Anti-sirpa monoclonal antibodies

Abstract

The application belongs to the technical field of biology and specifically relates to a monoclonal antibody or antigen binding fragment capable of being combined with human sirp alpha with high affinity, and further provides an amino acid sequence and a nucleotide sequence for coding the antibody or antigen binding fragment of the application; the antibody provided by the application can block the interaction between sirp alpha and CD47 without affecting the interaction between CD47 and sirpg.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, it relates to an anti-SIRPα monoclonal antibody, its preparation method, and its applications. Background Technology

[0002] Signal regulatory protein α, or SIRPα (also known as SIRPα / CD172a or SHPS-1), is a cell surface glycoprotein belonging to the immunoglobulin superfamily of transmembrane proteins. It is primarily expressed in myeloid cell lineages (including macrophages, dendritic cells, granulocytes, etc.) and is characterized by two juxtamembranous IgC domains and one distal IgV domain in its extracellular region; its intracellular tail contains two immunoreceptor tyrosine inhibitory motifs (ITIMs). Once the receptor is cross-linked, the tyrosine-phosphorylated ITIM sites recruit and activate SHP phosphatases to negatively regulate cellular functions such as phagocytosis or the release of inflammatory cytokines. Axel Ullrich et al. described the cloning and expression of human SIRPα in US6541615. Sarfati et al. in WO1999 / 040940 and Van den Berg et al. in WO00 / 66159 suggested that SIRPα and CD47 are involved in the pathogenesis of cancer and other diseases, and Van den Berg et al. also proposed the therapeutic application of SIRPα inhibitors. The interaction between SIRPα and CD47 plays a crucial role in regulating the phagocytosis of leukemia cells and leukemia stem cells (LSCs) by macrophages. Research from University Health Network (WO2013 / 056352), Eberhard's US6913894, and other institutions has also suggested that anti-SIRPα antibodies can effectively inhibit the interaction between SIRPα and CD47, thereby exerting anti-tumor effects.

[0003] CD47, as the major ligand of SIRPα, and its widespread expression in most cell types (including endothelial / epithelial cells, leukocytes, and erythrocytes), suggest that it mediates a “don’t eat me” signal to protect healthy cells from phagocytocyte-dependent clearance. To support this, several studies have shown rapid clearance of CD47-deficient cells in wild-type recipients through adoptive transfer of erythrocytes or leukocytes from CD47-knockout mice. Conversely, localized genetic analysis of immunocompromised mice receiving human hematopoietic stem cells from multiple strains identified the Sirpα allele in NOD mice as the cause of successful engraftment in xenotransplantation models. Subsequent studies demonstrated that allele variants of SIRPα expressed only in NOD mice retain the ability to bind human CD47 expressed on human hematopoietic stem cells, thereby suppressing macrophage-dependent transplant rejection.

[0004] Regulated expression of SIRPα and CD47 establishes homeostatic control mechanisms to modulate phagocytic activity. For example, apoptotic cells downregulate CD47 expression to promote phagocytosis by resident macrophages while keeping living cells unharmed. Similarly, inflammatory stimuli (such as LPS) reduce SIRPα expression in macrophages and dendritic cells (DCs) to enhance their activation during inflammation. However, as seen in cancer, dysregulation of SIRPα and CD47 expression contributes to immune-related diseases. Several tumors significantly increase CD47 expression relative to non-cancerous cells in order to evade immune surveillance mechanisms that eliminate malignant cells under normal conditions. Preclinical studies have revealed that CD47 knockdown in syngeneic tumor models (such as B16F10 melanoma) is sufficient to suppress tumor growth in immunocompetent mice. Similar results have been observed in human cancer cell lines with CD47 knockout transplanted into immunocompromised mice. Alternatively, biologics that disrupt the SIRPα-CD47 interaction (such as anti-CD47 antibodies) also enhance tumor clearance in mouse models. When combined with commercially available antitumor antigen antibodies (such as trastuzumab or rituximab), antiCD47 antibodies synergistically enhance antitumor responses compared to standard monotherapy.

[0005] However, given the widespread expression of CD47, anti-CD47 antibodies cause associated blood toxicity (anemia or thrombocytopenia), which limits their therapeutic efficacy.

[0006] Furthermore, CD47 also binds to another member of the sirp family, sirp-γ (also known as sirpg, sirpγ, CD172g, or sirpβ2), which is present on the surface of human T cells but not on human bone marrow cells. SIRPg is the result of replication of the sirp gene in ancient primates from nearly 35 million years ago and is expressed in a restricted manner on human T lymphocytes, in contrast to SIRPα expression on bone marrow cells. sirpg is absent in mice. It has been shown that the SIRPg-CD47 interaction mediates cell-cell adhesion, enhances superantigen-dependent T cell-mediated proliferation, and promotes co-stimulatory T cell activation.

[0007] Due to the high sequence similarity between SIRPα and sirpg (especially in the region where they interact with CD47), existing published anti-SIRPα antibodies also bind to sirpg and have adverse effects in humans, such as inhibiting T cell proliferation and reducing immune responses. Therefore, there remains a need in the art for novel antibodies with high affinity that can block the interaction between SIRPα and CD47 without affecting the interaction between CD47 and sirpg. Summary of the Invention

[0008] The purpose of this invention is to provide a high-affinity, therapeutic anti-SIRPα monoclonal antibody that can block the interaction between SIRPα and CD47 without affecting the binding between sirpg and CD47. The invention also provides a method for preparing the anti-SIRPα monoclonal antibody and its application.

[0009] In a first aspect, the present invention provides a murine monoclonal antibody capable of binding to SIRPα. The antibody is an anti-SIRPα monoclonal antibody or antigen-binding fragment having high affinity for SIRPα and low affinity for SIRPg, said antibody or antigen-binding fragment comprising a variable region of the antibody light chain containing LCDR1, LCDR2, and LCDR3, and a variable region of the antibody heavy chain containing HCDR1, HCDR2, and HCDR3.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain CDR selected from amino acid sequences of SEQ IN NO: 1-21 or any variant of such sequences; and / or a light chain CDR selected from amino acid sequences of SEQ IN NO: 22-39 or any variant of such sequences.

[0011] The amino acid sequence of HCDR1 is selected from SEQ IN NO: 1, 4, 7, 10, 13, 16 or SEQ IN NO: 19;

[0012] The amino acid sequence of HCDR2 is selected from SEQ IN NO: 2, 5, 8, 11, 14, 17 or SEQ IN NO: 20;

[0013] The amino acid sequence of HCDR3 is selected from SEQ IN NO: 3, 6, 9, 12, 15, 18 or SEQ IN NO: 21;

[0014] The amino acid sequence of LCDR1 is selected from SEQ IN NO: 22, 25, 28, 30, 33, 36 or SEQ IN NO: 38;

[0015] The amino acid sequence of LCDR2 is selected from SEQ IN NO: 23, 26, 29, 31, or SEQ IN NO: 34;

[0016] The amino acid sequence of LCDR3 is selected from SEQ IN NO: 24, 27, 32, 35, 37 or SEQ IN NO: 39.

[0017] In some preferred embodiments, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0018] (1) HCDR1 with amino acid sequence SEQ IN NO: 1, HCDR2 with amino acid sequence SEQ IN NO: 2, and HCDR3 with amino acid sequence SEQ IN NO: 3.

[0019] (2) HCDR1 with amino acid sequence SEQ IN NO: 4, HCDR2 with amino acid sequence SEQ IN NO: 5, and HCDR3 with amino acid sequence SEQ IN NO: 6.

[0020] (3) HCDR1 with amino acid sequence SEQ IN NO: 16, HCDR2 with amino acid sequence SEQ IN NO: 17, and HCDR3 with amino acid sequence SEQ IN NO: 18.

[0021] (4) HCDR1 with amino acid sequence SEQ IN NO: 7, HCDR2 with amino acid sequence SEQ IN NO: 8, and HCDR3 with amino acid sequence SEQ IN NO: 9.

[0022] (5) HCDR1 with amino acid sequence SEQ IN NO: 13, HCDR2 with amino acid sequence SEQ IN NO: 14, and HCDR3 with amino acid sequence SEQ IN NO: 15.

[0023] (6) HCDR1 with amino acid sequence SEQ IN NO: 10, HCDR2 with amino acid sequence SEQ IN NO: 11, and HCDR3 with amino acid sequence SEQ IN NO: 12.

