WO2022110922A1 - 抗SIRPα抗体或其抗原结合片段及应用 - Google Patents

抗SIRPα抗体或其抗原结合片段及应用 Download PDF

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WO2022110922A1
WO2022110922A1 PCT/CN2021/113416 CN2021113416W WO2022110922A1 WO 2022110922 A1 WO2022110922 A1 WO 2022110922A1 CN 2021113416 W CN2021113416 W CN 2021113416W WO 2022110922 A1 WO2022110922 A1 WO 2022110922A1
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cancer
antibody
sirpα
antigen
seq
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PCT/CN2021/113416
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French (fr)
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屈向东
潘琴
郑翰
都业杰
陈子硕
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启愈生物技术(上海)有限公司
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Priority claimed from CN202011602742.3A external-priority patent/CN115368455A/zh
Application filed by 启愈生物技术(上海)有限公司 filed Critical 启愈生物技术(上海)有限公司
Priority to JP2023532562A priority Critical patent/JP2024501134A/ja
Priority to US18/254,941 priority patent/US20240018255A1/en
Priority to EP21896427.8A priority patent/EP4253416A1/en
Publication of WO2022110922A1 publication Critical patent/WO2022110922A1/zh

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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to the technical field of biomedicine, in particular to an anti-SIRP ⁇ antibody or an antigen-binding fragment thereof and application thereof.
  • the relationship between tumor cells and the host immune system can be divided into three different stages: elimination, equilibrium, and escape.
  • the new tumor cells In the “cleared” state, the new tumor cells have strong antigenicity and are easily recognized and eliminated by the immune system; while the tumor cells that survived the clearance will enter a "balanced” state with the immune system, showing the host tumor-bearing state of survival.
  • tumor cells when tumor cells accumulate genetic mutations under the selection pressure of the host immune system to a certain extent, they will break the balance and enter the final "escape” state. Tumor cells at this stage can develop a series of malignant phenotypes, turn off tumor suppressor response mechanisms, induce normal immune responses, and thus be recognized as normal cells.
  • tumor cells due to the rapid unchecked growth of tumor cells, the tissue structures formed create a microenvironment that suppresses immune cells.
  • tumor cells release molecules with immunosuppressive functions, such as VEGF, TGF- ⁇ , IL-10, etc., which inhibit the activation and differentiation of bone marrow dendritic cells, thereby inhibiting the adaptive immune system.
  • immunosuppressive functions such as VEGF, TGF- ⁇ , IL-10, etc.
  • it can induce the production of regulatory T cells (Treg) expressing CTLA-4 in peripheral blood and lymph nodes, which can inhibit other immune cells, resulting in immune tolerance of the immune system to tumors.
  • CD47 protein is highly expressed on the surface of almost all tumor cells, which can interact with the signal regulatory protein ⁇ (signal regulatory protein ⁇ ) on the surface of bone marrow cells.
  • protein ⁇ , SIRP ⁇ signal regulatory protein ⁇
  • CD47 also known as integrin-associated protein (IAP)
  • IAP integrin-associated protein
  • CD47 has a molecular weight of 50kD, and its structure contains a large number of glycosylated N-terminal IgV variable domains, five highly hydrophobic transmembrane domains and a short C-terminal cytoplasmic tail, C-terminal cytoplasmic tail
  • the four alternatively spliced forms of the region determine the expression of CD47 in different tissues.
  • the corresponding SIRP ⁇ also known as SHPS-1, BIT or CD172a protein, is a transmembrane protein mainly expressed on myeloid cells, including macrophages, bone marrow dendritic cells, granulocytes cells, mast cells and their precursor cells.
  • SIRP ⁇ consists of three extracellular Ig-like domains and four intracytoplasmic tyrosine residues that are presumed to be phosphorylation sites. After phosphorylation, SIRP ⁇ activates the downstream signaling pathway by binding to the SH2 domain of SHP-1/2 protein and activating it.
  • SHP-1 and SHP-2 proteins The expression of SHP-1 and SHP-2 proteins is tissue-specific and thus SIRP ⁇ is a docked protein that recruits and activates downstream protein phosphatases in response to extracellular stimuli. Oldenborg first reported that mature red blood cells (RBC, red blood cells) protect themselves from the latter's clearance by binding to splenic macrophage SIRP ⁇ through CD47.
  • RBC can also bind to monocyte SIRP ⁇ to inhibit Fc ⁇ receptor-dependent phagocytosis, which is achieved by dephosphorylation of myosin-IIA, a key molecule in phagocytosis.
  • High expression of CD47 has been found in a variety of solid tumors and hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and non-Hodgkin's lymphoma. (NHL), breast cancer, bladder cancer, ovarian cancer, colon cancer, etc., the essence of which is that tumor cells evade the cellular clearance of macrophages through the regulatory mechanisms described above.
  • CD47 also has effects on other biological processes through binding to other receptors or through signaling through its intracellular cytoplasmic region. Interaction of CD47 with thrombospondin-1 (TSP-1, Thrombospondin-1) or vascular endothelial growth factor receptor 2 (VEGFR-2) inhibits angiogenesis, thereby limiting tumor growth.
  • TSP-1 thrombospondin-1
  • Thrombospondin-1 Thrombospondin-1
  • VEGFR-2 vascular endothelial growth factor receptor 2
  • CD47 antibody or SIRP ⁇ -Fc recombinant protein can play a role in different mouse PDX models, and it is not compatible with common chemotherapy drugs such as cytarabine, doxorubicin, paclitaxel, cisplatin or rituximab.
  • common chemotherapy drugs such as cytarabine, doxorubicin, paclitaxel, cisplatin or rituximab.
  • monoclonal antibody alemtuzumab, cetuximab, and trastuzumab with good results. Sockolosky et al. reported for the first time that the combination of CD47 nanobody and PD-L1 could exhibit good antitumor effect in the Syngeneic model of melanoma B16F10 cells.
  • CD47 The biological function of CD47 itself determines that the CD47 therapeutic antibody and SIRP ⁇ -Fc recombinant protein may have hematological toxicity or the risk of causing anemia, both in CD47 knockout NOD mice and in mouse models treated with CD47 antibody There are reports.
  • endothelial cell CD47 has been reported to interact with SIRP ⁇ through cell adhesion to promote the transendothelial migration of T cells, and SIRP ⁇ is mainly expressed on T cells but not myeloid cells.
  • SIRP ⁇ antibodies are a better choice for blocking the CD47-SIRP ⁇ signaling pathway.
  • the Weissman research group of Stanford University demonstrated that the combination of the screened humanized SIRP ⁇ antibody KWAR23 and rituximab can effectively inhibit Burkitt lymphoma in SRG mice (Rag2-/-Il2r ⁇ -/-) knocking in the human SIRP ⁇ gene tumor growth, but KWAR23 alone has no obvious efficacy.
  • the purpose of the present invention is to provide an anti-SIRP ⁇ antibody or its antigen-binding fragment and its application.
  • the anti-SIRP ⁇ antibody or its antigen-binding fragment can bind to human SIRP ⁇ protein and block the CD47-SIRP ⁇ signaling pathway.
  • an anti-SIRP ⁇ antibody or an antigen-binding fragment thereof comprising: a heavy chain variable region and a light chain variable region;
  • the heavy chain variable region comprises: the amino acid sequences are respectively as shown in SEQ ID NO: VHCDR1, VHCDR2 and VHCDR3 shown in NO: 3, 4 and 5;
  • the light chain variable region comprises: VLCDR1, VLCDR2 and VLCDR3 whose amino acid sequences are shown in any of the following sequences respectively;
  • VLCDR The sequence of VLCDR can be found in the table below:
  • variable region further comprises: a murine or human FR region.
  • sequence of the FR region is of murine origin; the sequence of the heavy chain variable region is shown in SEQ ID NO: 2 or has at least 85% sequence identity with it, and the sequence of the light chain variable region is As shown in SEQ ID NO: 6 or having at least 85% sequence identity therewith.
  • the human FR region includes: heavy chain FR region sequence; the heavy chain FR region sequence is derived from the combined sequence of human germline heavy chain IGHV1-18 and IGHJ2*01, including human germline heavy chain IGHV1 The FR1, FR2, FR3 regions of -18 and the FR4 region of IGHJ2*01.
  • the human FR region includes: a light chain FR region sequence; the light chain FR region sequence is derived from the combined sequence of human germline light chain IGKV4-1 and IGKJ2*01, including human germline light chain IGKV4 The FR1, FR2, FR3 regions of -1 and the FR4 region of IGKJ2*01.
  • the FR region sequence of the heavy chain variable region is derived from human germline, and the sequence of the heavy chain variable region is shown in SEQ ID NO: 17 or has at least 85% sequence identity therewith.
  • the FR region sequence of the light chain variable region is derived from human germline, and the sequence of the light chain variable region is selected from SEQ ID NOs: 16, 18, 19, 20, 21, 22, 23, 24, 25, or have at least 85% sequence identity therewith.
  • the anti-SIRP ⁇ antibody or antigen-binding fragment thereof further comprises: a heavy chain constant region selected from human IgG1, IgG2, IgG3, or IgG4 or a variant thereof; and a heavy chain constant region selected from human ⁇ , ⁇ chain or its The light chain constant region of the variant.
  • the heavy chain constant region comprises: an Fc fragment or a variant thereof.
  • the variant of the Fc fragment is derived from IgG1, according to EU count, including mutation sites: L234A, L235A, K338A.
  • the heavy chain sequence of the anti-SIRP ⁇ antibody or antigen-binding fragment thereof is shown in SEQ ID NO: 26, or has at least 85% sequence identity therewith.
  • the anti-SIRP ⁇ antibody or its antigen-binding fragment is a monoclonal antibody, a bispecific antibody, or a multi-specific antibody, or the antibody or its antigen-binding fragment is used to prepare an antibody-drug conjugate.
  • the structural form of the anti-SIRP ⁇ antibody or its antigen-binding fragment is Fab, F(ab')2, Fv, or ScFv.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned anti-SIRP ⁇ antibody or antigen-binding fragment thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the present invention also provides a nucleic acid molecule encoding the above-mentioned anti-SIRP ⁇ antibody or an antigen-binding fragment thereof.
  • the present invention also provides a vector comprising the above-mentioned nucleic acid molecule.
  • the present invention also provides a host cell transformed with the above-mentioned vector.
  • the present invention also provides the use of the above-mentioned anti-SIRP ⁇ antibody or antigen-binding fragment thereof in the preparation of a medicament for inhibiting or treating a disease, disorder or condition.
  • the medicament is prepared by combining the anti-SIRP ⁇ antibody or antigen-binding fragment thereof with one or more other cancer therapeutic agents.
  • the disease, disorder or condition includes: cancer, solid tumor, chronic infection, inflammatory disease, multiple sclerosis, autoimmune disease, neurological disease, brain injury, nerve injury, polycythemia, hemochromatosis disease, trauma, septic shock, fibrosis, atherosclerosis, obesity, type 2 diabetes, graft dysfunction or arthritis.
  • the cancer is selected from anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, gallbladder cancer, gastric cancer, lung cancer, bronchial cancer, bone cancer, hepatobiliary cancer, pancreatic cancer, breast cancer, liver cancer, ovary cancer, testicular cancer, kidney cancer, renal pelvis and ureter cancer, salivary gland cancer, small bowel cancer, urethra cancer, bladder cancer, head and neck cancer, spine cancer, brain cancer, cervical cancer, uterine cancer, endometrial cancer, colon cancer, colorectal cancer cancer, rectal cancer, esophageal cancer, gastrointestinal cancer, skin cancer, prostate cancer, pituitary cancer, vaginal cancer, thyroid cancer, laryngeal cancer, glioblastoma, melanoma, myelodysplastic syndrome, sarcoma, malformation Fetal tumor, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), acute lymphoblastic leukemia
  • the present invention also provides the use of the above-mentioned anti-SIRP ⁇ antibody or its antigen-binding fragment in the preparation of a preparation for blocking the binding of SIRP ⁇ and CD47, the preparation comprising a detection agent.
  • the present invention has the following beneficial effects:
  • the anti-SIRP ⁇ antibody or its antigen-binding fragment provided by the present invention can bind to human SIRP ⁇ protein, block the CD47-SIRP ⁇ signaling pathway, and is expected to be used for tumor treatment or preparation of tumor antibody drugs.
  • the anti-SIRP ⁇ antibody or its antigen-binding fragment provided by the present invention can bind to all subtypes of human SIRP ⁇ protein, which is beneficial to clinical development.
  • FIGS 1 to 6 show the results of Binding-ELISA.
