cells MC38-hPD-L1 were digested, the culture medium was removed, and the cells were washed twice with PBS. The cells were then counted and inoculated subcutaneously into the right side of C57BL / 6-hPD-L1 mice, with 5 × 10⁶ cells administered to each mouse. 5 Tumor cells were inoculated at a rate of 100 μL, with an average tumor volume of approximately 50 mm². 3 When the mice reached maturity, they were randomly divided into groups of 10, with the day of group division defined as day D0. On the day of group division, administration was started according to the experimental protocol design, with a dose volume of 10 μL / g. Detailed administration methods, doses, and routes are shown in Table 26 below.

[0212] [Table 27]

[0213] After starting drug administration, the body weight and tumor volume of the mice are measured twice a week. Tumor volume calculation formula: Tumor volume (mm) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the longest diameter of the tumor, and b represents the shortest diameter of the tumor). The experiment was stopped one week after the last drug administration, the mice were euthanized, the tumors were weighed, and photographs were taken.

[0214] The following analysis method is selected to perform data analysis and calculate the relative tumor growth rate (T / C) (%), i.e., the percentage of relative tumor volume or tumor weight between the treatment group and the control group at a specific time point. The formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: mean RTV of the treatment group, CRTV: mean RTV of the control group, RTV = Vt / V0, where V0 is the tumor volume of the mice at the time of group division, and Vt is the tumor volume of the mice after treatment). The relative tumor inhibition rate (TGI) is calculated based on tumor volume. TVThe percentage (TGI) is calculated using the following formula: TGITV% = (1 - T / C) × 100% (where T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively), and the tumor inhibition rate TGI is calculated based on the change in tumor weight. TW The percentage (%) is calculated using the following formula: TGITW% = (1 - TWtreat / TWvehicle) × 100% (TWtreat and TWvehicle are the average tumor weights of the treatment group and control group mice at the end of the experiment, respectively).

[0215] Experimental results: The average tumor volume in mice in the PBS control group was 402.47 mm² on day 19 after drug administration. 3 On day 19 after drug administration, the average tumor volume for the antibody molecule CHO44-L (7.5 mg / kg), CHO44-M (15 mg / kg), CHO44-H (30 mg / kg), and QP3447 (10 mg / kg) groups was 198.20 mm², respectively. 3 144.21mm 3 92.54mm 3 and 89.33mm 3 Compared to the control group PBS, CHO44-L (7.5 mg / kg, TGI=58.07%), CHO44-M (15 mg / kg, TGI=73.19%), CH044-H (30 mg / kg, TGI=87.94%), and QP3447 (10 mg / kg, TGI=88.92%) significantly inhibited tumor growth in a dose-gradient manner (P<0.05*, P<0.05*, P<0.01**, and P<0.01**).

[0216] The tumor inhibition rate (TGITV) was calculated based on mouse tumor volume, and the results are shown in Table 27. The results of the efficacy tests of the antibody molecules CHO44-L (7.5 mg / kg), CHO44-M (15 mg / kg), and CHO44-H (30 mg / kg) against the MC38-hPD-L1 tumor model are shown in Figure 59. Figure 60 shows the grouping of mice into the PBS group and the CHO44-L (7.5 mg / kg), CHO44-M (15 mg / kg), and CHO44-H (30 mg / kg) administration groups, as well as the tumor growth curves of each group after drug administration. Figure 61 shows the weight change curves of mice in each group after drug administration in the MC38-hPD-L1 colon cancer tumor model.

[0217] [Table 28]

[0218] Note: a: The data is expressed as the mean. b: Compared to the G1 group, an independent sample t-test was used, with *: P<0.05 and **: P<0.01.

[0219] In in vivo efficacy studies of an MC38-hPD-L1 tumor model in C57BL / 6-hPD-L1 mice, we found that both antibody molecules CHO44 and QP3447 significantly inhibited tumor growth. The inhibitory effect of CHO44 on tumor growth was dose-dependent, and mouse body weight did not decrease significantly during administration, indicating that the antibody molecules do not have any apparent toxic side effects on mice.