[0024] (7) HCDR1 with amino acid sequence SEQ IN NO: 19, HCDR2 with amino acid sequence SEQ IN NO: 20, and HCDR3 with amino acid sequence SEQ IN NO: 21.

[0025] More preferably, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0026] (4) HCDR1 with amino acid sequence SEQ IN NO: 7, HCDR2 with amino acid sequence SEQ IN NO: 8, and HCDR3 with amino acid sequence SEQ IN NO: 9.

[0027] (5) HCDR1 with amino acid sequence SEQ IN NO: 13, HCDR2 with amino acid sequence SEQ IN NO: 14, and HCDR3 with amino acid sequence SEQ IN NO: 15.

[0028] (6) HCDR1 with amino acid sequence SEQ IN NO: 10, HCDR2 with amino acid sequence SEQ IN NO: 11, and HCDR3 with amino acid sequence SEQ IN NO: 12.

[0029] (7) HCDR1 with amino acid sequence SEQ IN NO: 19, HCDR2 with amino acid sequence SEQ IN NO: 20, and HCDR3 with amino acid sequence SEQ IN NO: 21.

[0030] More preferably, the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0031] (6) HCDR1 with amino acid sequence SEQ IN NO: 10, HCDR2 with amino acid sequence SEQ IN NO: 11, and HCDR3 with amino acid sequence SEQ IN NO: 12.

[0032] (7) HCDR1 with amino acid sequence SEQ IN NO: 19, HCDR2 with amino acid sequence SEQ IN NO: 20, and HCDR3 with amino acid sequence SEQ IN NO: 21.

[0033] The above (1)-(7) are heavy chain CDRs of 36D6, 36G4, 55G4, 39D8, 52B3, 40F3 and 57B10 respectively.

[0034] In some preferred embodiments, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0035] (1) LCDR1 with amino acid sequence SEQ IN NO: 22, LCDR2 with amino acid sequence SEQ IN NO: 23, and LCDR3 with amino acid sequence SEQ IN NO: 24.

[0036] (2) CDR1 with amino acid sequence SEQ IN NO: 25, LCDR2 with amino acid sequence SEQ IN NO: 26, and LCDR3 with amino acid sequence SEQ IN NO: 27.

[0037] (3) CDR1 with amino acid sequence SEQ IN NO: 36, LCDR2 with amino acid sequence SEQ IN NO: 26, and LCDR3 with amino acid sequence SEQ IN NO: 37.

[0038] (4) CDR1 with amino acid sequence SEQ IN NO: 28, LCDR2 with amino acid sequence SEQ IN NO: 29, and LCDR3 with amino acid sequence SEQ IN NO: 27.

[0039] (5) CDR1 with amino acid sequence SEQ IN NO: 33, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 35.

[0040] (6) CDR1 with amino acid sequence SEQ IN NO: 30, LCDR2 with amino acid sequence SEQ IN NO: 31, and LCDR3 with amino acid sequence SEQ IN NO: 32;

[0041] (7) CDR1 with amino acid sequence SEQ IN NO: 38, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 39.

[0042] More preferably, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0043] (4) CDR1 with amino acid sequence SEQ IN NO: 28, LCDR2 with amino acid sequence SEQ IN NO: 29, and LCDR3 with amino acid sequence SEQ IN NO: 27.

[0044] (5) CDR1 with amino acid sequence SEQ IN NO: 33, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 35.

[0045] (6) CDR1 with amino acid sequence SEQ IN NO: 30, LCDR2 with amino acid sequence SEQ IN NO: 31, and LCDR3 with amino acid sequence SEQ IN NO: 32;

[0046] (7) CDR1 with amino acid sequence SEQ IN NO: 38, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 39.

[0047] More preferably, the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0048] (6) CDR1 with amino acid sequence SEQ IN NO: 30, LCDR2 with amino acid sequence SEQ IN NO: 31, and LCDR3 with amino acid sequence SEQ IN NO: 32;

[0049] (7) CDR1 with amino acid sequence SEQ IN NO: 38, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 39.

[0050] The above (1)-(7) are light chain CDRs of 36D6, 36G4, 55G4, 39D8, 52B3, 40F3 and 57B10 respectively.

[0051] In some preferred embodiments, the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0052] (1) HCDR1 with amino acid sequence SEQ IN NO: 1, HCDR2 with amino acid sequence SEQ IN NO: 2, and HCDR3 with amino acid sequence SEQ IN NO: 3; and LCDR1 with amino acid sequence SEQ IN NO: 22, LCDR2 with amino acid sequence SEQ IN NO: 23, and LCDR3 with amino acid sequence SEQ IN NO: 24.

[0053] (2) HCDR1 with amino acid sequence SEQ IN NO:4, HCDR2 with amino acid sequence SEQ IN NO:5, and HCDR3 with amino acid sequence SEQ IN NO:6; and LCDR1 with amino acid sequence SEQ IN NO:25, LCDR2 with amino acid sequence SEQ IN NO:26, and LCDR3 with amino acid sequence SEQ IN NO:27.

[0054] (3) HCDR1 with amino acid sequence SEQ IN NO: 16, HCDR2 with amino acid sequence SEQ IN NO: 17, and HCDR3 with amino acid sequence SEQ IN NO: 18; and LCDR1 with amino acid sequence SEQ IN NO: 36, LCDR2 with amino acid sequence SEQ IN NO: 26, and LCDR3 with amino acid sequence SEQ IN NO: 37.

[0055] (4) HCDR1 with amino acid sequence SEQ IN NO: 7, HCDR2 with amino acid sequence SEQ IN NO: 8, and HCDR3 with amino acid sequence SEQ IN NO: 9; and LCDR1 with amino acid sequence SEQ IN NO: 28, LCDR2 with amino acid sequence SEQ IN NO: 29, and LCDR3 with amino acid sequence SEQ IN NO: 27.

[0056] (5) HCDR1 with amino acid sequence SEQ IN NO: 13, HCDR2 with amino acid sequence SEQ IN NO: 14, and HCDR3 with amino acid sequence SEQ IN NO: 15; and LCDR1 with amino acid sequence SEQ IN NO: 33, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 35.

[0057] (6) HCDR1 with amino acid sequence SEQ IN NO: 10, HCDR2 with amino acid sequence SEQ IN NO: 11, and HCDR3 with amino acid sequence SEQ IN NO: 12; and LCDR1 with amino acid sequence SEQ IN NO: 30, LCDR2 with amino acid sequence SEQ IN NO: 31, and LCDR3 with amino acid sequence SEQ IN NO: 32.

[0058] (7) HCDR1 with amino acid sequence SEQ IN NO: 19, HCDR2 with amino acid sequence SEQ IN NO: 20, and HCDR3 with amino acid sequence SEQ IN NO: 21; and LCDR1 with amino acid sequence SEQ IN NO: 38, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 39.

[0059] More preferably, the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0060] (4) HCDR1 with amino acid sequence SEQ IN NO: 7, HCDR2 with amino acid sequence SEQ IN NO: 8, and HCDR3 with amino acid sequence SEQ IN NO: 9; and LCDR1 with amino acid sequence SEQ IN NO: 28, LCDR2 with amino acid sequence SEQ IN NO: 29, and LCDR3 with amino acid sequence SEQ IN NO: 27.

[0061] (5) HCDR1 with amino acid sequence SEQ IN NO: 13, HCDR2 with amino acid sequence SEQ IN NO: 14, and HCDR3 with amino acid sequence SEQ IN NO: 15; and LCDR1 with amino acid sequence SEQ IN NO: 33, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 35.

[0062] (6) HCDR1 with amino acid sequence SEQ IN NO: 10, HCDR2 with amino acid sequence SEQ IN NO: 11, and HCDR3 with amino acid sequence SEQ IN NO: 12; and LCDR1 with amino acid sequence SEQ IN NO: 30, LCDR2 with amino acid sequence SEQ IN NO: 31, and LCDR3 with amino acid sequence SEQ IN NO: 32.

[0063] (7) HCDR1 with amino acid sequence SEQ IN NO: 19, HCDR2 with amino acid sequence SEQ IN NO: 20, and HCDR3 with amino acid sequence SEQ IN NO: 21; and LCDR1 with amino acid sequence SEQ IN NO: 38, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 39.

[0064] More preferably, the amino acid sequences of HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0065] (6) HCDR1 with amino acid sequence SEQ IN NO: 10, HCDR2 with amino acid sequence SEQ IN NO: 11, and HCDR3 with amino acid sequence SEQ IN NO: 12; and LCDR1 with amino acid sequence SEQ IN NO: 30, LCDR2 with amino acid sequence SEQ IN NO: 31, and LCDR3 with amino acid sequence SEQ IN NO: 32.