  • Figure 7 shows the results of Blocking-ELISA detection.
  • FIG. 8 is the detection result of FACS detection of SIRP ⁇ antibody binding to human renal clear cell adenocarcinoma 786-O cells naturally expressing human SIRP ⁇ .
  • FIGS 9 to 11 are the ADCP results of the anti-SIRP ⁇ antibody in vitro functional experiments.
  • FIG. 12 is a graph showing the tumor growth curve and D18 imaging signal intensity results of tumor imaging signal values in each group.
  • Figure 13 is the survival curve of each group.
  • Figures 14 to 23 are graphs showing the results of ELISA detecting the binding of the antibody CHO71 of the present invention and the control antibodies 18D5 and KWAR23 to SIRP ⁇ V1/V2/V3/V4/V5/V6/V7/V8/V9/V10 subtypes.
  • Figure 24 is an amino acid sequence alignment of known human SIRP alpha binding domain alleles.
  • Antibody refers to an immunoglobulin (Ig) molecule that contains at least one antigen-binding site and can specifically bind to an antigen.
  • an “antigen” is a substance that can induce an immune response in the body and binds specifically to an antibody. Binding of antibodies to antigens is mediated by interactions formed between them, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region on the surface of an antigen to which an antibody binds is called an "antigenic determinant” or “epitope", and in general, there are multiple determinants per antigen.
  • antibody as referred to in the present invention is to be understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, antibodies comprising at least two different antigen-binding domains Multispecific antibodies (eg, bispecific antibodies).
  • Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies of other origins.
  • Antibodies of the present invention can be derived from any animal, including but not limited to humans, non-human primates, mice, rats, cows, horses, chickens, camels, llamas (Llama), alpacas (Alpaca), Immunoglobulin molecules of llama (Guanaco), llama (Vicunas), etc.
  • Antibodies may contain additional alterations such as unnatural amino acids, Fc effector function mutations and glycosylation site mutations.
  • Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of the antibody, and immunoglobulin molecules comprising any other modifications to the antigen recognition site, so long as these antibodies exhibit the desired biological activity.
  • the basic structure of an antibody is a Y-shaped monomer connected by two identical heavy chains (heavy chain, H) and two identical light chains (light chain, L) through disulfide bonds.
  • Each chain is composed of 2 to 5 domains (also known as functional regions) with similar sequences but different functions, containing about 110 amino acids.
  • the amino acid sequence of the light chain and the heavy chain near the N-terminus of the antibody molecule changes greatly, and the formed domain is called the variable region (variable region, V region);
  • the relatively constant region of the amino acid sequence near the C-terminus is called the constant region (constant region). , area C).
  • VH and VL The V regions of the heavy chain and light chain are called VH and VL, respectively.
  • VH and VL each have 3 regions with highly variable amino acid composition and arrangement sequence, called hypervariable region (HVR); this region forms an antigenic table.
  • the spatial conformation of bit-complementary is also called complementarity determining region (CDR).
  • the three CDRs of VH are represented by VHCDR1, VHCDR2, and VHCDR3, respectively, and the three CDRs of VL are represented by VLCDR1, VLCDR2, and VLCDR3, respectively.
  • a total of 6 CDRs of VH and VL together form an antigen-binding site.
  • the diversity of amino acids in the CDR regions is the molecular basis for the specific binding of antibodies to a large number of different antigens.
  • VH and VL each have four framework regions, which are represented by FR1, FR2, FR3, and FR4, respectively.
  • Each VH and VL is composed of three CDRs and four FRs, and the order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • human immunoglobulins can be divided into five classes: IgM, IgG, IgA, IgD, and IgE. It can also be further divided into different subclasses (isotypes), such as human IgG can be divided into IgG1, IgG2, IgG3, IgG4; IgA can be divided into IgA1 and IgA2. IgM, IgD, and IgE have not yet been found to have subclasses.
  • Light chains can be classified into kappa chains and lambda chains according to their amino acid sequences.
  • Antibodies of the invention can be of any class (eg, IgM, IgG, IgA, IgD, IgE) or subclass (eg, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2).
  • class eg, IgM, IgG, IgA, IgD, IgE
  • subclass eg, IgGl, IgG2, IgG3, IgG4, IgAl, IgA2
  • 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, and the heavy chain constant regions of IgM and IgE have four domains: CH1, CH2, CH3, and CH4.
  • CH1 and CH2 are a hinge region, which is rich in proline, so it is easy to stretch and bend, and can change the distance between the two arms of the Y-shape, which is conducive to the simultaneous binding of the two arms to the epitope.
  • Antigen-binding fragment refers to a Fab fragment, F(ab')2 fragment, Fv fragment, ScFv fragment, etc. having antigen-binding activity.
  • Fab fragment fragment of antigen binding, Fab means an antibody fragment consisting of VL, VH, CL and CH1 domains that binds (monovalently) a single epitope.
  • papain hydrolyzes IgG to form two identical Fab segments and one Fc segment; pepsin hydrolyzes IgG to form one F(ab')2 segment and several polypeptide fragments (pFc').
  • Fv fragments contain antibody heavy and light chain variable regions, but no constant regions.
  • Single chain variable fragment scFv single chain antibody fragment
  • Single chain antibody fragment is composed of antibody heavy chain variable region and light chain variable region connected by a linker.
  • Fc refers to a crystallizable fragment, which has no antigen-binding activity, and is the site of interaction of an antibody with an effector molecule or cell surface Fc receptor (FcR).
  • the Fc fragment comprises the constant region polypeptides of the antibody other than the heavy chain constant region CH1. Fc fragments bind to cells with corresponding Fc receptors on their surface, resulting in different biological effects.
  • ADCC effect antibody-dependent cell-mediated cytotoxicity, 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 associates with the killer cell (NK FcR binding on the surface of cells, macrophages, etc.) mediates the direct killing of target cells by killer cells.
  • ADCP is antibody-dependent cellular phagocytosis (antibody-dependent cellular phagocytosis). The mechanism of ADCP is that target cells acted on by antibodies activate the Fc ⁇ R mechanism on the surface of macrophages, induce phagocytosis, internalize target cells and be acidified and degraded by phagosomes.
  • Elimination of antibody Fc function may be beneficial in certain specific situations. These situations include the use of antibodies as: (1) receptor agonists, inducing cellular signaling; (2) receptor antagonists, blocking receptor and ligand binding, inhibiting signaling; or, (3) as drug carriers to deliver drugs to target cells expressing the corresponding antigen. If the Fc function is maintained, it will cause the antibody drug to accidentally injure the cells expressing the corresponding receptor, and cause the antibody-drug conjugate to accidentally injure important immune cells when it is off-target.
  • Combinations of Fc variants or mutations are not limited to the following formats (counted by EU).
  • the CDR amino acid residues of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering convention in number and position.
  • mouse-derived antibodies are a major source of antibody drugs. Due to the immunogenicity of murine antibodies, they are generally humanized.
  • Murine antibodies, chimeric antibodies, and humanized antibodies are provided in the following examples.
  • a "chimeric antibody” is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response induced by the murine antibody.
  • the constant region of the human antibody may be selected from heavy chain constant regions of human IgGl, IgG2, IgG3, IgG4 or variants thereof, and light chain constant regions selected from human kappa, lambda chains or variants thereof.
  • Humanized antibody refers to an antibody obtained by grafting the CDR sequence of a murine antibody into the framework of a human antibody variable region, which can overcome the strong reaction induced by a chimeric antibody that carries a large amount of mouse protein components.
  • Such framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • human antibody variable region framework sequences can be minimally reverse-mutated or back-mutated to maintain activity.
  • the humanized antibodies of the present invention also include humanized antibodies in which CDRs are further subjected to affinity maturation by phage display.
  • the rationale for antibody affinity maturation in vitro is to mimic the process of antibody affinity in vivo. By constructing a random mutation library to simulate the high-frequency mutation of B cells in vivo, high-affinity antibodies can be screened.
  • the medicaments provided by the present invention may contain a "therapeutically effective amount" of the antibody or antigen-binding fragment.
  • a “therapeutically effective amount” refers to an amount of a therapeutic agent effective to prevent or ameliorate a particular disease, and may vary depending on factors such as the patient's disease state, age and weight, and the drug's ability to produce the desired therapeutic effect in different patients.
  • Sequence identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides, and is the degree to which two polynucleotides or two polypeptides have the same bases or amino acids. "Having at least 85% sequence identity” as used herein refers to at least 85%, 90%, 95%, 97%, or 99% identity.
  • Antibody-Drug Conjugates refer to binding proteins linked to one or more chemical drugs (optionally may be therapeutic or cytotoxic agents).
  • Antibody drug conjugates can be obtained by linking small cytotoxic molecules (cytotoxins) and antibodies through permanent or unstable chemical linkers. ADCs selectively and consistently deliver cytotoxic drugs to tumors.
  • SIRP ⁇ The gene encoding SIRP ⁇ is a polymorphic gene, and 10 SIRP ⁇ variants are known to exist in the human population. sequenced the IgV-encoding SIRP alpha domains of 37 unrelated normal Caucasians, Africans, Chinese, and Japanese from the Human HapMap Genome Project and found 10 distinct SIRP alpha IgV-encoding alleles (Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells, NATURE IMMUNOLOGY VOLUME 8 NUMBER 12 DECEMBER 2007).
  • the 10 SIRP ⁇ variants are SIRP ⁇ V1/V2/V3/V4/V5/V6/V7/V8/V9/V10 subtypes, respectively.
  • SIRPalpha is highly polymorphic
  • the amino acid sequence alignment of the known human SIRPalpha alleles by ChiaChiM.Ho et al. showed that there are only two unique sequences at the binding interface of SIRP alpha and CD47, namely allele V1 (a2d1 ) and V2(a1d1).
  • FIG. 24 amino acid sequence alignment of known human SIRP alpha 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 significant human SIRP alpha allele, V1 (a2d1)
  • the second line of text in Figure 1 is the amino acid sequence of the most significant human SIRP allele, V2 (a1d1).
  • Black boxes indicate residues that interact with CD47, while shading indicates residues that differ from the V1 sequence.
  • Janet Sim et al. identified two SIRP ⁇ variants v1 and v2 representing three allelic groups: homozygous v1/v1, homozygous v2/v2, heterozygous v1 /v2.
  • the distribution and frequency of SIRP ⁇ v1 and v2 allele groups were determined in different populations and unrelated subpopulations. Among them, the distribution of v1/v2 heterozygotes in five superpopulations in Europe (EUR), America (AMR), East Asia (EAS), Africa (AFR) and South Asia (SAS) was similar, ranging from 42.0% to 47.2%. The number of v2/v2 in the East Asian population is significantly higher than that of v1/v1, and the frequency of occurrence is 42.3% and 13.3%, respectively.
  • v1/v1 is higher than that of v2/v2, v1 and v2 in African, European, American, and South Asian populations
  • the frequency ranges are 30.3-49.1% and 8.9-24.2%, respectively (see MABS, 2019, VOL.11, NO.6, 1036 ⁇ C1052 for references, https://doi.org/10.1080/19420862.2019.1624123).
  • Aduro Biotech also studied the East Asian population with a frequency of 41.3% v2/v2 homozygotes and 34.6% v1/v1 homozygotes, which also proved that 41.3% of East Asian populations were V2/V2 homozygotes (see references for details). Voets et al. Journal for ImmunoTherapy of Cancer (2019) 7:340).
  • anti-SIRP ⁇ antibodies are able to bind both SIRP ⁇ type v1 and SIRP ⁇ type v2 genes, which is crucial to facilitate clinical development.
  • Example 1 Obtaining anti-SIRP ⁇ mouse antibody
  • Anti-human SIRP ⁇ monoclonal antibodies were produced by immunizing mice. The experiment used Balb/c white mice, female, 6 weeks old. Breeding environment: SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12/12 hour light/dark cycle, the temperature was 20-25°C, and the humidity was 40-60%.
  • the recombinant protein QP009 (SIRP ⁇ ) 50 ⁇ g/mouse was first immunized with Freund's complete adjuvant (CFA) for two weeks, then QP009 (SIRP ⁇ ) plus incomplete Freund's adjuvant (IFA) or Alternate immunization with QP009 (SIRP ⁇ ) plus aluminum salt Alum+CpG ODN 1826, 25 ⁇ g/mouse, once a week.
