WO2021170077A1 - 抗cd47抗体和抗vegf抗体的组合及其用途 - Google Patents

抗cd47抗体和抗vegf抗体的组合及其用途 Download PDF

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WO2021170077A1
WO2021170077A1 PCT/CN2021/078075 CN2021078075W WO2021170077A1 WO 2021170077 A1 WO2021170077 A1 WO 2021170077A1 CN 2021078075 W CN2021078075 W CN 2021078075W WO 2021170077 A1 WO2021170077 A1 WO 2021170077A1
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seq
antibody
cancer
variable region
chain variable
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PCT/CN2021/078075
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English (en)
French (fr)
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陈炳良
曹蕾
伍伟伟
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信达生物制药(苏州)有限公司
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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention relates to the field of medicine. Specifically, the present invention relates to an anti-tumor drug combination and its use, in particular to a drug combination comprising an anti-CD47 antibody and an anti-VEGF antibody and its use in the prevention and/or treatment of tumors and/or cancers.
  • CD47 Cluster of Differentiation 47
  • IAP integrin-associated protein
  • anti-CD47 antibodies have been reported, and they have been developed for the treatment of various tumors and/or cancers.
  • WO2019042285A1 a series of recombinant fully human monoclonal anti-CD47 antibodies are disclosed.
  • the anti-CD47 antibody was administered to tumor-bearing mice produced by inoculating NOD-SCID mice with human Burkitt’s lymphoma Raji cells.
  • the results showed that the anti-CD47 antibody can improve the phagocytosis of macrophages and has significant effects. Its anti-tumor activity can significantly inhibit the growth of tumors and even make tumors disappear completely.
  • VEGF is an important factor in the process of angiogenesis and is over-expressed pathologically in the endothelial cells of most human tumors. VEGF greatly promotes the division, proliferation and migration of vascular endothelial cells by binding to its receptor VEGFR, improves vascular permeability, inhibits tumor cell apoptosis, and provides a good microenvironment for tumor growth and metastasis.
  • Anti-VEGF antibody can specifically bind to VEGF, by blocking the binding of VEGF to its receptors on the surface of vascular endothelial cells, blocking the conduction of PI3K-AKt/PKB and Ras-Raf-MEK-ERK signaling pathways, thereby inhibiting vascular endothelial cells
  • the growth, proliferation and migration as well as angiogenesis reduce the permeability of blood vessels, block the blood supply of tumor tissues, inhibit the proliferation and metastasis of tumor cells, and induce tumor cell apoptosis, so as to achieve the anti-tumor therapeutic effect.
  • Bevacizumab (trade name Avastin) has been approved for the market.
  • the present invention meets the above-mentioned needs by providing a drug combination of anti-CD47 antibody and anti-VEGF antibody.
  • the present invention provides a pharmaceutical combination comprising (i) an anti-CD47 antibody and/or an antigen-binding fragment thereof; and (ii) an anti-VEGF antibody and/or an antigen-binding fragment thereof.
  • the anti-CD47 antibody in the pharmaceutical combination of the present invention may be any anti-CD47 antibody, as long as it can specifically bind to CD47, thereby improving or enhancing the phagocytosis of macrophages.
  • the anti-CD47 antibody is a fully human monoclonal anti-CD47 antibody.
  • the anti-CD47 antibody comprises 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and 3 CDRs in the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2 3 CDRs.
  • the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 of the anti-CD47 antibody in the drug combination of the present invention are SYYWS (SEQ ID NO: 3) HCDR1, YIYYSGSTNYNPSLKS (SEQ ID NO: 4) shown in HCDR2 and GKTGSAA (SEQ ID NO: 5) shown in HCDR3; wherein the light chain variable region amino acid shown in SEQ ID NO: 2
  • the 3 CDRs in the sequence are LCDR1 shown in RASQGISRWLA (SEQ ID NO: 6) according to the Kabat numbering scheme, LCDR1 shown in AASSLQS (SEQ ID NO: 7) and LCDR3 shown in QQTVSFPIT (SEQ ID NO: 8) ; Or the anti-CD47 antibody in the drug combination of the present invention.
  • the three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:1 are GSISSYYWS (SEQ ID NO :9) HCDR1, YIYYSGSTNYNPSLKS (SEQ ID NO: 4) shown in HCDR2 and ARGKTGSAA (SEQ ID NO: 10) shown in HCDR3; wherein the light chain variable region shown in SEQ ID NO: 2
  • the 3 CDRs in the amino acid sequence are the LCDR1 shown in RASQGISRWLA (SEQ ID NO: 6) determined according to the combination of Kabat, AbM, and IMGT numbering schemes, LCDR1 shown in AASSLQS (SEQ ID NO: 7), and LCDR2 and QQTVSFPIT (SEQ ID NO) :8) LCDR3 shown in Table A below.
  • the anti-CD47 antibody in the pharmaceutical combination of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:1 or has at least 90% thereof, A sequence with 95%, 98% or 99% identity, and the light chain variable region includes the sequence of SEQ ID NO: 2 or a sequence with at least 90%, 95%, 98% or 99% identity with it.