[0066] (7) HCDR1 with amino acid sequence SEQ IN NO: 19, HCDR2 with amino acid sequence SEQ IN NO: 20, and HCDR3 with amino acid sequence SEQ IN NO: 21; and LCDR1 with amino acid sequence SEQ IN NO: 38, LCDR2 with amino acid sequence SEQ IN NO: 34, and LCDR3 with amino acid sequence SEQ IN NO: 39.

[0067] The above (1)-(7) are the heavy and light chain CDRs of 36D6, 36G4, 55G4, 39D8, 52B3, 40F3 and 57B10 respectively.

[0068] A second aspect of the present invention provides an amino acid sequence of the heavy chain variable region and / or light chain variable region of an antibody or antigen-binding fragment.

[0069] In some preferred embodiments, the amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment is selected from the heavy chain variable region of SEQ IN NO: 40, 41, 42, 43, 44, 45, 46 or any variant of the sequence; and / or the amino acid sequence of the light chain variable region is selected from the light chain variable region of SEQ IN NO: 47, 48, 49, 50, 51, 52, 53 or any variant of the sequence.

[0070] Preferably, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0071] (1) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 40, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 40; and the amino acid sequence of the light chain variable region is SEQ IN NO: 47, or a sequence having at least 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 47.

[0072] (2) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 41, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 41; and the amino acid sequence of the light chain variable region is SEQ IN NO: 48, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 48.

[0073] (3) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 45, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 45; and the amino acid sequence of the light chain variable region is SEQ IN NO: 52, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 52.

[0074] (4) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 42, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 42; and the amino acid sequence of the light chain variable region is SEQ IN NO: 49, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 49.

[0075] (5) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 44, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 44; and the amino acid sequence of the light chain variable region is SEQ IN NO: 51, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 51.

[0076] (6) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 43, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 43; and the amino acid sequence of the light chain variable region is SEQ IN NO: 50, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 50.

[0077] (7) The heavy chain amino acid sequence is SEQ IN NO: 46, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 46; and the light chain variable region amino acid sequence is SEQ IN NO: 53, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 53.

[0078] More preferably, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0079] (4) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 42, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 42; and the amino acid sequence of the light chain variable region is SEQ IN NO: 49, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 49.

[0080] (5) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 44, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 44; and the amino acid sequence of the light chain variable region is SEQ IN NO: 51, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 51.

[0081] (6) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 43, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 43; and the amino acid sequence of the light chain variable region is SEQ IN NO: 50, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 50.

[0082] (7) The heavy chain amino acid sequence is SEQ IN NO: 46, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 46; and the light chain variable region amino acid sequence is SEQ IN NO: 53, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 53.

[0083] More preferably, the heavy chain variable region and light chain variable region of the antibody or antigen-binding fragment are selected from a group of the following amino acid sequences:

[0084] (6) The amino acid sequence of the heavy chain variable region is SEQ IN NO: 43, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 43; and the amino acid sequence of the light chain variable region is SEQ IN NO: 50, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 50.

[0085] (7) The heavy chain amino acid sequence is SEQ IN NO: 46, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence of SEQ IN NO: 46; and the light chain variable region amino acid sequence is SEQ IN NO: 53, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence of SEQ IN NO: 53.

[0086] The above (1)-(7) are the heavy chain variable region and light chain variable region amino acid sequences of 36D6, 36G4, 55G4, 39D8, 52B3, 40F3, and 57B10, respectively.

[0087] A third aspect of the invention provides an isolated polynucleotide, wherein, according to embodiments of the invention, the described polynucleotide encodes the amino acid sequence of the variable region of the antibody heavy chain, having one of the following nucleotide sequences:

[0088] (1) The polynucleotide has a nucleotide sequence SEQ IN NO: 54 that encodes the amino acid sequence shown in SEQ ID NO: 40 for the heavy chain variable region.

[0089] (2) The polynucleotide has a nucleotide sequence SEQ IN NO: 55 that encodes the amino acid sequence shown in SEQ ID NO: 41 of the heavy chain variable region.

[0090] (3) The polynucleotide has a nucleotide sequence SEQ IN NO: 59 that encodes the amino acid sequence shown in SEQ ID NO: 45 for the heavy chain variable region.

[0091] (4) The polynucleotide has a nucleotide sequence SEQ IN NO: 56 that encodes the amino acid sequence shown in SEQ ID NO: 42 of the heavy chain variable region.

[0092] (5) The polynucleotide has a nucleotide sequence SEQ IN NO: 58 that encodes the amino acid sequence shown in SEQ ID NO: 44 of the heavy chain variable region.

[0093] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 57 that encodes the amino acid sequence shown in SEQ ID NO: 43 of the heavy chain variable region.

[0094] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 60 that encodes the amino acid sequence shown in SEQ ID NO: 46 of the heavy chain variable region.

[0095] More preferably, the polynucleotide encodes the amino acid sequence of the variable region of the antibody heavy chain of the present invention, having one of the following nucleotide sequences:

[0096] (4) The polynucleotide has a nucleotide sequence SEQ IN NO: 56 that encodes the amino acid sequence shown in SEQ ID NO: 42 of the heavy chain variable region.

[0097] (5) The polynucleotide has a nucleotide sequence SEQ IN NO: 58 that encodes the amino acid sequence shown in SEQ ID NO: 44 of the heavy chain variable region.

[0098] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 57 that encodes the amino acid sequence shown in SEQ ID NO: 43 of the heavy chain variable region.

[0099] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 60 that encodes the amino acid sequence shown in SEQ ID NO: 46 of the heavy chain variable region.

[0100] More preferably, the polynucleotide encodes the amino acid sequence of the variable region of the antibody heavy chain of the present invention, having one of the following nucleotide sequences:

[0101] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 57 that encodes the amino acid sequence shown in SEQ ID NO: 43 of the heavy chain variable region.

[0102] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 60 that encodes the amino acid sequence shown in SEQ ID NO: 46 of the heavy chain variable region;

[0103] The above (1)-(7) are the nucleotide sequences encoding the variable regions of the antibody heavy chain, namely 36D6, 36G4, 55G4, 39D8, 52B3, 40F3, and 57B10.

[0104] According to embodiments of the present invention, the polynucleotide described above encodes the amino acid sequence of the variable region of the antibody light chain of the present invention, having one of the following nucleotide sequences:

[0105] (1) The polynucleotide has a nucleotide sequence SEQ IN NO: 61 that encodes the amino acid sequence shown in SEQ ID NO: 47 of the light chain variable region.

[0106] (2) The polynucleotide has a nucleotide sequence SEQ IN NO: 62 that encodes the amino acid sequence shown in SEQ ID NO: 48 of the light chain variable region.

[0107] (3) The polynucleotide has a nucleotide sequence SEQ IN NO: 66 that encodes the amino acid sequence shown in SEQ ID NO: 52 of the light chain variable region.

[0108] (4) The polynucleotide has a nucleotide sequence SEQ IN NO: 63 that encodes the amino acid sequence shown in SEQ ID NO: 49 for the light chain variable region.

[0109] (5) The polynucleotide has a nucleotide sequence SEQ IN NO: 65 that encodes the amino acid sequence shown in SEQ ID NO: 51 of the light chain variable region.

[0110] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 64 that encodes the amino acid sequence shown in SEQ ID NO: 50 for the light chain variable region.

[0111] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 67 that encodes the amino acid sequence shown in SEQ ID NO: 53 for the light chain variable region.

[0112] More preferably, the polynucleotide encodes the amino acid sequence of the variable region of the antibody light chain of the present invention, having one of the following nucleotide sequences:

[0113] (4) The polynucleotide has a nucleotide sequence SEQ IN NO: 63 that encodes the amino acid sequence shown in SEQ ID NO: 49 for the light chain variable region.

[0114] (5) The polynucleotide has a nucleotide sequence SEQ IN NO: 65 that encodes the amino acid sequence shown in SEQ ID NO: 51 of the light chain variable region.

[0115] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 64 that encodes the amino acid sequence shown in SEQ ID NO: 50 for the light chain variable region.

[0116] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 67 that encodes the amino acid sequence shown in SEQ ID NO: 53 for the light chain variable region.