  • QP009 (SIRP ⁇ ) has the amino acid sequence shown below (SEQ ID NO: 1):
  • mice with high antibody titers in serum were selected for splenocyte fusion. Selected mice were immunized by intraperitoneal sprint 72 hours before fusion. Hybridoma cells were obtained by fusing spleen lymphocytes with myeloma Sp2/0 cells using an optimized PEG-mediated fusion procedure. The fused hybridoma cells were resuspended in HAT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI), and distributed in 96-well cell culture plates (1 ⁇ 10 5 cells/150 ⁇ l/ well) at 37°C, 5% CO 2 .
  • HAT complete medium IMDM medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • HT complete medium IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • ELISA was performed to screen the anti-SIRP ⁇ antibody in the supernatant of the hybridoma.
  • the supernatant of the hybridoma fusion well was taken, and the whole plate was screened by ELISA, and the anti-SIRP ⁇ antibody in the supernatant was detected to block the binding of SIRP ⁇ /CD47, which was the positive well of the preliminary screening.
  • the positive clones were expanded and transferred to 24/6-well plates in time, and the cloned wells that were positive for the binding of cell culture supernatant to SIRP ⁇ and could block the binding of SIRP ⁇ /CD47 were detected again by ELISA, which were the positive cloned wells of anti-SIRP ⁇ antibody. .
  • the positive clones were subjected to 2-3 rounds of limiting dilution to single-cell clones, and the positive single-cell strains were cryopreserved to obtain single-cell clone 71C10.
  • Hybridoma-positive monoclonal cell line 71C10 was taken, mRNA was extracted, mRNA was reverse transcribed into cDNA, and cDNA was used as a template for PCR amplification. PCR-positive clones were selected and sent for sequencing.
  • the heavy chain variable region sequence of 71C10 is SEQ ID NO: 2, as follows:
  • VHCDR1 SEQ ID NO:3
  • VHCDR2 SEQ ID NO:4
  • VHCDR3 SEQ ID NO:5
  • the light chain variable region sequence of 71C10 is SEQ ID NO:6, specifically as follows:
  • VLCDR1 SEQ ID NO:7
  • VLCDR2 SEQ ID NO:8
  • VLCDR3 SEQ ID NO:9
  • the murine variable region sequence of the monoclonal cell line 71C10 is fused with the human constant region gene to obtain a chimeric antibody molecule.
  • the antibody light chain uses the kappa light chain constant region CL.
  • different antigen sequences are designed for performance testing of antibody molecules.
  • the molecular clone design of antigen and chimeric antibody is shown in Table 1 and Table 2.
  • Antibodies with protein numbers of QP026027 and QP026249 were used as control antibodies, all of which used the variable region sequence of the known anti-SIRP ⁇ antibody KWAR23, with the difference in the constant region. Both QP163164 and QP163245 use the variable region of the monoclonal cell line 71C10, and the difference lies in the constant region.
  • the sequences shown in the above SEQ ID NOs show the heavy and light chain sequences of each antibody molecule, respectively.
  • pQD is the name of the vector with signal peptide and constant region gene (CH1-FC/CL) fragment, among which, pQDH is used for the connection and expression of heavy chain variable region, with signal peptide and constant region gene (CH1-FC) fragment ; pQDK is used for ligation and expression of light chain variable region, with signal peptide and constant region gene (CL) fragment.
  • H indicates heavy chain and "L” indicates light chain.
  • (IgG4) means that the heavy chain adopts the constant region of human IgG4. If not marked with "(IgG4)", the constant region of human IgG1 is used by default.
  • 180122VH represents the heavy chain variable region derived from the monoclonal cell line 71C10
  • 180122VL represents the light chain variable region derived from the monoclonal cell line 71C10.
  • pQDH-KWAR23-H means that the control sequence KWAR23 is fused to a pQDH vector with a signal peptide and a fragment of a constant region gene (CH1-FC), 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.
  • the double underlined part is the constant region sequence.
  • the double underlined part is the constant region sequence.
  • QP098 is cynomolgus monkey SIRP ⁇ sequence (uniprot database sequence number I7G9Z7)
  • QP100 is cynomolgus monkey SIRP ⁇ sequence (uniprot database sequence number G7PGS8)
  • QP271 is rhesus monkey SIRP ⁇ sequence, obtained by the inventor by sequencing monkey PBMC
  • QP273 The cynomolgus monkey SIRP ⁇ sequence was obtained by the inventors by sequencing monkey PBMC.
  • the 293E cell culture density was maintained between (0.2-3) ⁇ 10 6 /ml, and the maintenance phase medium (GIBCO Freestyle 293 expression medium) was cultured.
  • the cells to be transfected were centrifuged to change the medium, and the cell density was adjusted to ( 0.5-0.8) x 10 6 /ml.
  • the density of 293E cells was (1-1.5) x 106 /ml.
  • Prepare plasmid and transfection reagent PEI the amount of plasmid to be transfected is 100 ⁇ g/100ml cells, and the mass ratio of PEI and plasmid is 2:1. Mix the plasmid and PEI, let stand for 15min, not more than 20min.
  • the plasmid and PEI mixture was slowly added to the 293E cells, cultured in a shaker at 8% CO 2 , 120 rpm, and 37° C. On the fifth day of transfection, the cell supernatant was collected by centrifugation at 4700 rpm in a horizontal centrifuge for 20 min.
  • Protein A affinity chromatography purification use the equilibrium solution to pass through the column, at least 3CV, the actual volume is 20ml, to ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, the flow rate is 1ml/min; the supernatant of the culture medium after centrifugation is passed through the column , load 40ml, flow rate 0.33ml/min; pass through the column with equilibrium solution, at least 3CV, actual volume 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, flow rate 0.33ml/min; Column, when the UV280 rises to 15mAU, the elution peak (PAC-EP) starts to be collected, and when the UV280 drops to 15mAU, the collection is stopped, and the flow rate is 1 ml/min. After sample collection, PAC-EP was adjusted to neutrality with pH adjustment solution.
  • PAC-EP elution peak
  • the affinity of anti-SIRP ⁇ chimeric antibody QP163164 to human SIRP ⁇ type V1 (protein number QP094) and human SIRP ⁇ type V2 (protein number QP096) was determined by Biacore T200 (GE). Tables 3 and 4 show the detection results of QP163164 and QP026027. The results showed that the SIRP ⁇ chimeric antibody QP163164 had an affinity KD of 5.27E-10M for binding to human SIRP ⁇ V1, and a KD value of 6.78E-10M for binding to human SIRP ⁇ V2. The binding affinity of human SIRP ⁇ V1 and human SIRP ⁇ V2 was significantly better than that of the control antibody KWAR23 (QP026027).
  • variable region sequence is formed in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then select some important amino acid residues for back mutation combination. The amino acid residues are identified and annotated by the Kabat numbering system.
  • the heavy chain FR region sequence is derived from the combined sequence of human germline heavy chain IGHV1-18 and IGHJ2*01, which comprises the FR1, FR2, FR3 regions of human germline heavy chain IGHV1-18 and IGHJ2*01 FR4 region.
  • the light chain FR region sequence is derived from the combined sequence of human germline light chain IGKV4-1 and IGKJ2*01, which includes the FR1, FR2, FR3 regions of human germline light chain IGKV4-1 and the FR4 region of IGKJ2*01.
  • Design primers PCR to build each humanized antibody VH/VK gene fragment, and then carry out homologous recombination with the expression vector pQD (with 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.
  • pQD signal peptide and constant region gene
  • the expression vector pQD was constructed and digested, and the expression vector pQD was designed and constructed by using some special restriction enzymes, such as the BsmBI recognition sequence and the different characteristics of the restriction site. BsmBI digested the vector, and the gel was cut for recovery.
  • heavy chain expression vector pQD-VH-CH1-FC and light chain expression vector pQD-VL-CL heavy chain variable region VH gene fragment and BsmBI digested vector pQD (with signal peptide and heavy chain constant region (CH1 -FC) fragment) according to 3:1 ratio; light chain variable region VL gene fragment and BsmBI digested vector pQD (with signal peptide and light chain constant region (CL) fragment) according to 3:1 ratio;
  • the mixtures were transferred into DH5a competent cells, ice bathed at 0°C for 30 min, heat-shocked at 42°C for 90s, added with 5 volumes of LB medium, incubated at 37°C for 45min, coated with LB-Amp plates, incubated at 37°C overnight, and single clones were picked. The clones of interest were obtained by sequencing.
  • the table below shows specific information on the humanized design of QP163164.
  • the protein expression number is QP256253.
  • the light chain of the antibody adopts the constant region CL of the light chain of kappa
  • the heavy chain of the antibody adopts the constant region of human IgG4 (for the specific sequence of the constant region, please refer to Example 2).
  • the humanized design light and heavy chain variable region sequences are not limited to the sequences shown in the table below.
  • the light chain variable region of QP256253 is encoded by plasmid accession QD253.
  • the specific sequence of light chain variable region sequence SEQ ID NO:16 is:
  • the heavy chain variable region of QP256253 is encoded by plasmid accession QD256.
  • the specific sequence of the heavy chain variable region sequence SEQ ID NO: 17 is:
  • the 293E cell culture density was maintained between (0.2-3) ⁇ 10 6 /ml, and the maintenance phase medium (GIBCO Freestyle 293 expression medium) was cultured.
  • the cells to be transfected were centrifuged to change the medium, and the cell density was adjusted to ( 0.5-0.8) x 10 6 /ml.
  • the density of 293E cells was (1-1.5) x 106 /ml.
  • Prepare plasmid and transfection reagent PEI the amount of plasmid to be transfected is 100 ⁇ g/100ml cells, and the mass ratio of PEI and plasmid is 2:1. Mix the plasmid and PEI, let stand for 15min, not more than 20min.
  • the plasmid and PEI mixture was slowly added to the 293E cells, cultured in a shaker at 8% CO 2 , 120 rpm, and 37° C. On the fifth day of transfection, the cell supernatant was collected by centrifugation at 4700 rpm in a horizontal centrifuge for 20 min.
  • Protein A affinity chromatography purification use the equilibrium solution to pass through the column, at least 3CV, the actual volume is 20ml, to ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, the flow rate is 1ml/min; the supernatant of the culture medium after centrifugation is passed through the column , load 40ml, flow rate 0.33ml/min; pass through the column with equilibrium solution, at least 3CV, actual volume 20ml, ensure that the pH and conductivity of the solution flowing out of the final instrument are consistent with the equilibrium solution, flow rate 0.33ml/min; Column, when the UV280 rises to 15mAU, the elution peak (PAC-EP) starts to be collected, and when the UV280 drops to 15mAU, the collection is stopped, and the flow rate is 1 ml/min. After sample collection, PAC-EP was adjusted to neutrality with pH adjustment solution.
  • PAC-EP elution peak
  • Binding-ELISA experimental method QP094 (SIRP ⁇ V1-flag-his), QP096 (SIRP ⁇ V2-Flag-his), QP100 (cynoSIRP ⁇ -flag-his) were coated with 0.5 ⁇ g/ml, 50 ⁇ l/well, 4 degrees overnight.
  • the affinity of the humanized antibody to human SIRP ⁇ V1, human SIRP ⁇ V2 and cynomolgus monkey SIRP ⁇ was determined by biacore as shown in Table 6 below.
  • the results show that the anti-SIRP ⁇ humanized antibody QP256253 binds to human SIRP ⁇ V1 with an affinity KD of 3.36E-10M, and it binds to human SIRP ⁇ type V2 affinity KD value is 3.19E-10M.
  • the humanized QP256253 was constructed into a phagemid vector in scFv mode (VH-3 GGGGS-VL), respectively, as the wild-type sequence (ie, as the original or starting sequence, the mutant sequence obtained by affinity maturation screening).
  • VH, (GGGGS)3 linker, VL were spliced by over-lap PCR, and ligated into phagemid vector using NcoI and NotI restriction sites.
  • the codons in the mutant region have 50% wild-type codons and 50% NNK (the reverse primer is MNN), introducing mutations in all CDR regions to construct a mutant library.
  • the PCR fragment was digested with NcoI and NotI, ligated into a phagemid vector, and finally electrotransformed into E. coli TG1. Each codon-based primer creates an independent library.
  • liquid-phase panning was performed using biotinylated QP098 (cynoSIRP ⁇ (ECD)) antigen and streptavidin magnetic beads, and each round of screening was relative to the previous one. All rounds reduce antigen concentration.
  • 250 clones were picked for phage ELISA to detect binding activity, and positive clones were sequenced.
  • the non-redundant sequences were converted into full-length IG (CH1-CH2-CH3 of hIgG4 for the heavy chain constant region; ⁇ light chain CL for the light chain constant region) for lactation. Animal cell expression.
  • Full-length IG protein was obtained after affinity purification.