[0220] Example 27: Inhibition of in vivo growth of CT26-hPD-L1 & hCD47 tumor models in BALB / c-hPD-1 & hSIRPα mice. Experimental Objective: To evaluate the inhibitory activity of an anti-human SIRPα antibody against CT26 tumor growth through an in vivo drug efficacy study on CT26-hPD-L1 and hSIRPα tumors in BALB / c-hPD-1 and hSIRPα mice.

[0221] Experimental steps: Logarithmic growth phase mouse colon cancer cells CT26-hPD-L1&hCD47 were digested, the culture medium was removed, and the cells were counted after being washed twice with PBS. These cells were then inoculated subcutaneously into the right side of BALB / c-hPD-1&hSIRPα transgenic mice, with each mouse receiving 1.5 × 10⁶ tumor cells per 100 μL, resulting in an average tumor volume of approximately 40 mm². 3 Upon reaching the end of the study period, the mice were randomly divided into groups of 6, with the day of group division defined as day D0. On the day of group division, administration was initiated according to the experimental protocol design, with a dose volume of 10 μL / g. Detailed administration methods, doses, and routes are shown in Table 28.

[0222] [Table 29]

[0223] After starting drug administration, the body weight and tumor volume of the mice are measured twice a week. Tumor volume calculation formula: Tumor volume (mm) 3 ) = 1 / 2 × (a × b 2 (Here, a represents the longest diameter of the tumor, and b represents the shortest diameter of the tumor). After the test is complete, the experiment is stopped, the mice are euthanized, the tumors are removed, weighed, and photographs are taken.

[0224] The following analysis method is selected to perform data analysis and calculate the relative tumor growth rate (T / C) (%), i.e., the percentage of relative tumor volume or tumor weight between the treatment group and the control group at a specific time point. The formula is as follows: T / C% = TRTV / CRTV × 100% (TRTV: mean RTV of the treatment group, CRTV: mean RTV of the control group, RTV = Vt / V0, where V0 is the tumor volume of the mice at the time of group division, and Vt is the tumor volume of the mice after treatment). The relative tumor inhibition rate (TGI) is calculated based on tumor volume. TV The percentage (%) is calculated using the following formula: TGI TV % = (1 - T / C) × 100% (where T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively), tumor inhibition rate TGI based on change in tumor weight. TWThe percentage (%) is calculated using the following formula: TGI TW %=(1-TW treat / TW vehicle ) × 100% (TW treat and TW vehicle (These are the average tumor weights of the mice in the treatment group and the control group at the end of the experiment, respectively.)

[0225] Experimental results: The average tumor volume in mice in the PBS control group was 841.5 mm³ at 30 days post-drug administration. 3 On day 30 after drug administration, the average tumor volume in the antibody molecule CHO44-L (15 mg / kg) and CHO44-H (30 mg / kg) groups was 158.3 mm², respectively. 3 128.1mm 3 The tumor inhibition rate (TGI) is based on the tumor volume of the mouse. TV The TGI was calculated, and compared to the control group PBS, CHO44-L (15 mg / kg, TGI=80.85%) and CHO44-H (30 mg / kg, TGI=83.9%) all significantly inhibited tumor growth (P<0.05*, P<0.05*, P<0.01**, and P<0.01**). See Table 29 and Figure 62. Figure 63 shows the grouping of mice administered PBS, CHO44-L (15 mg / kg), and CHO44-H (30 mg / kg), and the tumor growth curves for each group after drug administration. In the CHO-L group, tumors disappeared in 3 mice on D14 (2 mice) and D23 (1 mouse), respectively. In the CHO44-H group, tumors disappeared in 4 mice on D12 (2 mice), D14 (1 mouse), and D21 (1 mouse), respectively. Figure 64 shows the body weight change curves of mice in each group after drug administration in the CT26-hPD-L1&hCD47 colon cancer tumor model.