[0117] More preferably, the polynucleotide encodes the amino acid sequence of the variable region of the antibody light chain of the present invention, having one of the following nucleotide sequences:

[0118] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 64 that encodes the amino acid sequence shown in SEQ ID NO: 50 for the light chain variable region.

[0119] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 67 that encodes the amino acid sequence shown in SEQ ID NO: 53 for the light chain variable region.

[0120] The above (1)-(7) are the nucleotide sequences encoding the variable regions of the light chain of antibodies 36D6, 36G4, 55G4, 39D8, 52B3, 40F3, and 57B10, respectively.

[0121] According to embodiments of the present invention, the polynucleotide described above encodes the antibody or its antigen-binding fragment described herein, having one of the following nucleotide sequences:

[0122] (1) The polynucleotide has a nucleotide sequence SEQ IN NO: 54 encoding the amino acid sequence shown in SEQ ID NO: 40 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 61 encoding the amino acid sequence shown in SEQ ID NO: 47 of the light chain variable region.

[0123] (2) The polynucleotide has a nucleotide sequence SEQ IN NO: 55 encoding the amino acid sequence shown in SEQ ID NO: 41 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 62 encoding the amino acid sequence shown in SEQ ID NO: 48 of the light chain variable region.

[0124] (3) The polynucleotide has a nucleotide sequence SEQ IN NO: 59 encoding the amino acid sequence shown in SEQ ID NO: 45 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 66 encoding the amino acid sequence shown in SEQ ID NO: 52 of the light chain variable region.

[0125] (4) The polynucleotide has a nucleotide sequence SEQ IN NO: 56 encoding the amino acid sequence shown in SEQ ID NO: 42 of the heavy chain variable region; and the polynucleotide has a nucleotide sequence SEQ IN NO: 63 encoding the amino acid sequence shown in SEQ ID NO: 49 of the light chain variable region.

[0126] (5) The polynucleotide has a nucleotide sequence SEQ IN NO: 58 encoding the amino acid sequence shown in SEQ ID NO: 44 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 65 encoding the amino acid sequence shown in SEQ ID NO: 51 of the light chain variable region.

[0127] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 57 encoding the amino acid sequence shown in SEQ ID NO: 43 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 64 encoding the amino acid sequence shown in SEQ ID NO: 50 of the light chain variable region.

[0128] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 60 encoding the amino acid sequence shown in SEQ ID NO: 46 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 67 encoding the amino acid sequence shown in SEQ ID NO: 53 of the light chain variable region.

[0129] More preferably, the polynucleotide encodes the antibody or its antigen-binding fragment described in this invention and has one of the following nucleotide sequences:

[0130] (4) The polynucleotide has a nucleotide sequence SEQ IN NO: 56 encoding the amino acid sequence shown in SEQ ID NO: 42 of the heavy chain variable region; and the polynucleotide has a nucleotide sequence SEQ IN NO: 63 encoding the amino acid sequence shown in SEQ ID NO: 49 of the light chain variable region.

[0131] (5) The polynucleotide has a nucleotide sequence SEQ IN NO: 58 encoding the amino acid sequence shown in SEQ ID NO: 44 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 65 encoding the amino acid sequence shown in SEQ ID NO: 51 of the light chain variable region.

[0132] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 57 encoding the amino acid sequence shown in SEQ ID NO: 43 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 64 encoding the amino acid sequence shown in SEQ ID NO: 50 of the light chain variable region.

[0133] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 60 encoding the amino acid sequence shown in SEQ ID NO: 46 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 67 encoding the amino acid sequence shown in SEQ ID NO: 53 of the light chain variable region.

[0134] More preferably, the polynucleotide encodes the antibody or its antigen-binding fragment described in this invention and has one of the following nucleotide sequences:

[0135] (6) The polynucleotide has a nucleotide sequence SEQ IN NO: 57 encoding the amino acid sequence shown in SEQ ID NO: 43 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 64 encoding the amino acid sequence shown in SEQ ID NO: 50 of the light chain variable region.

[0136] (7) The polynucleotide has a nucleotide sequence SEQ IN NO: 60 encoding the amino acid sequence shown in SEQ ID NO: 46 of the heavy chain variable region; and a nucleotide sequence SEQ IN NO: 67 encoding the amino acid sequence shown in SEQ ID NO: 53 of the light chain variable region.

[0137] The above (1)-(7) are the nucleotide sequences encoding the heavy chain variable region and light chain variable region of antibodies 36D6, 36G4, 55G4, 39D8, 52B3, 40F3, and 57B10, respectively.

[0138] A fourth aspect of the invention provides a method for preparing anti-SIRPα antibodies, wherein the monoclonal antibody can be obtained using various methods well known to those skilled in the art. For example, the monoclonal antibody can be prepared using the classic hybridoma method (first proposed by Kohler et al., Nature, 256:495 (1975)) or the recombinant DNA method (US4816567). The monoclonal antibody can also be isolated from a phage antibody library using, for example, the techniques described in Clackson et al., Nather, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991). One preparation method may include the following steps:

[0139] (1) Preparation of immunogen and immunization of animals. The amino acid sequence of SIRPα was found from Uniprot (Uniprot: P78324). Amino acids 31-373 were selected and the extracellular region of SIRPα was linked to the mouse heavy chain constant region by three linker peptides (GGGGS). The sequence was optimized and DNA was synthesized by Genscript Biotech Co., Ltd. A eukaryotic expression vector was constructed, transfected into CHO-S cells, and the fusion protein was expressed and purified. It was used as an antigen to immunize mice.

[0140] (2) SIRPα monoclonal antibody was prepared using the classic hybridoma technique.

[0141] (3) Antibodies that cross-bind with CHO-hSIRPα and CHO-cyno-SIRPα were screened using indirect ELISA and cross-flow cytometry.

[0142] (4) The antibody was purified by affinity chromatography, the molecular weight and purity of the antibody were determined by SDS-PAGE, and the affinity of the purified antibody was determined by Biacore T200.

[0143] The method for determining the heavy chain and light chain variable regions of the anti-SIRPα antibody in this invention is as follows: specific primers are synthesized based on the constant region sequence of the antibody gene; the heavy chain and light chain variable regions of the monoclonal antibody SIRPα are amplified by PCR; the target fragment is recovered, cloned into the pMD19-T (simple) vector, transformed into E. coli DH5α, positive clones are selected, plasmids are extracted and sequenced to determine the heavy chain and light chain variable region sequences of the SIRPα monoclonal antibody.

[0144] After obtaining the nucleotide sequences encoding the heavy chain variable region and light chain variable region of the monoclonal antibody of the present invention, the anti-SIRPα monoclonal antibody of the present invention can also be prepared by the following method. The specific operation is as follows:

[0145] First, an expression vector containing a nucleotide sequence encoding the anti-SIRPα monoclonal antibody of the present invention is provided.

[0146] The DNA sequence encoding the anti-SIRPα antibody of the present invention can be obtained using conventional methods well known to those skilled in the art. For example, nucleotide sequences encoding the heavy chain variable region and light chain variable region of the monoclonal antibody can be artificially synthesized or amplified by PCR according to the sequence disclosed in the present invention. These nucleotide sequences are then inserted into a suitable expression vector by selecting appropriate restriction enzyme sites using various methods well known in the art, such that they precede the heavy chain constant region coding sequence and the light chain constant region coding sequence carried by the expression vector, respectively, and are placed within the same reading frame.

[0147] The expression vectors used in this invention are various commercially available expression vectors known to those skilled in the art. These include, but are not limited to: plasmids; phage particles; Cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0148] Subsequently, the expression vector obtained above is transformed into suitable host cells. "Host cells" generally include prokaryotic and eukaryotic cells. Common examples of prokaryotic host cells include *Escherichia coli* and *Bacillus subtilis*. Common eukaryotic host cells include yeast cells, insect cells, and mammalian cells. In this invention, CHO cells are preferred host cells.

[0149] There are many methods for transforming host cells using expression vectors, and the transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene (1,5-dimethyl-1,5-diazadecylmethylene polymethyl bromide)-mediated transfection, protoplast fusion, electroporation, liposome-mediated transfection, and direct microinjection of DNA into the cell nucleus.

[0150] Then, the transformed host cells were cultured under conditions suitable for the expression of the anti-SIRPα monoclonal antibody of the present invention. The antibody of the present invention was purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, and other conventional separation and purification methods well known to those skilled in the art.

[0151] The obtained monoclonal antibodies can be identified using conventional methods. The binding specificity of monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of monoclonal antibodies can be determined using analytical methods such as SPR.