  • the specific sequence is shown in the table below.
  • the light chain of the antibody adopts the constant region CL of the light chain of kappa
  • the heavy chain of the antibody adopts the constant region of human IgG4 (for the specific sequence of the constant region, please refer to Example 2).
  • the nomenclature of protein numbers is a combination of heavy chain plasmid number and light chain plasmid number.
  • its heavy chain plasmid number is QD256
  • its light chain plasmid number is QD279.
  • the sequences indicated by the SEQ ID NOs in the table are the heavy chain variable region or light chain variable region sequences of different antibodies.
  • the specific sequence of the light chain variable region is as follows:
  • Binding-ELISA experimental method coat QP094 (SIRP ⁇ V1-flag-his), QP096 (SIRP ⁇ V2-Flag-his), QP098 (cynoSIRP ⁇ -flag-his), QP100 (cynoSIRP ⁇ -flag-his) 0.5 ⁇ g/ml, 50 ⁇ l respectively /hole, 4 degrees overnight.
  • Blocking-ELISA experimental method coat QP001.2 2 ⁇ g/ml, 4 degrees overnight, wash 3 times with PBS, block 5% milk 250 ⁇ l/well, incubate Biotin-QP002 0.05 ⁇ g/ml+Abs 50 ⁇ g/ml 1:1 mixing, 25 Incubate for 1 h with HRP-Strepavidin (1:5000). The results are shown in Figure 7.
  • the affinity of the anti-SIRP ⁇ antibody with human SIRP ⁇ V1, human SIRP ⁇ V2 and cynomolgus monkey SIRP ⁇ was determined by biacore, and some of the results are shown in Table 10.
  • the anti-SIRP ⁇ antibodies QP2561589, QP2561586, QP2561581, QP256279, and QP2561770 all bound to human SIRP ⁇ type V1 and human SIRP ⁇ type V2.
  • QP2561589, QP2561586, QP256279, QP2561770, and QP256253 all bind to different cynomolgus monkey and rhesus monkey SIRP ⁇ proteins.
  • Example 5 FACS detection of anti-SIRP ⁇ antibody binding to human renal clear cell adenocarcinoma 786-O cells naturally expressing human SIRP ⁇
  • the anti-SIRP ⁇ antibody is made into different IgG subtypes, and the molecular cloning design is as follows:
  • the nomenclature of protein numbers is a combination of heavy chain plasmid number and light chain plasmid number.
  • the sequences shown in the SEQ ID NOs of the heavy chains are the heavy chain sequences of antibodies of different subtypes.
  • the sequences shown in the SEQ ID NOs of the light chains are the light chain or light chain variable region sequences of antibodies of different subtypes.
  • the sequence of the light chain variable region of QP32700279 is shown in SEQ ID NO:18.
  • Macrophage Preparation of macrophages (Macrophage): revive PBMC, separate monocytes with EasySep TM Human Monocyte Isolation Kit (Stemcell-19359), add Human Recombinant M-CSF (final concentration of 50ng/mL), mix well The cells were cultured at 37°C for 6 days to induce Macrophage; the cells were collected and counted for use. CFSE labeled Raji cells.
  • Example 7 Evaluation of the inhibitory effect of anti-SIRP ⁇ antibody on Raji-Luc tumor growth by QP32700279 in the B-NDG-hSIRP ⁇ mouse model
  • the Raji-Luc tumor model was intravenously inoculated with B-NDG-hSIRP ⁇ to evaluate the growth inhibitory effect of SIRP ⁇ antibody and Rituximab on tumor.
  • Raji-Luc cells were cultured in RPMI1640 medium containing 10% fetal bovine serum.
  • Raji-Luc cells resuspended in PBS were inoculated into the tail vein of B-NDG-hSIPRa mice at a concentration of 5 ⁇ 10 5 cells/0.2 mL and a volume of 0.2 mL/cell.
  • a small animal imager was used to measure the tumor imaging signal value.
  • the administration volume is calculated according to the body weight of the experimental animal at 10 ⁇ L/g;
  • Q3D means dosing once every 3 days
  • Q2W means dosing once every 2 weeks.
  • the day of group administration was counted as D0, and as of D18, the tumor growth curve and D18 imaging signal intensity data of each group of tumor imaging signal values are shown in Figure 12 and the following table:
  • mice Due to the characteristics of the model, the mice will appear abnormal movement or paralysis in the later stage of the experiment. At this time, the mice are euthanized and the survival curve is recorded. By the end of the G1 group of mice all dead (D25), the survival curves of each group are shown in Figure 13
  • Example 8 ELISA detection of anti-SIRP ⁇ antibody binding to all subtypes of human SIRP ⁇
  • SIRP alpha Signal Regulatory Protein
  • the SIRP ⁇ antibody QP256279 was stably expressed in CHOS cells, and the CHOS stably expressed protein was numbered as CHO71.
  • the SIRP ⁇ antibody CHO71 of the present invention binds to all subtypes of SIRP ⁇ V1/V2/V3/V4/V5/V6/V7/V8/V9/V10.
  • OSE's SIRPa antibody 18D5 does not bind SIRPa V2/V3/V7/V8/V10.

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Abstract

一种抗SIRPα抗体或其抗原结合片段及应用。通过免疫小鼠获得抗人SIRPα单克隆抗体，进一步获得嵌合抗体和人源化抗体。提供的抗SIRPα抗体或其抗原结合片段，能够结合人SIRPα蛋白，阻断CD47-SIRPα信号通路，具有增强吞噬等免疫调控作用。还提供了一种药物组合物及一种核酸分子，以及抗SIRPα抗体或其抗原结合片段在制备用于抑制或治疗疾病的药物中的用途。

Description

抗SIRPα抗体或其抗原结合片段及应用 技术领域
本发明涉及生物医药技术领域，具体涉及抗SIRPα抗体或其抗原结合片段及应用。
背景技术
据AACR预计，2018年美国新确诊恶性肿瘤病例将达到180万例，由于癌症死亡人数将超过60万，仅次于心脏病。据中国国家癌症中心公布的数据，2014年中国新确诊癌症共计380万例，而当年由于癌症死亡人数达到230万人。细胞增殖失控是导致恶性肿瘤形成的关键因素，肿瘤细胞为了加速自身的生长，保护自己免于受到机体免疫系统的攻击，在漫长的进化过程中获得了一系列途径逃逸宿主对肿瘤细胞的免疫监控。根据Schreiber提出的癌症免疫编辑理论，肿瘤细胞与宿主免疫系统的关系可以分为3种不同阶段：清除(elimination)、平衡(equilibrium)以及逃逸(escape)。在“清除”状态下，新生肿瘤细胞具有较强抗原性，易被免疫系统识别并清除；而逃过清除作用存活下来的肿瘤细胞则会与免疫系统进入“平衡”状态，表现为宿主荷瘤生存的状态。但肿瘤细胞在宿主免疫系统的选择压力下的基因突变积累到一定程度时就会打破平衡，进入到最终的“逃逸”状态。在这个阶段的肿瘤细胞可以产生一系列恶性表型，关闭抑癌响应机制，诱导正常的免疫反应，从而被识别为正常细胞。肿瘤细胞中的细胞凋亡信号通路也发生变化，使免疫细胞诱导的肿瘤细胞凋亡机制失效。此外，由于肿瘤细胞不受抑制的快速生长，形成的组织结构会产生抑制免疫细胞的微环境。在肿瘤微环境中，肿瘤细胞会释放具有免疫抑制功能的分子，如VEGF、TGF-β、IL-10等，抑制骨髓树突状细胞(dentritic cell)的激活和分化，从而抑制适应性免疫系统。同时能在外周血和淋巴结诱导产生表达CTLA-4的调节性T细胞(Treg)，对其他免疫细胞产生抑制作用，导致免疫系统产生对肿瘤的免疫耐受。
肿瘤微环境中的肿瘤相关巨噬细胞(TAM，tumor associated macrophage)的吞噬作用受到抑制，其原因在于几乎所有肿瘤细胞表面都高表达CD47蛋白，能够与骨髓细胞表面的信号调节蛋白α(signal regulatory proteinα,SIRPα)结合向机体发出“don’t eat me”或“self”的信号，从而抑制吞噬作用。CD47又名整合素相关蛋白(IAP，intergin-associated protein)，是一种广泛表达的跨膜糖蛋白，属于免疫球蛋白(Ig)超家族。CD47分子量为50kD，结构包含了大量的糖基化的N-末端IgV可变结构域，5个高度疏水的跨膜结构域和一个短的C-末端胞质尾区，C-末端胞质尾区的4种选择性拼接形式决定了CD47在不同组织的表达。与之相对应的SIRPα又被称为SHPS-1、BIT或CD172a蛋白，它是一种跨膜蛋白，主要在骨髓细胞(myeloid cell)上表达，包括巨噬细胞、骨髓树突状细胞、粒细胞、肥大细胞及其前体细胞。SIRPα由胞外的3个Ig样结构域和胞质内的4个酪氨酸残基组成，该4个酪氨酸残基被推测认为是磷酸化位点。 当发生磷酸化后，SIRPα通过结合SHP-1/2蛋白的SH2结构域并使之激活，从而激活下游信号通路。SHP-1和SHP-2蛋白的表达具有组织特异性，因此SIRPα是一种停靠蛋白，通过响应胞外刺激招募及激活下游蛋白磷酸酶。Oldenborg首先报道了成熟血红细胞(RBC，red blood cell)通过CD47与脾脏巨噬细胞SIRPα结合保护自身免遭后者的清除。随后研究人员发现RBC上的也能与单核细胞SIRPα结合从而抑制依赖Fcγ受体的吞噬作用，这是通过对吞噬作用中的关键分子肌球蛋白-IIA(myosin-IIA)去磷酸化作用实现的。临床上多种实体瘤和血液恶性肿瘤中都发现了CD47高表达的现象，包括急性髓样白血病(AML)、急性淋巴白血病(ALL)、慢性髓样白血病(CML)、非霍奇金淋巴瘤(NHL)、乳腺癌、膀胱癌、卵巢癌、结肠癌等，其本质是肿瘤细胞通过上文所述调节机制逃避巨噬细胞的细胞清除作用。CD47还通过与其他受体结合或通过其胞内细胞质区域的信号传导对其他生物过程产生影响。CD47与血小板反应蛋白-1(TSP-1，Thrombospondin-1)或血管内皮生长因子受体2(VEGFR-2)的相互作用抑制血管生成，从而限制肿瘤生长。