[0226] [Table 30]

[0227] In pharmacodynamic evaluation of BALB / c-hPD1 / hSIRPα mice subcutaneously inoculated with CT26-hPDL1hCD47, some mice showed tumor regression. These mice were then re-inoculated with CT26-hPDL1&hCD47 to evaluate tumor growth.

[0228] The cell processing, tumor volume weighing, and TGI calculation methods in the study will all refer to the in vivo efficacy evaluation methods used for the initial vaccination. The number of cells administered will be the same as the initial number, the injection site will be on the left side opposite to the initial injection site, and tumor volume will be measured twice a week.

[0229] The results showed that, after cell reinoculation, the average tumor volume 14 days after inoculation in the PBS group was 113.56 mm². 3 This shows that tumors do not grow. Tumors in the antibody molecule CHO44-L (15 mg / kg) and CHO44-H (30 mg / kg) groups did not grow 14 days after reinoculation. See Figure 65 for the results, and Figures 66 and 67 for the growth curves and body weight of the mice in each group. The results show that mice develop immunological memory during the initial treatment, and when mice with regressed tumors are reinoculated, the tumors do not grow.

[0230] Example 28: Pharmacodynamic evaluation of a PBMC reconstitution model in female NCG mice subcutaneously transplanted with non-small cell lung cancer HCC827 cell line. Human non-small cell lung cancer (HCC827) cells were subcutaneously inoculated into female NCG mice at a rate of 3.0E+06 cells / 100μl. The average tumor volume was 100 mm². 3When the tumor volume reaches a certain point, it is defined as day D0. On day D0, PBMC 5.5E+06 cells / mouse are intraperitoneally inoculated, and 7 days later (D7), the mice are randomly divided into four groups of 8 each according to tumor volume: G1 / PBS, G2 / CHO44-10mg / kg, G3 / CHO44-25mg / kg, and G4 / CHO71-8mg / kg+QP3447-4mg / kg. After the start of drug administration, the body weight of the mice is measured twice a week and the tumor size is measured 2-3 times a week. Based on statistical analysis of the tumor volume data on day D24, compared to the control group PBS, the G2 / CHO44-10mg / kg, G3 / CHO44-25mg / kg, and G4 / CHO71-8mg / kg+QP3447-4mg / kg groups all showed significant inhibitory activity against tumor growth, and CHO44 showed a dose-gradient dependence. The TGI rates were G2: 72.98% (p<0.0001), G3: 86.28% (p<0.0001), and G4: 58.02% (p=0.0023), respectively (Figure 68, Table 30). For changes in body weight during the administration period, see Figure 69.

[0231] [Table 31]

[0232] Although the present invention has been described in detail through the preferred embodiments described above, it should be understood that the present invention is not limited to the above description. Those skilled in the art will see that various modifications and changes to the present invention are possible upon reading the above description. Accordingly, the scope of protection of the present invention should be limited by the appended claims.

Claims

1. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1, It includes a SIRPα-binding domain and a PD-L1-binding domain, where, The SIRPα-binding domain includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes VHCDR1, VHCDR2, and VHCDR3 whose amino acid sequences are shown in SEQ ID NO: 3, 4, and 5, respectively, and the light chain variable region includes VLCDR1, VLCDR2, and VLCDR3 whose amino acid sequences are shown in SEQ ID NO: 37, 38, and 9, respectively. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1, characterized in that the PD-L1 binding domain comprises a VHH fragment, and the VHH fragment comprises CDR1, CDR2, and CDR3 whose amino acid sequences are shown in SEQ ID NO: 63, 64, and 65, respectively.

2. The sequence of the heavy chain variable region of the SIRPα-binding domain is as shown in SEQ ID NO: 17, or has at least 85% sequence identity thereto, or the sequence of the light chain variable region of the SIRPα-binding domain is selected from SEQ ID NO: 18, or has at least 85% sequence identity thereto. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 1.

3. The sequence of the VHH fragment is as shown in SEQ ID NO: 62, or has at least 85% sequence identity thereto. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 1.