[0152] In some embodiments, the present invention may also employ another method to prepare the antibody, the method comprising:

[0153] (1) Constructing an expression vector containing the DNA molecule described in the third aspect: digesting the expression vector pGS with EcoRI and NotI restriction enzymes, ligating the sequence encoding the antibody of the present invention to the digested expression vector to construct the expression vector.

[0154] (2) Transform host cells using the expression vector described in step (1): Transform the expression vector constructed in step (1) into Escherichia coli DH5a, amplify and culture, collect cells, extract plasmids, and transfect CHO cells with the above plasmids by electroporation.

[0155] (3) Culture the host cells obtained in step (2): The transfected CHO cells were screened for clones on MTX selective medium, cultured using the limiting dilution method, and single clone cell lines were selected and cultured in CD FortiCHO medium.

[0156] (4) The monoclonal antibody obtained is isolated, purified and detected.

[0157] Advantages and beneficial effects of the present invention:

[0158] The anti-SIRPα monoclonal antibody of this invention has a purity of over 95% after purification; the antibody affinity reaches 10. -11M, with strong blocking efficacy, the anti-SIRPα monoclonal antibody can bind to SIRPα with high affinity, blocking the interaction between SIRPα and CD47 without binding to SIRPg, while not affecting the binding of CD47 to SIRPg. This invention lays a good foundation for constructing anti-human SIRPα chimeric antibodies and humanized antibodies with good pharmaceutical value.

[0159] the term

[0160] As used herein, unless otherwise expressly stated, the singular forms “an” and “the” include plural indicators. Thus, for example, the term “antibody” optionally includes a combination of two or more such molecules, etc.

[0161] As used herein, the term “about” refers to the typical range of error for each value that is readily known to those skilled in the art.

[0162] The term “antibody” is used in the broadest sense herein and includes a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), and antibody fragments, provided they exhibit the desired antigen-binding activity.

[0163] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homologous group of antibodies, i.e., the individual antibodies comprising that group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring mutations or variant antibodies that appear during the production of a monoclonal antibody formulation), which are typically present in small quantities. In contrast to polyclonal antibody formulations (which typically comprise different antibodies targeting different determinants (epitaxes)), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates the characteristic of an antibody derived from a substantially homologous group of antibodies and is not interpreted as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to the present invention can be manufactured using a variety of techniques, including but not limited to hybridoma methods (first proposed by Kohler et al., Nature, 256:495 (1975)), recombinant DNA methods (US4816567), and isolation from a phage antibody library, as described above. Examples include phage display methods, such as those described in Clackson et al., Nather, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991), and methods using transgenic animals containing all or part of the human immunoglobulin loci.

[0164] As used in this article, "antibody" and "monoclonal antibody" refer to heterotetraglycoproteins of approximately 150,000 Daltons with the same structural characteristics, composed of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. The variable region of each light chain is opposite to the variable region of the heavy chain. Specific amino acids form interfaces between the variable regions of the light and heavy chains.

[0165] An "antibody fragment" is a molecule that is distinct from the intact antibody, containing a portion of the intact antibody and binding to an antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules, such as scFv molecules; and multispecific antibodies formed from antibody fragments.

[0166] "Antibody that binds to the same epitope as the reference antibody" or "antibody that has the same binding specificity as the reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competitive assay. An antibody that binds to the same epitope may bind to the same epitope as the reference antibody, or may bind to a portion of the epitope. Exemplary competitive assays are provided herein.

[0167] As used herein, “variable region” refers to the antibody variable region containing segments of frame 1, CDR1, frame 2, CDR2, and frame 3 (including CDR3 and frame 4), which are added to the V segment due to rearrangement of heavy and light chain V-region genes during B cell differentiation.

[0168] As used herein, “complementarity-determining regions (CDRs)” refer to the three highly variable regions (HVRs) in each chain that break the four “framework regions” established by the variable regions of the light and heavy chains. CDRs are the major contributors to the epitopes that bind to the antigen. The CDRs of each chain are designated CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus and identified by the chain in which a particular CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain from which the antibody found it, while VL CDR1 is CDR1 from the variable domain of the light chain from which the antibody found it. The term “CDR” is used interchangeably with “HVR”. As used herein, heavy chain complementarity-determining regions or heavy chain variable region CDRs are labeled HCDR1, HCDR2, and HCDR3, respectively, and light chain complementarity-determining regions or light chain variable region CDRs are labeled LCDR1, LCDR2, and LCDR3, respectively.

[0169] The amino acid sequences of the CDR and framework regions can be determined using a variety of known definitions in the art, such as Kabat, Chothia, the International Immunogenetic Database (IMGT), and AbM (see, for example, Johnson et al., supra; Chothia and Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196: 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342: 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments, J. Mol. Biol. 227: 799-817; Al Lazikani et al., J. Mol. Biol. 1997, 273(4)). The definition of antigen combination sites is also described in the following: Ruiz et al., IMGT, International Immunogenetic Database, Nucleic Acids Res. 28:219-221 (2000); and Lefranc, MPIMGT, International Immunogenetic Database, Nucleic Acids Res. Jan 1; 29(1):207-9 (2001); MacCallum et al., Antibody-antigen interactions: Contact analysis and binding sitetopography, J. Mol. Biol. 262(5):732-745 (1996); and Martin et al., Proc. Natl Acad. Sci. USA 86:9268-9272 (1989); Martin et al., Methods Enzymol. 203:121-153 (1991); Pedersen et al., Immunomethods 1:126 (1992); and Rees et al., in Sternberg MJE ed., Protein Structure Prediction, Oxford University Press, Oxford, 141-172, 1996).The CDR determined by the Kabat number is based, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institute of Health, Bethesda, MD (1991)). The Chothia CDR is determined by the definition of Chothia (see, for example, Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)).

[0170] "Host cell" refers to a cell that can be used to introduce the vector. Host cells include the offspring of a single host cell. Due to natural, accidental, or intentional mutations, the offspring need not be identical to the original parent cell (morphologically or in terms of genomic or total DNA complementarity). Host cells include cells transfected in vivo with the vector of the present invention. "Host cell" can refer to prokaryotic cells, such as bacterial cells, Escherichia coli, or Bacillus subtilis; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0171] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally refers to a suitable host cell containing an expression vector capable of producing the desired expression product.

[0172] The term "equilibrium dissociation constant" (KD) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. It is the ratio of the dissociation rate constant (kd) to the binding rate constant (Ka, time⁻¹M⁻¹). The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies exhibit an equilibrium dissociation constant of less than approximately 10⁻¹. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The antigen is bound by a dissociation equilibrium constant (KD) of M or smaller. The equilibrium dissociation constant can be determined using methods known to those skilled in the art.

[0173] When referring to proteins or peptides, the phrases "specific (or selective) binding" to an antigen or target or "specific (or selective) immune response" refer to a binding reaction in which the antibody binds to the antigen or target of interest. In the context of this invention, the antibody typically binds SIRPα with a KD at least 100 times greater affinity for other antigens than it has for them. In some embodiments, the antibody binds human SIRPα with a KD at least 100 times greater affinity for other antigens than it has for them. In some embodiments, the antibody binds mouse and human SIRPα. Therefore, "specific binding" or "selective binding" as used herein does not necessarily require (even if it may include) specific binding. Antibodies that specifically bind to a target may have a binding constant of at least about 10. -3 M or 10 - 4 M, sometimes about 10 -5 M or 10 -6 M, in other cases, is approximately 10. -6 M or 10 -7 M, approximately 10 -8 M to 10 -9 M, or about 10 -10 M to 10 -11 M or better. Various forms of immunoassays can be used to select antibodies that specifically react with a particular protein. For example, solid-phase ELISA is routinely used to select monoclonal antibodies that specifically react with a protein. See, for example, Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for descriptions of the forms and conditions of immunoassays that can be used to determine specific immunoreactivity. Attached Figure Description

[0174] Figure 1 SDS-PAGE assay of anti-SIRPα antibody.

[0175] From right to left: antibody protein marker, 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10

[0176] Figure 2a The binding activity of anti-SIRPα antibodies 36D6, 36G4, 40F3, and commercial antibody SE5A5 to hu-SIRPα-his was determined.

[0177] Figure 2bThe binding activity of anti-SIRPα antibodies 39D8, 52B3, 55G4, 57B10, and commercial antibody SE5A5 to hu-SIRPα-his was determined.