已有多项研究证明CD47抗体或者SIRPα-Fc重组蛋白能够在不同的小鼠PDX模型中发挥作用，而与阿糖胞苷、多柔比星、紫杉醇、顺铂等常见化疗药物或利妥昔单抗、阿仑单抗、西妥昔单抗、曲妥珠单抗联合使用的报道中也具有良好的效果。Sockolosky等则首次报道了联合使用CD47纳米抗体和PD-L1能够在黑色素瘤B16F10细胞的Syngeneic模型中表现出良好的抗肿瘤作用。近年来也有数个作用于CD47-SIRPα通路的抗体进入临床I期，包括Hu5F9-G4(恶性实体瘤：NCT02216409，恶性血液瘤：NCT02678338、NCT03248479，结肠癌：NCT02953782，B细胞非霍奇金淋巴瘤：NCT02953509)，CC-90002(AML和骨髓增生异常综合症：NCT02641002，晚期实体瘤和血液瘤：NCT02367196)，SRF231(晚期实体瘤和血液瘤：NCT03512340)，SIRPα-Fc重组蛋白TTI-662(实体瘤：NCT02890368，恶性血液瘤和实体瘤：NCT02663518，骨髓瘤和淋巴瘤：NCT03530683)，ALX148(晚期实体瘤和淋巴瘤：NCT03013218)。
CD47本身的生物学功能决定了CD47治疗性抗体和SIRPα-Fc重组蛋白可能具有血液系统毒性或者有引发贫血的风险，这在CD47基因敲除NOD鼠和使用CD47抗体进行治疗的小鼠模型中均有报道。此外，内皮细胞CD47被报道能够通过细胞黏附作用与SIRPγ相互作用促进T细胞的跨内皮迁移(transendothelial migration)，而SIRPγ主要表达于T细胞而非骨髓细胞。
因此，使用SIRPα抗体作为阻断CD47-SIRPα信号通路的更优选择。Stanford大学Weissman研究组证明其筛选出的人源化SIRPα抗体KWAR23与利妥昔单抗联用能够在敲入人源SIRPα基因的SRG鼠(Rag2-/-Il2rγ-/-)中有效抑制Burkitt淋巴瘤生长，但KWAR23单独使用则无明显药效。
发明的公开
本发明的目的是提供一种抗SIRPα抗体或其抗原结合片段及应用，抗SIRPα抗体或其抗原结合片段能够结合人SIRPα蛋白，阻断CD47-SIRPα信号通路。
为了达到上述目的，本发明提供了一种抗SIRPα抗体或其抗原结合片段，其包含：重链可变区和轻链可变区；所述重链可变区包含：氨基酸序列分别如SEQ ID NO:3、4、5所示的VHCDR1、VHCDR2和VHCDR3；所述轻链可变区包含：氨基酸序列分别如以下任意一组序列所示的VLCDR1、VLCDR2和VLCDR3；
(1)SEQ ID NO:37、38、9；
(2)SEQ ID NO:39、38、9；
(3)SEQ ID NO:7、40、9；
(4)SEQ ID NO:7、8、41；
(5)SEQ ID NO:7、8、42；
(6)SEQ ID NO:7、8、43；
(7)SEQ ID NO:37、38、41；
(8)SEQ ID NO:44、38、41；
(9)SEQ ID NO:7、8、9。
VLCDR的序列可参阅下表：
Figure PCTCN2021113416-appb-000001
可选的，所述可变区还包含：鼠源或人源FR区。
可选的，所述FR区的序列为鼠源；所述重链可变区的序列如SEQ ID NO:2所示或与其具有至少85％序列同一性，所述轻链可变区的序列如SEQ ID NO:6所示或与其具有至少85％序列同一性。
可选的，所述人源FR区包括：重链FR区序列；所述重链FR区序列来源于人种系重链IGHV1-18及IGHJ2*01的组合序列，包括人种系重链IGHV1-18的FR1、FR2、FR3区和IGHJ2*01的FR4区。
可选的，所述人源FR区包括：轻链FR区序列；所述轻链FR区序列来源于人种系轻链IGKV4-1及IGKJ2*01的组合序列，包括人种系轻链IGKV4-1的FR1、FR2、FR3区和IGKJ2*01的FR4区。
可选的，所述重链可变区的FR区序列来源于人种系，所述重链可变区的序列如SEQ ID NO:17所示或与其具有至少85％序列同一性。
可选的，所述轻链可变区的FR区序列来源于人种系，所述轻链可变区的序列选自SEQ ID NO:16、18、19、20、21、22、23、24、25中任意一项，或与其具有至少85％序列同一性。
可选的，所述抗SIRPα抗体或其抗原结合片段还包含：选自人源IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区；以及选自人源κ、λ链或其变体的轻链恒定区。
可选的，所述重链恒定区包含：Fc片段或其变体。
可选的，所述Fc片段的变体来源于IgG1，根据EU计数，包括突变位点：L234A、L235A、K338A。
可选的，所述抗SIRPα抗体或其抗原结合片段的重链序列如SEQ ID NO:26所示，或与其具有至少85％序列同一性。
可选的，所述的抗SIRPα抗体或其抗原结合片段为单克隆抗体、双特异抗体、或多特异抗体，或者，所述的抗体或其抗原结合片段用于制备抗体药物偶联物。
可选的，所述的抗SIRPα抗体或其抗原结合片段的结构形式为为Fab、F(ab’)2、Fv、或ScFv。
本发明还提供了一种药物组合物，其含有上述的抗SIRPα抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
本发明还提供了一种核酸分子，其编码上述的抗SIRPα抗体或其抗原结合片段。
本发明还提供了一种载体，其包含上述的核酸分子。
本发明还提供了一种采用上述的载体转化得到的宿主细胞。
本发明还提供了上述的抗SIRPα抗体或其抗原结合片段在制备用于抑制或治疗疾病、病症或状况的药物中的用途。
可选的，所述的药物由所述的抗SIRPα抗体或其抗原结合片段与一种或多种其他癌症治疗剂联合制备。
可选的，所述疾病、病症或状况包括：癌症、实体瘤、慢性感染、炎性疾病、多发性硬化、自身免疫性疾病、神经系统疾病、脑损伤、神经损伤、红细胞增多症、血色素沉着病、创伤、感染性休克、纤维化、动脉粥样硬化、肥胖症、II型糖尿病、移植功能障碍或关节炎。
可选的，所述癌症选自肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆囊癌、胃癌、肺癌、支气管癌、骨癌、肝胆管癌、胰腺癌、乳腺癌、肝癌、卵巢癌、睾丸癌、肾癌、肾盂和输尿管癌、唾液腺癌、小肠癌、尿道癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、直肠癌、食道癌、胃肠道癌、皮肤癌、前列腺癌、垂体癌、阴道癌、甲状腺癌、喉癌、胶质母细胞瘤、 黑素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、慢性淋巴细胞白血病(CLL)、慢性髓性白血病(CML)、急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、霍奇金淋巴瘤、非霍奇金淋巴瘤、多发性骨髓瘤、T或B细胞淋巴瘤、胃肠道间质瘤、软组织肿瘤、肝细胞癌或腺癌。
本发明还提供了上述的抗SIRPα抗体或其抗原结合片段在制备阻断SIRPα和CD47结合的制剂中的应用，所述制剂包括检测剂。
相对于现有技术，本发明具有以下有益效果：
(1)本发明提供的抗SIRPα抗体或其抗原结合片段，能够结合人SIRPα蛋白，阻断CD47-SIRPα信号通路，有望用于肿瘤治疗或制备肿瘤抗体药物。
(2)本发明提供的抗SIRPα抗体或其抗原结合片段，能够结合人SIRPα蛋白的所有亚型，有利于临床开发。
附图的简要说明
图1至图6为Binding-ELISA检测结果。
图7为Blocking-ELISA检测结果。
图8为FACS检测SIRPα抗体结合天然表达人SIRPa的人肾透明细胞腺癌细胞786-O细胞的检测结果。
图9至图11为抗SIRPα抗体体外功能实验ADCP结果。
图12为各组肿瘤成像信号值反应的肿瘤生长曲线及D18成像信号强度结果图。
图13为各组生存曲线。
图14至图23为ELISA检测本发明的抗体CHO71及对照抗体18D5、KWAR23结合SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10亚型的结果图。
图24为已知的人类SIRP alpha结合域等位基因的氨基酸序列比对图。
实现本发明的最佳方式
以下结合附图和实施例对本发明的技术方案做进一步的说明。
本实验中未注明具体条件的实验方法，通常是按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
术语：
“抗体”(antibody，Ab)指包含至少一个抗原结合位点并能特异性结合抗原的免疫球蛋白分子(immunoglobulin，Ig)。
“抗原”是在机体内能诱发免疫应答且与抗体特异性结合的物质。抗体与抗原的结合依靠二者间形成 的相互作用来介导，包括氢键、范德华力、离子键以及疏水键。抗原表面与抗体结合的区域为“抗原决定簇”或“表位”，一般来说，每个抗原有多个决定簇。
本发明所提及的术语“抗体”以其最广泛的含义理解，并包含单克隆抗体(包括全长单克隆抗体)、多克隆抗体、抗体片段、包含至少两个不同的抗原结合结构域的多特异性抗体(例如，双特异性抗体)。抗体还包括鼠源抗体、人源化抗体、嵌合抗体、人抗体以及其它来源的抗体。本发明的抗体可以来源于任何动物，包括但不限于人、非人灵长类动物、小鼠、大鼠、牛、马、鸡、骆驼、美洲驼羊(Llama)、羊驼(Alpaca)、大羊驼(Guanaco)、骆马(Vicunas)的免疫球蛋白分子等。抗体可以含有另外的改变，如非天然氨基酸，Fc效应子功能突变和糖基化位点突变。抗体还包括翻译后修饰的抗体、包含抗体的抗原决定簇的融合蛋白，以及包含对抗原识别位点的任何其它修饰的免疫球蛋白分子，只要这些抗体展现出所期望的生物活性。
抗体的基本结构是由两条完全相同的重链(heavy chain，H)和两条完全相同的轻链(light chain，L)通过二硫键连接的呈Y形的单体。每条链分别由2～5个约含110个氨基酸，序列相似但功能不同的结构域(又称功能区)组成。抗体分子中轻链和重链靠近N端的氨基酸序列变化较大，形成的结构域称为可变区(variable region，V区)；靠近C端的氨基酸序列相对恒定的区域称为恒定区(constant region，C区)。
重链和轻链的V区分别称为VH和VL，VH和VL各有3个区域的氨基酸组成和排列顺序高度可变，称为高变区(hypervariable region，HVR)；该区域形成与抗原表位互补的空间构象，又被称为互补决定区(complementarity determining region，CDR)。VH的3个CDR分别用VHCDR1、VHCDR2、VHCDR3表示，VL的3个CDR分别用VLCDR1、VLCDR2、VLCDR3表示。VH和VL共6个CDR共同组成抗原结合部位(antigen-binding site)。CDR区氨基酸的多样性是抗体与数量庞大的不同抗原特异性结合的分子基础。V区中CDR之外的氨基酸组成和排列顺序相对变化不大，称为骨架区或框架区(framework region，FR)。VH和VL各有4个骨架区，分别用FR1、FR2、FR3、FR4表示。每个VH和VL有三个CDR和四个FR组成，从氨基端到羧基端排列顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
根据抗体重链恒定区的氨基酸序列，可以将人免疫球蛋白分为5类：IgM、IgG、IgA、IgD、IgE。其还可以进一步分成不同的亚类(同种型)，如人IgG可以分为IgG1、IgG2、IgG3、IgG4；IgA可分为IgA1和IgA2。IgM、IgD、IgE尚未发现有亚类。根据轻链氨基酸序列，可将轻链分类为κ链和λ链。本发明的抗体可以是任何种类(如IgM、IgG、IgA、IgD、IgE)或亚类(如IgG1、IgG2、IgG3、IgG4、IgA1、IgA2)。
重链和轻链的恒定区分别称为CH和CL。IgG、IgA、IgD的重链恒定区有CH1、CH2、CH3三个结构域，IgM、IgE的重链恒定区有CH1、CH2、CH3、CH4四个结构域。
CH1和CH2之间为铰链区(hinge region)，含有丰富的脯氨酸，因此易伸展弯曲，能改变Y形两个臂之间的距离，有利于两臂同时结合抗原表位。
“抗原结合片段”指具有抗原结合活性的Fab片段、F(ab’)2片段、Fv片段、ScFv片段等。“Fab片段” (fragment of antigen binding,Fab)，意指由VL、VH、CL和CH1结构域组成的抗体片段，与单个抗原表位结合(单价)。本领域技术人员可知，木瓜蛋白酶水解IgG形成2个相同的Fab段和1个Fc段；胃蛋白酶水解IgG形成1个F(ab’)2段和若干多肽碎片(pFc’)。若F(ab’)2重链间二硫键断裂，可形成2个Fab’片段，后者可进一步被酶解成Fv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区。单链可变片段scFv(single chain antibody fragment)，或称单链抗体，由抗体重链可变区和轻链可变区通过连接片段(linker)连接而成。
术语“Fc”、“Fc段”或“Fc片段”是指可结晶片段(fragment crystallizable)，无抗原结合活性，是抗体与效应分子或细胞表面Fc受体(FcR)相互作用的部位。Fc片段包含抗体除重链恒定区CH1之外的恒定区多肽。Fc片段与表面具有相应Fc受体的细胞结合，产生不同的生物学作用。在ADCC效应中(抗体依赖的细胞介导的细胞毒性作用，antibody-dependent cell-mediated cytotoxicity)，抗体的Fab段结合病毒感染的细胞或肿瘤细胞的抗原表位，其Fc段与杀伤细胞(NK细胞、巨噬细胞等)表面的FcR结合，介导杀伤细胞直接杀伤靶细胞。ADCP为抗体依赖性细胞吞噬作用(antibody-dependent cellular phagocytosis)，ADCP的机制是抗体作用的靶细胞激活巨噬细胞表面上的FcγR机制，诱导吞噬，使靶细胞内化和被吞噬体酸化降解。在某些特定情形下消除抗体Fc功能更有益。这些情形包括抗体用作：(1)受体激动剂，诱导细胞信号；(2)受体拮抗剂，阻断受体和配体的结合，抑制信号；或，(3)作为药物载体递送药物到表达相应抗原的靶细胞。如果保持着Fc功能将导致抗体药物误伤表达相应受体的细胞，以及造成抗体偶联药物脱靶情况下误伤重要的免疫细胞。