  • the anti-CD47 antibody comprises SEQ ID NO: 11 or a heavy chain sequence having at least 90%, 95%, 98% or 99% identity with SEQ ID NO: 12 or at least 90% with SEQ ID NO: 12, 95%, 98% or 99% identity of the light chain sequence.
  • the anti-VEGF antibody in the pharmaceutical combination of the present invention may be any anti-VEGF antibody, as long as it can specifically bind to VEGF and thereby block or inhibit the binding of VEGF to its receptor VEGFR.
  • the anti-VEGF monoclonal antibody is Ranibizumab, brolucizumab, varisacumab or Bevacizumab and biological analogs thereof.
  • the anti-VEGF monoclonal antibody is bevacizumab.
  • the anti-VEGF antibody in the drug combination of the present invention includes 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable shown in SEQ ID NO: 14 3 CDRs in the amino acid sequence of the region.
  • the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 13 of the anti-VEGF antibody in the drug combination of the present invention are NYGMN (SEQ ID NO: 15) HCDR1, HCDR2 shown by WINTYTGEPTYAADFKR (SEQ ID NO: 16) and HCDR3 shown by YPHYYGSSHWYFDV (SEQ ID NO: 17); wherein the amino acid of the light chain variable region shown by SEQ ID NO: 14
  • the 3 CDRs in the sequence are LCDR1 shown in SASQDISNYLN (SEQ ID NO: 18) according to the Kabat numbering scheme, LCDR1 shown in FTSSLHS (SEQ ID NO: 19), and LCDR3 shown in QQYSTVPWT (SEQ ID NO: 20) Or wherein the three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 13 are HCDR1 and NTYTGE (SEQ ID NO: GYTFTNY (SEQ ID NO:
  • the anti-VEGF antibody in the pharmaceutical combination of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 13 or has at least 90% thereof, 95%, 98%, or 99% identical sequence, and the light chain variable region comprises the sequence of SEQ ID NO: 14 or a sequence with at least 90%, 95%, 98% or 99% identity;
  • the anti-VEGF antibody in the drug combination of the present invention comprises SEQ ID NO: 23 or a heavy chain sequence having at least 90%, 95%, 98% or 99% identity thereto, and SEQ ID NO: 24 or a light chain sequence with at least 90%, 95%, 98% or 99% identity with it.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutical combination of an anti-CD47 antibody and an anti-VEGF antibody, and a pharmaceutically acceptable carrier.
  • the present invention provides a kit of medicines comprising an anti-CD47 antibody and an anti-VEGF antibody drug combination.
  • the kit is in the form of a drug dosage unit, so that the drug can be provided according to the dosage regimen. Dosage unit.
  • kit of the present invention contains in the same package:
  • -A second container containing anti-VEGF antibodies and/or antigen-binding fragments thereof for parenteral administration.
  • the present invention provides the use of a drug combination of anti-CD47 antibody and anti-VEGF antibody for the preparation of drugs for the prevention and/or treatment of tumors and/or cancers.
  • the tumor and/or cancer are not particularly limited.
  • the tumor and/or cancer may be various blood cancers and solid tumors, for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia, acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), Multiple myeloma (MM), lymphoma, breast cancer, stomach cancer, lung cancer, esophageal cancer, bowel cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, bladder cancer, glioma, melanoma, skin cancer Wait.
  • the tumor and/or cancer is a solid tumor, and further is breast cancer or skin cancer.
  • the present invention provides the use of anti-CD47 antibodies and/or antigen-binding fragments thereof in combination with anti-VEGF antibodies and/or antigen-binding fragments to prepare drugs for preventing and/or treating tumors and/or cancers combination.
  • the anti-CD47 antibody is the anti-CD47 antibody described in the first aspect of the invention.
  • the anti-VEGF antibody is the anti-VEGF antibody described in the first aspect of the invention.
  • the tumor and/or cancer are not particularly limited.
  • the tumor and/or cancer may be various blood cancers and solid tumors, for example, acute myelogenous leukemia (AML), chronic myelogenous leukemia, acute lymphocytic leukemia (ALL), non-Hodgkin lymphoma (NHL), Multiple myeloma (MM), lymphoma, breast cancer, stomach cancer, lung cancer, esophageal cancer, bowel cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, bladder cancer, glioma, melanoma, skin cancer Wait.
  • AML acute myelogenous leukemia
  • ALL acute lymphocytic leukemia
  • NHL non-Hodgkin lymphoma
  • MM Multiple myeloma
  • lymphoma lymphoma
  • breast cancer stomach cancer
  • lung cancer esophageal cancer
  • bowel cancer ovarian cancer
  • cervical cancer ovarian cancer
  • kidney cancer pancreatic cancer
  • bladder cancer glioma, mel
  • the present invention provides a method for preventing and/or treating tumors and/or cancers, the method comprising administering to a subject in need an effective amount of the pharmaceutical combination, pharmaceutical composition or kit of the present invention Pill box.
  • the drug combination of the anti-CD47 antibody and the anti-VEGF antibody of the present invention can provide better anti-tumor efficacy.
  • the anti-CD47 antibody in the drug combination is the fully human monoclonal anti-CD47 antibody disclosed in WO2019042285A1.