[0178] Figure 3a The determination of the competitive binding of anti-SIRPα antibodies 39D8, 40F3, 55G4, mIgG1, and commercial antibody SE5A5 to CD47.

[0179] Figure 3b The assay of anti-SIRPα antibodies 52B3, 36D6, 36G4, 57B10, mIgG1, and commercial antibody SE5A5 competing with CD47 for binding to SIRPα.

[0180] Figure 4 ELISA assay to determine the binding of anti-SIRPα antibody to SIRPg.

[0181] Figure 5 Does the SIRPα antibody affect the assay of CD47 binding to sirpg? Detailed Implementation

[0182] The present invention will be further explained and described below with reference to the embodiments. It should be understood that the following embodiments are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention.

[0183] In the following embodiments, all materials used in the experiments can be purchased or prepared with reference to existing publicly available techniques; materials whose source and specifications are not specified are commercially available; and various processes and methods not described in detail are conventional methods known in the art.

[0184] In the following examples, commercial antibodies were used for comparison. The first antibody was a sirpa / β antibody, named SE5A5 (Biolegengd BLE323802); the second was a mouse anti-human sirpa antibody, named Kwar23 (CreativeBiolabs); and the third was a mouse monoclonal specific anti-sirpg antibody, named LSB2-20 (Santa Cruz sc-53604).

[0185] Example 1: Obtaining anti-SIRPα antibody

[0186] (1) Preparation of antigen, the specific steps are as follows:

[0187] a. Obtaining the target gene

[0188] In this embodiment, the protein sequence of SIRPα (Uniprot: P78324) was retrieved from Uniprot, and amino acids 31-373 were selected. The extracellular region of SIRPα was ligated to the mouse heavy chain constant region using three linker peptides (GGGGS). The sequence was optimized and the DNA synthesized by Genscript Biotech Co., Ltd. The optimized and synthesized PUC-57-SIRPa-mFC plasmid and PGS plasmid were double-digested with HindIII and EcoRI, and the target fragment was recovered using an agarose gel extraction kit. Ligation was performed overnight at 16°C using T4 DNA ligase.

[0189] b. Constructing recombinant eukaryotic expression vectors

[0190] Both the sequence and the PGS vector were digested with restriction endonucleases HindIII and EcoRI, and the target fragment was recovered and ligated with T4 ligase to construct the PGS-SIRPα-mFc vector.

[0191] c. Obtaining strains containing recombinant expression plasmids and obtaining recombinant expression plasmids The recombinant plasmid obtained in step b was transformed into E. coli DH5α competent cells, and selected using solid LB medium containing ampicillin. Single clones were picked, cultured, and a small amount of plasmid was extracted. Preliminary identification was performed by double enzyme digestion. The sequence of the target gene was verified by sequencing. A large quantity of correctly sequenced recombinant plasmids was extracted, and some plasmids and the bacterial strain were preserved.

[0192] d. Obtain a cell library expressing recombinant proteins

[0193] Plasmid PGS-SIRPα-mFc was extracted, linearized with restriction endonuclease PvuI, transfected into CHOS cells, and selected by MSX under pressure to obtain a cell bank expressing SIRPα-mFc.

[0194] (2) Protein expression and purification, the specific steps are as follows:

[0195] The cell bank was expanded to 2L, and cultured under MSX pressure at 37°C, 100 rpm, and 8% CO2 for 10 days. After centrifugation at 4500 rpm for 5 min, the cell culture supernatant was collected, and SIRPα-mFc protein was purified using Protein A affinity chromatography as an antigen.

[0196] (3) Preparation and purification of monoclonal antibodies, the specific steps are as follows:

[0197] a. Immunized animals

[0198] Six 6-8 week old female Balb / c mice were selected and immunized three times. One mouse was not immunized and served as a control.

[0199] First immunization: SIRPα-mFc antigen protein, diluted with PBS, mixed with complete Freund's adjuvant at a 1:1 ratio, emulsified, 100 μg / animal, subcutaneously injected for immunization.

[0200] Secondary immunization: A secondary immunization is performed 10-14 days after the initial immunization. SIRPα-mFc antigen protein is diluted with PBS, mixed with incomplete Freund's adjuvant at a ratio of 1:1, emulsified, and administered as 150 μg / animal via subcutaneous injection.

[0201] Four days after the second immunization, blood was collected from the tail of the animal and measured for antibody titers using ELISA.

[0202] Three immunizations: A second immunization was performed 14 days after the second immunization. SIRPα-mFc antigen protein was diluted with PBS, mixed with incomplete Freund's adjuvant at a ratio of 1:1, emulsified, and administered as 150 μg / animal via subcutaneous injection.

[0203] Three days after the third immunization, blood was collected from the tail of the animal and measured for antibody titers using ELISA. The antibody with the highest titer was selected for cell fusion.

[0204] Enhanced immunity: Two days before cell fusion, mice were injected intraperitoneally with SIRPα-mFc antigen protein, 50 μg / mouse. Two days later, the spleen of the mice was harvested for cell fusion.

[0205] b. Preparation of mouse peritoneal cells

[0206] Feeder cells were prepared 24 hours before cell fusion. Mice were euthanized by dislocation and disinfected with 70% alcohol. An abdominal incision was made, the incision was opened, and the abdominal wall was cleaned with 70% alcohol. The abdominal wall was lifted with forceps, and 4.5–5.5 ml of DMEM culture medium was injected intraperitoneally. The abdomen was massaged, and 4–5 ml of fluid was aspirated. The cells were centrifuged at 1000 rpm for 7 minutes, and the supernatant was discarded. The cell culture medium was resuspended to 10⁻⁶. 5 / ml cells, seed the cells in a 96-well plate, 0.1ml / well.

[0207] c. Cell fusion

[0208] Three days after booster immunization, the mice were enucleated and blood was collected. The mice were euthanized by cervical dislocation and disinfected with 70% alcohol. An abdominal incision was made, tearing open to fully expose the abdominal wall, which was then washed with 70% alcohol. The peritoneum was cut open and the spleen removed. The spleen was placed in 8 ml of serum-free culture medium and squeezed using a syringe core. A spleen cell suspension was prepared, filtered through a 70µm filter, and the cells were transferred to a 50 ml centrifuge tube. The cells were pipetted three times and allowed to stand for 10 minutes to allow the tissue fragments to precipitate. The supernatant was transferred to another tube. 30 ml of DMEM was added, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and 3-5 times the volume of erythrocyte lysis buffer was added. The cells were allowed to stand for 1-2 minutes. After centrifugation at 1000 rpm for 5 minutes, the cells were washed three times with 20 ml of DMEM and the cells were counted. The prepared syngeneic myeloma cells SP2 / 0 were mixed with mouse spleen cells at a certain ratio (1:5-1:10), and polyethylene glycol was added. After adding polyethylene glycol, incubate at 37°C for 90 seconds. Within 2-4 minutes, add 15 ml of serum-free RPMI-1640 medium, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, add 35 ml of HAT selective medium, and aliquot into 96-well plates already containing macrophages for culture. Change the HAT selective medium every 2-3 days, and observe for hybridoma appearance for two consecutive weeks. After 2 weeks, culture in HT medium.

[0209] (4) Screening for positive hybridoma cells

[0210] a. ELISA detection of hybridoma cell culture supernatant in a 96-well plate: 100 μl of huSIRPα-his protein was coated onto each well of the ELISA plate and incubated overnight at 4°C. The coating was removed, and each well was blocked with 200 μl of 5% skim milk powder at 37°C. After washing three times with PBST, 100 μl of hybridoma cell culture supernatant was added and incubated at 37°C for 1–2 h. After washing three times with PBST, secondary antibody (goat anti-mouse IgG-HRP) was added and incubated at 37°C for 1 h. After washing three times with PBST, chromogenic buffer was added and incubated for 5 min, then the incubation was terminated. The OD value at 450 nm was measured using an ELISA reader. In this screening, clones with an OD value greater than 0.5 times were used for the next round of testing. The screened antigen was used to retest positive cultures to confirm antibody secretion, and an unrelated antigen (human transferrin) was used to eliminate nonspecific or "sticky" monoclonal antibodies and exclude false positives.

[0211] b. Flow cytometry was used to detect the binding ability of CHO-huSIRPα and CHO-cyno-SIRPα in the supernatant of 96-well hybridoma cell culture.