4. The bispecific antibody or its antigen-binding fragment is characterized by further comprising a heavy chain constant region selected from human IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and a light chain constant region selected from human κ chain, λ chain or a variant thereof. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 1.

5. The heavy chain constant region comprises an Fc fragment or a variant thereof, wherein the variant of the Fc fragment is derived from IgG1 and, according to EU counts, comprises the mutation sites: L234A, L235A, and K338A. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 4.

6. The bispecific antibody or its antigen-binding fragment comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises a heavy chain variable region of the SIRPα-binding domain, a heavy chain constant region, and a VHH fragment, wherein the VHH fragment is fused to the N-terminus of the heavy chain variable region of the SIRPα-binding domain, or the VHH fragment is fused to the C-terminus of the heavy chain constant region. The second polypeptide chain is characterized by including the light chain variable region of the SIRPα-binding domain and the light chain constant region. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 4.

7. The bispecific antibody or its antigen-binding fragment comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain includes the heavy chain variable region and the heavy chain constant region of the SIRPα-binding domain. The second polypeptide chain comprises a light chain variable region of the SIRPα-binding domain, a light chain constant region, and a VHH fragment, wherein the VHH fragment is fused to the N-terminus of the light chain variable region of the SIRPα-binding domain. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 4.

8. The bispecific antibody or its antigen-binding fragment is characterized by having a symmetric structure comprising two first polypeptide chains and two second polypeptide chains. A bispecific antibody or antigen-binding fragment thereof targeting SIRPα and PD-L1 according to claim 6 or 7.

9. The bispecific antibody or its antigen-binding fragment further comprises a linking sequence, wherein the linking sequence is selected as (GGGGS)n, and n is an integer from 1 to 4. A bispecific antibody or antigen-binding fragment thereof targeting SIRPα and PD-L1 according to claim 6 or 7.

10. The amino acid sequence of the first polypeptide chain is as shown in any of SEQ ID NO: 66, 26, 69, 84, or 85, or the amino acid sequence of the second polypeptide chain is as shown in any of SEQ ID NO: 67, 68, 82, or 83. A bispecific antibody or antigen-binding fragment thereof targeting SIRPα and PD-L1 according to claim 6 or 7.

11. The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 66, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:

67. A bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 10.

12. It is a drug, The drug is characterized by comprising a bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 1.

13. The drug is characterized by further comprising one or more other cancer treatment agents. The drug according to claim 12.

14. nucleic acid molecules, The nucleic acid molecule characterized by encoding a bispecific antibody or antigen-binding fragment thereof that targets SIRPα and PD-L1 as described in claim 1.

15. It is a vector, The vector, characterized by comprising the nucleic acid molecule described in claim 14.

16. A host cell transformed with the vector described in claim 15.

17. Use of a bispecific antibody or antigen-binding fragment thereof targeting SIRPα and PD-L1 according to claim 1 in the preparation of a drug for inhibiting or treating a disease, symptom, or condition.

18. The aforementioned diseases, symptoms, or conditions are characterized by including cancer, solid tumors, chronic infections, inflammatory diseases, multiple sclerosis, autoimmune diseases, nervous system disorders, brain injuries, nerve injuries, polycythemia, hemochromatosis, trauma, septic shock, fibrosis, atherosclerosis, obesity, type II diabetes, graft dysfunction, or arthritis. The use described in claim 17.

19. The aforementioned cancers include anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, stomach cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovarian cancer, testicular cancer, kidney cancer, renal pelvis and ureteral cancer, salivary gland cancer, small intestine cancer, urethral cancer, bladder cancer, head and neck cancer, spinal cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, and prostate cancer. The treatment is characterized by being selected from pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, teratoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, T-cell or B-cell lymphoma, gastrointestinal stromal tumor, soft tissue tumor, hepatocellular carcinoma, or adenocarcinoma. The use described in claim 18.

20. The aforementioned drug is characterized by being used in combination with one or more other drugs. The use described in claim 17.

21. The aforementioned other drugs are characterized by containing rituximab. The use described in claim 20.

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