Fc变体或突变的组合不限于以下形式(根据EU计数)。
Figure PCTCN2021113416-appb-000002
本发明所述抗体或抗原结合片段的CDR氨基酸残基在数量和位置上符合已知的Kabat编号规则。
目前鼠源抗体是抗体药物的一大来源。由于鼠源抗体的免疫原性，一般会对其作人源化处理。以下实施例中提供了鼠源抗体、嵌合抗体、人源化抗体。“嵌合抗体”是将鼠源抗体的可变区和人抗体的恒定区融合而成的抗体，可以减轻鼠源抗体诱发的免疫应答反应。人抗体的恒定区可选自人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，以及选自人源κ、λ链或其变体的轻链恒定区。“人源化抗体”是指将鼠源抗体的CDR序列移植到人的抗体可变区框架得到的抗体，可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导强烈反应。此类框架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时引起的活性下降，可对人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
理论上，抗体亲和力的提高有助于改善抗体的特异性和效力，有助于减少用药剂量，降低毒副作用等。虽然实际的研究工作证明亲和力的提高与抗体效价的提高并不总是线性的关系，尤其在实体瘤的治疗上，但很多情况下，这个线性关系是明显存在的。本发明的人源化抗体也包括进一步由噬菌体展示对 CDR进行亲和力成熟后的人源化抗体。体外的抗体亲和力成熟的理论基础是模仿了体内抗体亲和力的过程。通过构建随机突变库，模拟B细胞在体内的高频突变，可以筛选到高亲和的抗体。
本发明提供的药物可含有“治疗有效量”的抗体或抗原结合片段。“治疗有效量”指有效预防或缓解具体疾病的治疗剂的量，可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在不同患者体内产生需要疗效的能力。
“序列同一性”指两个多核苷酸序列之间或两个多肽之间的序列相似性，是两个多核苷酸或两个多肽之间具有相同碱基或氨基酸的程度。本发明所述的“具有至少85％序列同一性”是指至少达到85％、90％、95％、97％、或99％同一性。
Antibody-Drug Conjugates(ADCs，抗体药物偶联物)，是指与一个或多个化学药物(任选地可以是治疗剂或细胞毒性剂)连接的结合蛋白。可通过永久的或者不稳定的化学连接体(linker)将细胞毒小分子(cytotoxin)和抗体连接，获得抗体药物偶联物。ADC可选择和持续性地将细胞毒性药物递送至肿瘤。
编码SIRPα的基因是多态性基因(polymorphic gene)，已知人类群体中有10种SIRPα变体。Katsuto Takenaka等对来自人类HapMap基因组计划的37个不相关的正常高加索人、非洲人、中国人和日本人的IgV编码SIRP alpha域进行了测序，发现了10个不同的SIRP alpha IgV编码等位基因(Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells，NATURE IMMUNOLOGY VOLUME 8 NUMBER 12 DECEMBER 2007)。10种SIRPα变体分别为SIRPα V1/V2/V3/V4/V5/V6/V7/V8/V9/V10亚型。虽然SIRPalpha具有高度多态性，但是ChiaChiM.Ho等对已知的人类SIRPalpha等位基因的氨基酸序列比对显示，SIRP alpha与CD47结合界面只有两个独特的序列，分别为等位基因V1(a2d1)型和V2(a1d1)型。(“Velcro”Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein(SIRP alpha)Antagonists That Enhance Antibody-dependent Cellular Phagocytosis，JOURNAL OF BIOLOGICAL CHEMISTRY，VOLUME 290·NUMBER 20·MAY 15,2015)。
如图24所示，对已知的人类SIRP alpha结合域等位基因的氨基酸序列比对显示，在CD47接触界面只有两个变异：a1d1和a2d1。图24的第一行文本为人类最显著的SIRP alpha等位基因V1(a2d1)的氨基酸序列，图1的第二行文本为人类最显著的SIRP等位基因V2(a1d1)的氨基酸序列。黑盒子表示与CD47相互作用的残基，而阴影表示与V1序列不同的残基。Janet Sim等对2535个个体的SIRPα序列和510个样本的Sanger测序鉴定出两个SIRPα变体v1和v2，代表三个等位基因组：纯合子v1/v1,纯合子v2/v2,杂合子v1/v2。通过对在不同的群体和不相关的亚群体中确定了SIRPαv1和v2等位基因群的分布和频率。其中欧洲(EUR)、美国(AMR)、东亚(EAS)、非洲(AFR)和南亚(SAS)5个超级群体的v1/v2杂合子分布相似，分布范围为42.0％～47.2％。东亚人口v2/v2数量明显高于v1/v1，发生频率分别为42.3％和13.3％，在非洲、欧洲人、美国人、和南亚人群v1/v1数量高于v2/v2，v1和v2的发生频率范围分别为30.3-49.1％和8.9-24.2％(参考文献详见MABS，2019,VOL.11,NO.6,1036¨C1052， https://doi.org/10.1080/19420862.2019.1624123)。Aduro Biotech也研究了东亚人群v2/v2纯合子发生频率为41.3％，v1/v1纯合子发生频率为34.6％，同样证明东亚人群中有41.3％的人是V2/V2纯合子(参考文献详见Voets et al.Journal for ImmunoTherapy of Cancer(2019)7:340)。
基于SIRPα多态分析的结果，抗SIRPα抗体能够同时结合SIRPα v1型和SIRPa v2型基因，这是至关重要的，以便于临床开发。
实施例1：获得抗SIRPα小鼠抗体
(1)小鼠免疫：
抗人SIRPα单克隆抗体通过免疫小鼠产生。实验用Balb/c白小鼠，雌性，6周龄。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。免疫Balb/c小鼠，首次用弗式完全佐剂(CFA)免疫重组蛋白QP009(SIRPα)50μg/只小鼠两周后，此后用QP009(SIRPα)加弗式不完全佐剂(IFA)或者QP009(SIRPα)加铝盐Alum+CpG ODN 1826交替免疫，25μg/只小鼠，一周一次。
QP009(SIRPα)具有如下所示氨基酸序列(SEQ ID NO:1)：
EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSDYKDDDDKHHHHHH。序列参考自UNIPROT编号P78324(31-149)( SIRPA-Tyrosine-protein phosphatase non-receptor type substrate  1 precursor-Homo sapiens(Human)-SIRPA gene&protein(uniprot.org))。
(2)细胞融合：
选择血清中抗体滴度高的小鼠进行脾细胞融合。融合前72小时，腹腔注射冲刺免疫所选小鼠。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤Sp2/0细胞进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的IMDM培养基)重悬，分装于96孔细胞培养板中(1×10 5个/150μl/孔)，37℃、5％CO 2培养。融合后的第5天加入20％FBS的IMDM培养基(含2×HAT和1×OPI)，50μl/孔，37℃，5％CO 2培养。融合后第7天～8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，250μl/孔，37℃、5％CO 2培养。
(3)杂交瘤细胞筛选：
根据细胞生长密度，融合后第10-14天，进行ELISA检测筛选杂交瘤上清中抗SIRPα抗体。取杂交瘤融合孔上清，通过ELISA进行96孔板整板初筛，检测上清中抗SIRPα抗体能阻断SIRPα/CD47的结合，即为初筛阳性孔。再取初筛阳性孔上清ELISA检测与QP009(SIRPα)的结合，选取结合SIRPα且能阻断SIRPα/CD47结合均为阳性的克隆，即为抗SIRPα抗体阳性克隆孔。将阳性克隆扩培并及时转移至24/6孔板中，通过ELISA再次检测细胞培养上清结合SIRPα且能阻断SIRPα/CD47的结合均为阳性的克隆孔，即为抗SIRPα抗体阳性克隆孔。将阳性克隆进行2-3轮有限稀释至单细胞克隆，冻存阳性单细胞株，获 得单细胞克隆71C10。
(4)杂交瘤单抗测序获得抗体序列：
取杂交瘤阳性单克隆细胞株71C10，提取mRNA，mRNA反转录成cDNA，以cDNA为模板PCR扩增，挑选PCR阳性克隆送测序，通过序列分析得到单克隆抗体轻重链可变区序列。
71C10的重链可变区序列为SEQ ID NO:2，具体如下：
Figure PCTCN2021113416-appb-000003
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。加粗并下划线部分分别为VHCDR1(SEQ ID NO:3)、VHCDR2(SEQ ID NO:4)、VHCDR3(SEQ ID NO:5)。
71C10的轻链可变区序列为SEQ ID NO:6，具体如下所示：
Figure PCTCN2021113416-appb-000004
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。加粗并下划线部分分别为VLCDR1(SEQ ID NO:7)、VLCDR2(SEQ ID NO:8)、VLCDR3(SEQ ID NO:9)。
实施例2：抗SIRPα嵌合抗体SPR检测亲和力
(1)将单克隆细胞株71C10的鼠源可变区序列与人恒定区基因融合，获得嵌合抗体分子。抗体轻链采用kappa轻链恒定区CL。同时设计不同的抗原序列，用于抗体分子的性能测试。抗原及嵌合抗体分子克隆设计见表1和表2。
表1嵌合抗体分子克隆设计
Figure PCTCN2021113416-appb-000005
注：
蛋白编号为QP026027、QP026249的抗体作为对照抗体，其均采用已知抗SIRPα抗体KWAR23的可变区序列，区别在于恒定区不同。QP163164、QP163245均采用单克隆细胞株71C10的可变区，区别在于恒定区不同。上述序列编号所示的序列分别给出各个抗体分子的重、轻链序列。
pQD为带信号肽及恒定区基因(CH1-FC/CL)片段的载体名称，其中，pQDH用于重链可变区的连接和表达，带有信号肽及恒定区基因(CH1-FC)片段；pQDK用于轻链可变区的连接和表达，带有信号肽及恒 定区基因(CL)片段。“H”表示重链，“L”表示轻链。“(IgG4)”表示重链采用人IgG4的恒定区。如果未标注有“(IgG4)”，则默认采用人IgG1的恒定区。180122VH代表来源于单克隆细胞株71C10的重链可变区，180122VL代表来源于单克隆细胞株71C10的轻链可变区。
示例性的，“pQDH-KWAR23-H”表示对照序列KWAR23融合到pQDH载体上，pQDH带有信号肽及恒定区基因(CH1-FC)片段，采用人IgG1的恒定区。“pQDH-180122VH”表示重链可变区序列180122VH融合到pQDH载体上，采用人IgG1的恒定区。上述序列编号所示的序列具体如下：
>QD026(SEQ ID NO:10)
Figure PCTCN2021113416-appb-000006
>QD027(SEQ ID NO:11)
MEFGLSWLFLVAILKGVQCEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。其中， MEFGLSWLFLVAILKGVQC为信号肽。
>QD249(SEQ ID NO:12)
MEFGLSWLFLVAILKGVQCEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK。其中， MEFGLSWLFLVAILKGVQC为信号肽。
>QD163(SEQ ID NO:13)
Figure PCTCN2021113416-appb-000007
Figure PCTCN2021113416-appb-000008
Figure PCTCN2021113416-appb-000009
其中，双下划线部分是恒定区序列。
>QD164(SEQ ID NO:14)
Figure PCTCN2021113416-appb-000010
Figure PCTCN2021113416-appb-000011
其中，双下划线部分是恒定区序列。
>QD245(SEQ ID NO:15)
Figure PCTCN2021113416-appb-000012
Figure PCTCN2021113416-appb-000013
其中，
Figure PCTCN2021113416-appb-000014
为信号肽，双下划线部分为重链恒定区序列。
表2抗原克隆设计
Figure PCTCN2021113416-appb-000015
注：QP098为食蟹猴SIRPα序列(uniprot数据库序列编号I7G9Z7)，QP100为食蟹猴SIRPα序列(uniprot数据库序列编号G7PGS8)，QP271为恒河猴SIRPα序列，是发明人通过测序猴子PBMC获得，QP273 为食蟹猴SIRPα序列，是发明人通过测序猴子PBMC获得。
(2)抗原及嵌合抗体表达、纯化
293E细胞培养密度维持在(0.2-3)×10 6/ml之间，维护阶段培养基(GIBCO Freestyle 293 expression medium)进行培养，转染前一天待转染细胞离心换液，调整细胞密度为(0.5-0.8)×10 6/ml。转染当天，293E细胞密度为(1-1.5)×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100μg/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