[0212] The supernatant from 328 positive clones screened by ELSIIA was analyzed by flow cytometry to detect the binding capacity of human receptor (CHO-huSIRPα) and monkey receptor (CHO-cyno-SIRPα) ectopically expressed in rodent Chinese hamster ovary cell lines. CHO-huSIRPα and CHO-cyno-SIRPα cells were harvested and cultured at 10⁻⁶ cells per cell line. 5 Cells were seeded per well in 96-well plates, washed, and incubated in 100 μl of FACS buffer containing Fc blocking reagent and monoclonal antibody supernatant. Cells were then washed twice and incubated on ice for 30 min in FACS buffer containing 1:200 diluted conjugated PE anti-mouse IgG Fc secondary antibody. Cells were washed twice in ice-cold FACS buffer and obtained on BD FACS Canto. Mean fluorescence intensity (MFI) values ​​were analyzed and calculated using FlowJo (TreeStar) software version 10.0.7. The results are shown in Table 1.

[0213] Table 1. MFI values ​​of antibodies binding to CHO-huSIRPα and CHO-cyno-SIRPα

[0214] The results showed that the antibodies secreted by the seven hybridoma cell lines 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10 exhibited strong cross-binding with CHO-huSIRPα and CHO-cyno-SIRPα.

[0215] (5) Identification, subcloning and large-scale preparation of anti-SIRPα murine antibodies

[0216] a. Subtype identification: Take the hybridoma cell culture supernatant and use IsoStrip blotting. TM The antibody subtypes were identified using a mouse monoclonal antibody subtype identification kit. The results are shown in Table 2. All anti-SIRPα antibodies in this invention were IgG1,κ.

[0217] Table 2 Anti-human SIRPα antibody subtypes

[0218] b. Hybridoma cell subcloning:

[0219] After transfer to 96-well plates, the seven hybridoma cell lines (36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10) were cultured in 24-well plates for 32 days. This period was designated as the stationary phase, during which the clones' ability to maintain stable secretion was tested. During the re-stabilization period, a temporary frozen backup of the cell lines, consisting of all clones of interest, was stored at -80°C (where they can survive for up to 6 months). During this period, the secretion and specificity of the hybridomas were periodically monitored.

[0220] Subcloning of hybridoma cell lines (clones) 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10 was performed to ensure monoclonal integrity. Clonalization was carried out using limiting dilution culture, and subclones were screened by indirect ELISA and antibody capture ELISA. The top-performing subclones from each parent were used for expansion and preservation. A second round of subcloning was performed on parental clones with <50% cloning.

[0221] c. Large-scale preparation and purification of monoclonal antibodies

[0222] The cell lines 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10 obtained above were expanded and cultured, and the cell culture supernatant was collected. Antibodies were purified using Protein A affinity chromatography. First, a Protein A affinity column was prepared. After equilibrating the column with PBS, the cell culture supernatant, centrifuged and filtered through a 0.4 µm membrane, was passed through the column. The column was then washed with PBS until the OD value was close to zero, followed by elution with 50 mmol / L glycine-HCl solution at pH 7.5. The eluent from the peak region was collected, dialyzed, and used for later use.

[0223] Example 2: SDS-PAGE detection of antibody molecular weight

[0224] SDS-PAGE electrophoresis was performed according to the methods described in Appendix III of the Chinese Pharmacopoeia to identify its molecular weight and expression level. The SDS-PAGE pattern of the purified antibody is shown below. Figure 1 As shown, the molecular weights are approximately 50 kDa and 25 kDa, respectively, which are consistent with the molecular weights of the antibody heavy and light chains, and the purity is above 95%.

[0225] Example 3: Variable region amplification of anti-SIRPα murine monoclonal antibody

[0226] Candidate hybridoma cells (36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10) were cultured to the logarithmic growth phase. Cells were collected by centrifugation at 1000 rpm for 10 min, and total RNA was extracted using a kit (Takara). The RNA was then processed using a reverse transcription kit (Prime Script). TMFirst-strand cDNA was synthesized by RT-PCR, and the variable region DNA sequence of the antibody corresponding to hybridoma cells was amplified using the first-strand cDNA as a template. Based on the subtype identification results, the heavy and light chain constant region sequences of the antibody subtype were obtained, involving specific nested PCR primers. The primer sequences used in this amplification reaction were complementary to the first frame region and constant region of the antibody variable region.

[0227] (1) Cloning of the heavy chain variable regions of murine antibodies 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10

[0228] mRNA was isolated from mouse hybridoma cells 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10 using an mRNA purification kit (Takara), and cDNA was prepared accordingly. TM RT-PCR kit (Takara). Heavy chain variable region DNA fragments were isolated from cDNA by PCR using degenerate primer 1: 5'-CCTAGGAGGTSMARCTGCAGSAGTCWGG-3' and primer 2: 5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3'. The purified DNA fragments were cloned into the PMDA19-T (simple) vector and sequenced to obtain the nucleotide and amino acid sequences of the variable regions encoding the heavy chains of mouse antibodies 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10, as well as the amino acid sequences of their complementarity-determining regions.

[0229] (2) Cloning of the light chain variable regions of murine antibodies 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10

[0230] Using a similar PCR method, primer 5'-CCTAGGGACATTCAGCTGACCCAGTCTCCA-3' (primer 3) and another 3' primer homologous to and antisense the constant region of the mouse immunoglobulin light chain, namely 5'-CATATGGTTAGATCTCCAGCTTGGTCCC-3' (primer 4), were used to isolate light chain variable region DNA fragments from cDNA. These obtained DNA fragments were cloned into the TOPO-TA vector and sequenced to obtain the nucleotide and amino acid sequences of the variable regions encoding the mouse hybridoma light chains 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10, as well as the amino acid sequences of their complementarity-determining regions.

[0231] Using standard techniques, the amino acid sequences of the heavy chain variable region and light chain variable region encoding the generated antibody were determined. The antibody heavy chain CDR sequence, light chain CDR, and antibody heavy chain and light chain variable region sequences are shown in Tables 3-1 and 3-2, where the CDRs are determined according to the Kabat number.

[0232] Table 3-1 Amino acid sequences of antibody heavy chain, light chain variable region, and CDR

[0233]

[0234] Table 3-2 Nucleotide sequences of antibody variable regions

[0235] Example 4: Assay of the binding activity of anti-SIRPα antibody to SIRPα

[0236] Recombinant hu-SIRPα-his was coated onto an ELISA plate at 0.5 μg / ml with carbonate buffer (pH 9.2) overnight. 300 μl of 2% BSA was added to each well, and the plate was blocked at 37°C for 1 h. After washing three times with 0.1% PBST, 100 μl of serially diluted purified antibody was added to each well, and the plate was incubated at 37°C for 1 h. SE5A5 was used as a positive control antibody. After washing three times with 0.1% PBST and drying, 100 μl of 1:10000 diluted anti-mouse IgG-HRP secondary antibody was added to each well, and the plate was incubated at 37°C for 1 h. The secondary antibody was discarded, and the plate was washed three times with 0.1% PBST, dried, and 50 μl of TMB was added to each well for 20 min. 50 μl of 1M HCl was added to each well to terminate the incubation. The OD450 value was read using a multi-mode microplate reader, and the raw data were retrieved. The data were imported into Graphpad Prism 8.0 to analyze the EC50 value of the antibody binding to SIRPα. The results are shown in Table 4. Figure 2a and Figure 2b As shown. These results indicate that the antibodies detected in this invention are good SIRPα binders compared to other known anti-SIRPα antibodies SE5A5 (22.47 ng / ml). The binding of the antibodies of this invention is superior to that of control antibodies, especially antibodies 40F3 and 57B10, with EC50 values ​​of 5.866 ng / ml and 5.457 ng / ml, respectively.