Protein A亲和层析纯化：用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
(3)表面等离子体共振(SPR)检测亲和力
通过Biacore T200(GE)测定抗SIRPα嵌合抗体QP163164与人SIRPαV1型(蛋白编号QP094)及人SIRPαV2型(蛋白编号QP096)亲和力。表3和表4展示QP163164以及QP026027的检测结果。结果显示SIRPα嵌合抗体QP163164结合人SIRPαV1型亲和力KD为5.27E-10M，结合人SIRPαV2型亲和力KD值为6.78E-10M。结合人SIRPαV1型及人SIRPαV2型亲和力明显优于对照抗体KWAR23(QP026027)。
表3抗SIRPα嵌合抗体与SIRPα V1及SIRPα V2亲和力
Figure PCTCN2021113416-appb-000016
通过biacore测定嵌合抗体与食蟹猴SIRPα亲和力如下表所示：
表4嵌合抗体与食蟹猴SIRPα亲和力
Figure PCTCN2021113416-appb-000017
实施例3：抗SIRPα杂交瘤单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，挑选与QP163164同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为 FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号系统确定并注释。以下实施例中，重链FR区序列来源于人种系重链IGHV1-18及IGHJ2*01的组合序列，其包含人种系重链IGHV1-18的FR1、FR2、FR3区和IGHJ2*01的FR4区。轻链FR区序列来源于人种系轻链IGKV4-1及IGKJ2*01的组合序列，其包含人种系轻链IGKV4-1的FR1、FR2、FR3区和IGKJ2*01的FR4区。
(1)抗SIRPα抗体人源化分子克隆
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pQD/VK-CL-pQD。
利用在线软件DNAWorks(v3.2.2)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH/VK+30bp CH1/CL-3’。按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。表达载体pQD构建及酶切，利用一些特殊的限制性内切酶，如BsmBI识别序列与酶切位点不同的特性设计构建表达载体pQD。BsmBI酶切载体，切胶回收备用。构建重链表达载体pQD-VH-CH1-FC及轻链表达载体pQD-VL-CL：重链可变区VH基因片段与经BsmBI酶切的载体pQD(带信号肽及重链恒定区(CH1-FC)片段)按照3:1比例混合；轻链可变区VL基因片段与经BsmBI酶切的载体pQD(带信号肽及轻链恒定区(CL)片段)按照3:1比例混合；将混合物分别转入DH5a感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
下表所示为QP163164的人源化设计的具体信息。蛋白表达编号为QP256253。此表中抗体轻链采用kappa轻链恒定区CL，抗体重链采用人IgG4恒定区(恒定区具体序列可参阅实施例2)。人源化设计轻重链可变区序列并不限于下表所示的序列。
表5人源化设计轻重链序列及蛋白表达编号
Figure PCTCN2021113416-appb-000018
注：
QP256253的轻链可变区由编号为QD253的质粒编码。轻链可变区序列SEQ ID NO:16具体序列为：
Figure PCTCN2021113416-appb-000019
QP256253的重链可变区由编号为QD256的质粒编码。重链可变区序列SEQ ID NO:17具体序列为：
Figure PCTCN2021113416-appb-000020
Figure PCTCN2021113416-appb-000021
(2)抗SIRPα抗体人源化蛋白表达
293E细胞培养密度维持在(0.2-3)×10 6/ml之间，维护阶段培养基(GIBCO Freestyle 293 expression medium)进行培养，转染前一天待转染细胞离心换液，调整细胞密度为(0.5-0.8)×10 6/ml。转染当天，293E细胞密度为(1-1.5)×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100μg/100ml细胞，使用PEI和质粒的质量比为2:1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
(3)抗SIRPα抗体人源化蛋白纯化
Protein A亲和层析纯化：用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
(4)人源化SIRPα抗体活性鉴定(Binding-ELISA)
Binding-ELISA实验方法：分别包被QP094(SIRPαV1-flag-his)、QP096(SIRPαV2-Flag-his)、QP100(cynoSIRPα-flag-his)0.5μg/ml，50μl/孔，4度过夜。PBS洗3遍，3％BSA/PBS 200μl/孔，RT孵育2h；PBST洗3遍；加入不同浓度的抗体，RT孵育1h，PBST洗3遍，PBS洗3遍；孵育二抗HRP-anti Fab 1：2500稀释，RT孵育1h，PBST洗3遍，PBS洗3遍，TMB显示，2M的H 2SO 4终止，450nm读数。
(5)人源化SIRPα抗体SPR鉴定亲和力
通过biacore测定人源化抗体与人SIRPαV1型、人SIRPαV2型及食蟹猴SIRPα亲和力如下表6所示，结果显示抗SIRPα人源化抗体QP256253结合人SIRPαV1型亲和力KD为3.36E-10M，结合人SIRPαV2型亲和力KD值为3.19E-10M。
表6人源化抗体与人SIRPαV1型及SIRPαV2型及食蟹猴SIRPα亲和力
Figure PCTCN2021113416-appb-000022
实施例4：抗SIRPα抗体QP163164亲和力成熟
(1)构建人源化噬菌粒载体
人源化后的QP256253分别以scFv模式(VH-3个GGGGS-VL)构建到噬菌粒载体中，作为野生型序列(即作为原始或起始序列，亲和力成熟筛选得到的为突变序列)。利用重叠PCR(over-lap PCR)拼接VH、 (GGGGS)3接头、VL，采用NcoI和NotI酶切位点连接入噬菌粒载体。
(2)构建噬菌体展示文库
利用构建好的野生型scFv为模板，采用基于密码子的(codon-based)引物，在引物合成过程中，突变区域密码子有50％野生型的密码子和50％的NNK(反向引物为MNN)，在所有CDR区引入突变构建突变文库。PCR片段经过NcoI和NotI酶切，连接到噬菌粒体载体中，最后电转化大肠杆菌TG1。每条基于密码子的引物建立一个独立的文库。
(3)文库淘筛
文库经过拯救包装出淘筛用的噬菌体颗粒后，利用生物素化的QP098(cynoSIRPα(ECD))抗原和链霉亲和素磁珠进行液相法淘筛，并且每一轮筛选相对于上一轮都降低抗原浓度。三轮淘筛之后，挑取250克隆进行噬菌体ELISA检测结合活性，阳性克隆进行测序。经过对测序克隆进行比对分析，去除冗余序列之后，将非冗余序列转换成全长IG(重链恒定区选择hIgG4的CH1-CH2-CH3；轻链恒定区选择κ轻链CL)进行哺乳动物细胞表达。亲和纯化之后得到全长IG蛋白。具体序列如下表所示。此表中抗体轻链采用kappa轻链恒定区CL，抗体重链采用人IgG4恒定区(恒定区具体序列可参阅实施例2)。
表7阳性克隆序列
Figure PCTCN2021113416-appb-000023
注：蛋白编号的命名规则为重链质粒编号和轻链质粒编号的组合。示例性的，蛋白编号为QP256279的抗体分子，其重链质粒编号为QD256，轻链质粒编号为QD279。表中序列编号所示的序列为不同抗体的重链可变区或轻链可变区序列。轻链可变区具体序列如下：
>QD279(SEQ ID NO:18)
Figure PCTCN2021113416-appb-000024
>QD291(SEQ ID NO:19)
Figure PCTCN2021113416-appb-000025
>QD1581(SEQ ID NO:20)
Figure PCTCN2021113416-appb-000026
Figure PCTCN2021113416-appb-000027
>QD1586(SEQ ID NO:21)
Figure PCTCN2021113416-appb-000028
>QD1589(SEQ ID NO:22)
Figure PCTCN2021113416-appb-000029
>QD1594(SEQ ID NO:23)
Figure PCTCN2021113416-appb-000030
>QD1770(SEQ ID NO:24)
Figure PCTCN2021113416-appb-000031
>QD1771(SEQ ID NO:25)
Figure PCTCN2021113416-appb-000032
以上加粗并下划线部分分别为各抗体分子的VLCDR1、VLCDR2、VLCDR3，与野生型序列QP256253具体比较如下：
表8各抗体分子的LCDR区序列与野生型序列的比较
Figure PCTCN2021113416-appb-000033
注：“/”表示序列与QP256253相同，黑体并加粗表示与QD253不同的氨基酸。
(4)ELISA检测
Binding-ELISA实验方法：分别包被QP094(SIRPαV1-flag-his)、QP096(SIRPαV2-Flag-his)、QP098(cynoSIRPα-flag-his)、QP100(cynoSIRPα-flag-his)0.5μg/ml，50μl/孔，4度过夜。PBS洗3遍，3％BSA/PBS 200μl/孔，RT孵育2h；PBST洗3遍；加入不同浓度的抗体，RT孵育1h，PBST洗3遍，PBS洗3遍；孵育二抗HRP-anti Fab 1：2500稀释，RT孵育1h，PBST洗3遍，PBS洗3遍，TMB显示，2M的H 2SO 4终止，450nm读数。EC50值如下表所示。下表中还展示了人源化抗体QP256253、嵌合抗体QP163245、对照抗体QP026249的检测结果。结果如图1至图6所示。
表9 ELISA检测EC50值
conc.(μg/ml) QP094 QP096 QP098 QP100 QP271 QP273
QP2561589 0.001366 0.001171 0.001598 0.002009 0.0009365 0.001429
QP256291 0.001859 0.001429 0.00157 0.001178 0.001173 0.001618
QP2561586 0.001046 0.001108 0.001199 0.001235 0.0009672 0.001027
QP2561581 0.001075 0.0008623 0.0009179 0.0007578 0.0005554 0.0009209
QP2561594 0.001115 0.001055 0.001134 0.0009833 0.0008066 0.002348
QP256279 0.0006508 0.0006239 0.000619 0.0005584 0.0006008 0.0007207
QP2561770 0.001615 0.001363 0.001302 0.001005 0.001207 0.001675
QP2561771 0.001145 0.001127 0.001082 0.001128 0.001127 0.001217
QP256253 0.001189 0.001185 0.001255 0.00172 0.0009472 0.001514
QP163245 0.001106 0.001235 0.001526 0.001166 0.0008822 0.0016
QP026249 0.005285 0.003311 0.02366 0.001925 0.00193 0.002028
Blocking-ELISA实验方法：coat QP001.2 2μg/ml，4度过夜，PBS洗3遍，5％milk 250μl/孔封闭，孵育Biotin-QP002 0.05μg/ml+Abs 50μg/ml 1：1混合，25度孵育1h，HRP-Strepavidin(1:5000)。结果如图7所示。
(5)表面等离子体共振(SPR)检测亲合力
通过biacore测定抗SIRPα抗体与人SIRPαV1型、人SIRPαV2型及食蟹猴SIRPα亲和力，部分结果如表10所示。由表10可知，抗SIRPα抗体QP2561589、QP2561586、QP2561581、QP256279、QP2561770均结合人SIRPαV1型和人SIRPαV2型。同时QP2561589、QP2561586、QP256279、QP2561770、QP256253均结合不同食蟹猴及恒河猴SIRPα蛋白。
表10 SPR检测亲合力结果
Figure PCTCN2021113416-appb-000034
Figure PCTCN2021113416-appb-000035
由上表可知，亲和力成熟抗体QP2561589、QP2561586、QP256279蛋白对人SIRPα V1型及SIRPα V2型亲和力比对照抗体KWAR23(QP026249)的高50倍以上。
实施例5：FACS检测抗SIRPα抗体结合天然表达人SIRPα的人肾透明细胞腺癌细胞786-O细胞
实验步骤：收集786-O细胞2E5/孔，PBS洗1遍，300g离心3min弃上清。封闭：用2％FBS重悬，2E5/孔，200μl/孔种96孔U底板，冰浴1h。300g离心3min弃上清。抗体孵育：孵育抗体10μg/ml 1:3稀释，100μl/孔，冰浴1h。离心，弃上清。加入预冷的PBS 200μl/孔，300g离心5min弃上清，重复2次。二抗：PE-anti human FC(1:200)50μl/孔，冰浴0.5h。离心，弃上清。加入预冷的PBS 200μl/孔，300g离心5min弃上清，重复3次。FACS读取平均荧光值。结果如图8所示，SIRPα抗体QP163245、QP256253、QP256279、QP2561586、QP2561589均结合天然表达人SIRPα的人肾透明细胞腺癌细胞786-O细胞，且结合亲和力优于对照抗体QP026249(KWAR23)。