[0237] Table 4 ELISA combined with EC50 values

[0238] Example 5: Affinity determination of anti-SIRPα antibody with hSIRPα

[0239] In this embodiment, the affinity of antibodies 36D6, 36G4, 39D8, 40F3, 52B3, 55G4, and 57B10 for SIRPα antigen was determined using a Biacore (T200) and standard surface plasmon resonance (SPR) technology. A capture method was employed, with humanized anti-SIRPα monoclonal antibodies used as ligands captured on a Protein A chip. Different concentrations of SIRPα protein were injected as analytes for affinity analysis and calculation. The specific operating procedure is as follows:

[0240] Filter ultrapure water through a 0.22 μm filter membrane to prepare HBS-EP+ buffer; dilute the SIRPα monoclonal antibody stock solution to 6 μg / ml using HBS-EP+ buffer as the ligand; dilute SIRPα protein starting at 100 nM with HBS-EP+ buffer in eight 2-fold gradients, then add a zero point as the analyte; use an appropriate amount of Glycine 1.5 as the regeneration buffer; place the ligand, analyte, regeneration buffer, and HBS-EP+ buffer onto the sample tray; set the program: ligand flow rate 10 μl / min, 20 s; analyte flow rate 30 μl / min, binding time 100 s, dissociation time 600 s; regeneration buffer flow rate 30 μl / min, 30 s. Start the program. Acquire and save the SPR signal using Biacore T200 Control Software, and then process the data using Biacore T200 Evaluation analysis software. Subtract the corresponding reference channel signal value from the detected channel signal value to obtain the corrected signal curve. Affinity evaluation employed a double-subtraction method. A zero-concentration cycle (Sample 0) with identical parameters was added before the sample cycle. The sample cycle minus the zero-concentration cycle (Sample - Sample 0) yielded the second-corrected signal curve. The affinity kinetics curve was fitted using a 1:1 Langmuir binding model, and the KD value was calculated. The results are shown in Table 5.

[0241] The results show that the antibodies 40F3 and 57B10 of the present invention have an affinity on the order of 10E-11, which is superior to SE5A5.

[0242] Table 5. Binding rate, dissociation rate, and affinity constant of anti-huSIRPα antibody

[0243] Example 6: Competitive ELISA assay to identify SIRPα antibodies that block and do not block CD47

[0244] Recombinant hSIRPα was coated onto ELISA plates overnight at 1 μg / ml with carbonate buffer (pH 9.2). Blocking was performed at 37°C for 1 h with 300 μl / w 2% BSA. After washing, purified antibodies (at different concentrations) and biotinylated recombinant human biotinylated CD47Fc protein were added to a final concentration of 6 μg / ml (fixed concentration). The plates were incubated at 37°C for 2 h. SE5A5 was the positive control antibody, and mIgG1 was the negative control antibody. After culturing and washing, 100 μl / well of streptavidin labeled with peroxidase was added as a 1:1000 solution, and the plates were incubated at 37°C for 1 h. After washing, 50 μl / well of TMB was added for color development for 15 min, followed by 50 μl / well of HCl to terminate the reaction. The OD450 value was read using a multi-mode microplate reader, and the raw data were saved. The data were imported into Graphpad Prism 8.0 for analysis, and the results are shown in Table 6 and Figure 3.

[0245] The results showed that antibodies 40F3 (IC50 value 65.31 ng / ml) and 57B10 (IC50 value 63.29 ng / ml) had stronger blocking effects than the commercial antibody SE5A5 (IC50 value 121.2 ng / ml); 52B3 (IC50 value 120.6 ng / ml) and 39D8 (IC50 value 113.2 ng / ml) had blocking effects comparable to the commercial antibody SE5A5; 36D6 (IC50 value 247.9 ng / ml) was weaker than the control antibody; and 55G4 did not have the function of blocking the binding of CD47 to SIRPα.

[0246] Table 6 IC50 values ​​of SIRPα antibodies blocking CD47

[0247] Note: "NA" indicates no dose-response relationship.

[0248] Example 7: Detection of the binding of anti-SIRPα antibody and SIRPg by ELISA

[0249] Recombinant hsirpg-his was coated onto an ELISA plate at 1 μg / ml with carbonate buffer (pH 9.2) overnight; 300 μl / w of 2% BSA was added, and the plate was blocked at 37°C for 1 h. After washing, 100 μl / well of purified antibody was added, and the plate was incubated at 37°C for 1 h. Kwar23 and LSB2-20 were commercially available antibodies, and IgG1 was used as a negative control antibody. The plate was washed three times with 0.1% PBST, patted dry, and 100 μl / w of anti-mouse IgG-HRP secondary antibody diluted 1:10000 or goat anti-human IgG-HRP secondary antibody was added to the selection plate. The plate was incubated at 37°C for 1 h. The secondary antibody was discarded, and the plate was washed three times with 0.1% PBST, patted dry, and 50 μl of TMB was added to each well. The plate was incubated for 20 min, and then 50 μl of 1M HCl was added to each well to terminate the reaction. The OD450 value was read using a multi-mode microplate reader, and the raw data were read. The data were imported into Graphpad Prism 8.0 to analyze the binding activity of the antibodies to sirpg. Kwar23 and LSB2-20 were commercially available antibodies, and IgG1 was a negative control antibody.

[0250] The results are as follows Figure 4 As shown, anti-SIRPα antibodies 40F3 and 57B10 do not bind to sirpg, while known antibodies Kwar23 and LSB2-20 show significant binding to sirpg.

[0251] Example 8: ELISA to determine whether anti-SIRPα antibody affects the binding of CD47 to SIRPg.

[0252] Recombinant hsirpg-his was coated on ELISA plates overnight with 1 μg / ml carbonate buffer (pH 9.2). After washing three times, the plates were blocked with 300 μl / w of 2% BSA at 37°C for 1 h. After washing three times, 50 μl of purified antibody (different concentrations) was added and incubated with sirpg for 30 min, followed by the addition of biotinylated recombinant human biotinylated CD47-Fc to a final protein concentration of 6 μg / ml (fixed concentration). The plates were then incubated at 37°C for 1 h. After washing three times, 100 μl / well of the secondary antibody peroxidase-labeled streptavidin was added at a 1:1000 ratio and the plates were incubated at 37°C for 1 h. After washing four times, 50 μl / well of TMB was added for color development for 15 min, followed by 50 μl / well of HCl to terminate the reaction. The OD450 values ​​were read using a multi-mode microplate reader, and the original data were saved. Kwar23 was a commercially available antibody. Import the data into Graphpad Prism 8.0 to analyze the results, as shown in Table 7. Figure 5 As shown, the results indicate that the anti-sirpa antibodies 40F3 and 50B10 of the present invention do not affect the binding of CD47 to SIRPg at all.

[0253] Table 7. IC50 values ​​of CD47 and SIRPα antibody competitively binding to sirpg.

[0254] Note: "NA" indicates no dose-response relationship.

Claims

1. An anti-SIRPα monoclonal antibody or antigen-binding fragment, characterized in that, The monoclonal antibody or antigen-binding fragment comprises a variable region of antibody light chain containing LCDR1, LCDR2, and LCDR3, and a variable region of antibody heavy chain containing HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 is shown in SEQ ID NO:10, the amino acid sequence of HCDR2 is shown in SEQ ID NO:11, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:12; the amino acid sequence of LCDR1 is shown in SEQ ID NO:30, the amino acid sequence of LCDR2 is shown in SEQ ID NO:31, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:32; wherein the CDRs are determined according to the Kabat number.

2. The anti-SIRPα monoclonal antibody or antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody or antigen-binding fragment is as shown in SEQ ID NO:43, or a sequence having at least about 85% or 92% or 98% or 99% homology with the heavy chain variable region sequence shown in SEQ ID NO:43; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:50, or a sequence having at least about 85% or 92% or 98% or 99% homology with the light chain variable region sequence shown in SEQ ID NO:

50.

3. A polynucleotide molecule, characterized in that, It encodes the monoclonal antibody or antigen-binding fragment as described in any one of claims 1-2.

4. A polynucleotide molecule according to claim 3, characterized in that, The polynucleotide molecule comprises a nucleotide sequence SEQ ID NO:57 encoding the heavy chain variable region as shown in SEQ ID NO:43; and a nucleotide sequence SEQ ID NO:64 encoding the light chain variable region as shown in SEQ ID NO:

50.

5. A method for preparing the monoclonal antibody according to any one of claims 1-2, characterized in that, The method includes: Step (1): Synthesize a nucleotide sequence containing SIRPα and mouse Fc, construct a eukaryotic expression vector, transfect host cells, express and purify the fusion protein as an antigen; Step (2): After immunizing mice with the antigen provided by the present invention, mouse spleen cells are obtained. Hybridoma cell lines that can produce high-titer monoclonal antibodies are prepared and screened using cell fusion technology. After in vitro culture, anti-SIRPα monoclonal antibodies are obtained.

6. The use of the anti-SIRPα monoclonal antibody or antigen-binding fragment according to any one of claims 1-2 in the preparation of a medicament for treating tumors that blocks the binding of CD47 to SIRPα without affecting the interaction between CD47 and sirpg.

Images