实施例6：抗SIRPα抗体体外功能实验ADCP
(1)将抗SIRPα抗体做成不同IgG亚型，分子克隆设计如下：
表11抗SIRPα抗体体外功能实验ADCP分子克隆设计
Figure PCTCN2021113416-appb-000036
Figure PCTCN2021113416-appb-000037
注：蛋白编号的命名规则为重链质粒编号和轻链质粒编号的组合。重链的序列编号所示的序列为不同亚型抗体的重链序列。轻链的序列编号所示的序列为不同亚型抗体的轻链或轻链可变区序列。
其中，QP32700279的重链(SEQ ID NO:26)的具体序列如下：
Figure PCTCN2021113416-appb-000038
QP32700279的轻链可变区的序列如SEQ ID NO:18所示。
(2)抗SIRPα抗体体外功能实验ADCP
准备巨噬细胞(Macrophage)：复苏PBMC，用试剂盒EasySep TM Human Monocyte Isolation Kit(Stemcell-19359)分离单核细胞(monocytes)，加入Human Recombinant M-CSF(终浓度为50ng/mL)，充分混匀；37℃培养细胞6天诱导为Macrophage；收集细胞，计数备用。CFSE标记Raji细胞。将Raji重悬为2×10 6cells/ml，然后按50μl/well(1×10 5/well)加入已有巨噬细胞的96孔板中；稀释抗体：将Rituximab(利 妥昔单抗)用完全培养基稀释为80μg/ml，再3倍稀释9个梯度，将anti-SIRPα用完全培养基稀释为20μg/ml；抗体混合：Combination组将稀释好的2个抗体1:1混合，Rituximab组用等体积的培养基混合，按50μl/well加入之前铺好的细胞的96孔板中；37℃培养2h；FACS检测：通过对活CFSE+/CD14+细胞进行门控来测量吞噬作用(Phagocytosis was measured by gating live CFSE+/CD14+cells)。
将亲和力成熟分子和对照抗体做了协同Rituximab的ADCP assay，实验结果显示SIRPα抗体和Rituximab联用与Rituximab单用相比，EC50变小，ADCP协同效果显著增强。结果如图9、图10、图11所示。
实施例7：QP32700279在B-NDG-hSIRPΑ鼠模型中评价抗SIRPα抗体对Raji-Luc肿瘤生长的抑制作用
为了考察抗SIRPα抗体对肿瘤的杀伤作用，采用B-NDG-hSIRPΑ静脉接种Raji-Luc肿瘤模型评价SIRPα抗体和Rituximab对肿瘤的生长抑制作用。Raji-Luc细胞培养在含10％胎牛血清的RPMI1640培养液中。将PBS重悬的Raji-Luc细胞以5×10 5个/0.2mL浓度，0.2mL/只体积接种到B-NDG-hSIPRa小鼠尾静脉。在接种后第0天、第3天使用小动物成像仪测量肿瘤成像信号值，当平均成像信号强度达到1×10 6P/S左右，根据肿瘤成像信号值、动物体重选择合适动物入组，平均分配到4个实验组中，每个实验组8只。分组当天开始给药，具体给药方案见下表：
Figure PCTCN2021113416-appb-000039
注：
a：给药体积依实验动物体重按10μL/g计算；
b：Q3D指每3天给药1次，Q2W指每2周给药1次。
分组给药当天计为D0，截止至D18，各组肿瘤成像信号值反应的肿瘤生长曲线及D18成像信号强度数据如图12和下表所示：
Figure PCTCN2021113416-appb-000040
Figure PCTCN2021113416-appb-000041
肿瘤生长曲线结果显示，Rituximab、QP32700279及QP32700279、Rituximab联合用药组均能显著抑制Raji-Luc肿瘤生长，肿瘤抑制率(TGI)分别为58.6％、46.4％和84.5％，而且联合用药组比单独用药组表现出更强的抗肿瘤活性。
由于模型特征，试验后期小鼠会出现行动异常或瘫痪的现象，此时给予小鼠安乐死处理，并记录生存曲线。截止至G1组小鼠全部死亡(D25)，各组生存曲线如图13所示
生存分析采用Kaplan-Meier法，组间比较采用Log rank检验，p<0.05视为差异显著。与对照组相比，QP32700279和联合给药组(QP32700279+Rituximab)均可以显著延长Raji-Luc荷瘤小鼠的生存期(p＝0.0445*,p<0.001**)，而Rituximab组不能有效地延长荷瘤小鼠的生存期(p＝0.23)。该试验结果提示，QP32700279和QP32700279+Rituximab联合给药可以有效地抑制Raji-Luc荷瘤小鼠的肿瘤生长情况，进而改善小鼠的生存情况。
实施例8：ELISA检测抗SIRPα抗体对人SIRPα所有亚型结合
根据已有文献(“Velcro”Engineering of High Affinity CD47 Ectodomain as Signal Regulatory Protein(SIRP alpha)Antagonists That Enhance Antibody-dependent Cellular Phagocytosis，JOURNAL OF BIOLOGICAL CHEMISTRY，VOLUME 290·NUMBER 20·MAY 15,2015)报道的SIRPαV1/V2/V3/V4/V5/V6/V7/V8/V9/V10序列，通过基因合成将上面SIRPαC端融合小鼠IgG2a亚型的Fc(mouse IgG2a)，构建到真核表达载体pQD，通过293E瞬时转染，Protein A纯化瞬转第五天上清，分别得到SIRPα V1/V2/V3/V4/V5/V6/V7/V8/V9/V10融合Fc(mouse IgG2a)蛋白，进一步进行ELISA检测SIRPα抗体与SIRPα所有亚型的结合。序列如下所示。
>SIRPα V1(SEQ ID NO:51)
Figure PCTCN2021113416-appb-000042
>SIRPαV2(SEQ ID NO:52)
Figure PCTCN2021113416-appb-000043
>SIRPα V3(SEQ ID NO:53)
Figure PCTCN2021113416-appb-000044
>SIRPα V4(SEQ ID NO:54)
Figure PCTCN2021113416-appb-000045
Figure PCTCN2021113416-appb-000046
>SIRPα V5(SEQ ID NO:55)
Figure PCTCN2021113416-appb-000047
>SIRPα V6(SEQ ID NO:56)
Figure PCTCN2021113416-appb-000048
>SIRPα V7(SEQ ID NO:57)
Figure PCTCN2021113416-appb-000049
>SIRPα V8(SEQ ID NO:58)
Figure PCTCN2021113416-appb-000050
>SIRPα V9(SEQ ID NO:59)
Figure PCTCN2021113416-appb-000051
>SIRPα V10(SEQ ID NO:60)
Figure PCTCN2021113416-appb-000052
>FC(mouse IgG2a)(SEQ ID NO:61)
Figure PCTCN2021113416-appb-000053
待检测SIRPα抗体：
将SIRPα抗体QP256279在CHOS细胞中稳定表达，CHOS稳转表达蛋白编号为CHO71。
根据专利WO2017178653提供的序列，分子克隆构建并表达纯化OSE公司抗SIRPα抗体18D5，用于实验对照。同时如前所述QP026249即为47公司(Forty Seven)的抗SIRPα抗体KWAR23，这里用KWAR23表示。
ELISA检测SIRPα抗体结合SIRPα V1/V2/V3/V4/V5/V6/V7/V8/V9/V10实验步骤：
包被SIRPα V1/V2/V3/V4/V5/V6/V7/V8/V9/V10，1μg/ml，60μl/孔，4℃过夜，PBST洗2遍；5％non-fat  milk(生工)封闭，200μl/孔，室温孵育1h，PBST洗2遍；孵育抗体10μg/ml，5倍稀释，10个梯度，60μ/孔，室温孵育1h，PBST洗5遍；孵育二抗：anti-hFab1：10000，60μl/孔，室温孵育1h，PBST洗5遍；显色：TMB提前1h平衡室温，100μl/孔，显色10min，2M H 2SO 4 50ul/孔终止，酶标仪450nm读数。
实验结果如图14至图23所示，本发明的SIRPα抗体CHO71结合SIRPα V1/V2/V3/V4/V5/V6/V7/V8/V9/V10所有亚型。OSE公司的SIRPa抗体18D5不结合SIRPa V2/V3/V7/V8/V10。
尽管本发明的内容已经通过上述优选实施例作了详细介绍，但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后，对于本发明的多种修改和替代都将是显而易见的。因此，本发明的保护范围应由所附的权利要求来限定。

Claims (14)

  1. 一种抗SIRPα抗体或其抗原结合片段，其特征在于，包含：重链可变区和轻链可变区；所述重链可变区包含：氨基酸序列分别如SEQ ID NO:3、4、5所示的VHCDR1、VHCDR2和VHCDR3；所述轻链可变区包含：氨基酸序列分别如以下任意一组序列所示的VLCDR1、VLCDR2和VLCDR3；
    (1)SEQ ID NO:37、38、9；
    (2)SEQ ID NO:39、38、9；
    (3)SEQ ID NO:7、40、9；
    (4)SEQ ID NO:7、8、41；
    (5)SEQ ID NO:7、8、42；
    (6)SEQ ID NO:7、8、43；
    (7)SEQ ID NO:37、38、41；
    (8)SEQ ID NO:44、38、41；
    (9)SEQ ID NO:7、8、9。
  2. 根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述可变区还包含：鼠源或人源FR区。
  3. 根据权利要求2所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述FR区的序列为鼠源；所述重链可变区的序列如SEQ ID NO:2所示或与其具有至少85％序列同一性，所述轻链可变区的序列如SEQ ID NO:6所示或与其具有至少85％序列同一性。
  4. 根据权利要求2所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述人源FR区包括：重链FR区序列和轻链FR区序列；所述重链FR区序列来源于人种系重链IGHV1-18及IGHJ2*01的组合序列，包括人种系重链IGHV1-18的FR1、FR2、FR3区和IGHJ2*01的FR4区；所述轻链FR区序列来源于人种系轻链IGKV4-1及IGKJ2*01的组合序列，包括人种系轻链IGKV4-1的FR1、FR2、FR3区和IGKJ2*01的FR4区。
  5. 根据权利要求2所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述重链可变区的序列如SEQ ID NO:17所示或与其具有至少85％序列同一性；所述轻链可变区的序列选自SEQ ID NO:16、18、19、20、21、22、23、24、25中任意一项，或与其具有至少85％序列同一性。
  6. 根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述抗SIRPα抗 体或其抗原结合片段还包含：选自人源IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区；以及选自人源κ、λ链或其变体的轻链恒定区。
  7. 根据权利要求6所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述重链恒定区包含：Fc片段或其变体；所述Fc片段的变体来源于IgG1，根据EU计数，包括突变位点：L234A、L235A、K338A。
  8. 根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，所述抗SIRPα抗体或其抗原结合片段的重链序列如SEQ ID NO:26所示，或与其具有至少85％序列同一性。
  9. 根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，其为单克隆抗体、双特异抗体、或多特异抗体，或者，所述的抗体或其抗原结合片段用于制备抗体药物偶联物。
  10. 根据权利要求1所述的抗SIRPα抗体或其抗原结合片段，其特征在于，其结构形式为Fab、F(ab’)2、Fv、或ScFv。
  11. 一种药物组合物，其含有权利要求1至10中任意一项所述的抗SIRPα抗体或其抗原结合片段，以及一种或多种药学上可接受的载体、稀释剂或赋形剂。
  12. 一种核酸分子，其编码权利要求1至10中任意一项所述的抗SIRPα抗体或其抗原结合片段。
  13. 权利要求1至10中任意一项所述的抗SIRPα抗体或其抗原结合片段在制备用于抑制或治疗疾病、病症或状况的药物中的用途。
  14. 根据权利要求13所述的用途，其特征在于，所述疾病、病症或状况包括：癌症、实体瘤、慢性感染、炎性疾病、多发性硬化、自身免疫性疾病、神经系统疾病、脑损伤、神经损伤、红细胞增多症、血色素沉着病、创伤、感染性休克、纤维化、动脉粥样硬化、肥胖症、II型糖尿病、移植功能障碍或关节炎；所述癌症选自肛门癌、阑尾癌、星形细胞瘤、基底细胞癌、胆囊癌、胃癌、肺癌、支气管癌、骨癌、肝胆管癌、胰腺癌、乳腺癌、肝癌、卵巢癌、睾丸癌、肾癌、肾盂和输尿管癌、唾液腺癌、小肠癌、尿道癌、膀胱癌、头颈癌、脊柱癌、脑癌、宫颈癌、子宫癌、子宫内膜癌、结肠癌、结直肠癌、直肠癌、食道癌、胃肠道癌、皮肤癌、前列腺癌、垂体癌、阴道癌、甲状腺癌、喉癌、胶质母细胞瘤、黑素瘤、骨髓增生异常综合征、肉瘤、畸胎瘤、慢性淋巴细胞白血病(CLL)、慢性髓性白血病(CML)、急性淋巴细胞白血病(ALL)、急性髓性白血病(AML)、霍奇金淋巴瘤、非霍奇金淋巴瘤、多发性骨髓瘤、T或B细胞淋巴瘤、胃肠道间质瘤、软组织肿瘤、肝细胞癌或腺癌。
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