  • the fully human monoclonal anti-CD47 antibody disclosed in WO2019042285A1 is combined with the anti-VEGF antibody described in US Patent No. 6,884,879 or WO 2005/044853, preferably with Bevacizumab.
  • the anti-tumor efficacy obtained by the drug combination of the present invention is significantly better than the anti-tumor efficacy achieved by the single-drug administration of the anti-CD47 antibody or the anti-VEGF antibody.
  • Figure 1 Shows the weight change rate of MDA-MB-231 tumor-bearing mice in each administration group.
  • Figure 2 shows the changes in tumor volume of MDA-MB-231 tumor-bearing mice in each administration group.
  • Figure 3A shows the effect of anti-CD47 antibody 1 mg/kg combined with anti-VEGF antibody 0.25 mg/kg on the change of tumor volume in tumor-bearing mice compared with single drug administration.
  • Figure 3B Shows the effect of anti-CD47 antibody 1 mg/kg combined with anti-VEGF antibody 0.5 mg/kg on the change of tumor volume in tumor-bearing mice compared with single drug administration.
  • Figure 4A shows the effect of the administration of 2 mg/kg of anti-CD47 antibody in combination with 0.25 mg/kg of anti-VEGF antibody on the change of tumor volume in tumor-bearing mice compared with single-drug administration.
  • Figure 4B shows the effect of the anti-CD47 antibody 2 mg/kg combined with the anti-VEGF antibody 0.5 mg/kg on the change of tumor volume in tumor-bearing mice compared with single drug administration.
  • Figure 5 shows the weight change rate of A-431 tumor-bearing mice in each administration group.
  • Figure 6 Shows the changes in tumor volume of A-431 tumor-bearing mice in each administration group.
  • Figure 7 Shows the effect of anti-CD47 antibody 0.1 mg/kg combined with anti-VEGF antibody 1 mg/kg on the change of tumor volume in tumor-bearing mice compared with single drug administration.
  • Figure 8 Shows the effect of anti-CD47 antibody 0.3 mg/kg combined with anti-VEGF antibody 1 mg/kg on the change of tumor volume in tumor-bearing mice compared with single drug administration.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” when used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence when referring to an antibody variable region that "comprises” a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.
  • the terms “whole antibody”, “full-length antibody”, “full antibody” and “whole antibody” are used interchangeably herein to refer to at least two heavy chains (H) and two Light chain (L) glycoprotein.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three structural domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • variable region refers to the domain of the heavy chain of the antibody or the domain of the light chain that participates in the binding of the antibody to the antigen.
  • the variable domains of the heavy and light chains of natural antibodies usually have similar structures, where each variable domain contains four conserved framework regions (FR) and three complementarity determining regions. (See, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. 91 (2007)). A single VH or VL domain may be sufficient to give antigen binding specificity.
  • VH or VL domains from antibodies that bind to a specific antigen can be used to isolate antibodies that bind to the antigen to screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • CDR region or “CDR” or “hypervariable region” (herein can be used interchangeably with hypervariable region “HVR”) is an antibody variable domain that is hypervariable in sequence and A structurally defined loop ("hypervariable loop") and/or a region containing antigen contact residues ("antigen contact point”) is formed.
  • CDR is mainly responsible for binding to antigen epitopes.
  • the CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus.
  • the CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.
  • CDR of the antibody of the present invention can be artificially evaluated and determined according to any scheme in the art or a combination thereof.
  • the term "CDR” or "CDR sequence” encompasses CDR sequences determined in any of the above-mentioned ways.
  • Antibodies with different specificities have different CDRs.
  • CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding.
  • the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding.
  • the minimum binding unit can be a sub-portion of the CDR.
  • the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia, etc. can be replaced by conserved amino acid residues.
  • variable region residues in an antibody refers to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues).
  • the sequences are aligned for optimal comparison purposes (for example, the first and second amino acid sequences or nucleic acid sequences may be used for optimal alignment. Gaps can be introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the compared reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at this position.
  • Mathematical algorithms can be used to achieve sequence comparison between two sequences and calculation of percent identity.
  • the Needlema and Wunsch ((1970) J.Mol.Biol.48:444-453) algorithm (at http://www.gcg.com) that has been integrated into the GAP program of the GCG software package is used. Available), use Blossum 62 matrix or PAM250 matrix and gap weight 16, 14, 12, 10, 8, 6 or 4 and length weight 1, 2, 3, 4, 5 or 6, to determine the difference between two amino acid sequences Percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 80 are used. Length weights 1, 2, 3, 4, 5, or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and a parameter set that should be used unless otherwise specified) is a Blossom 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • antigen-binding fragment is a part or section of a complete or complete antibody that has fewer amino acid residues than a complete or complete antibody, which can bind to the antigen or compete with the complete antibody (ie, the complete antibody from which the antigen-binding fragment is derived) Binding antigen.
  • the antigen-binding fragment can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of the intact antibody.
  • Antigen-binding fragments include but are not limited to Fab, Fab', F(ab') 2 , Fv, single chain Fv, diabody, single domain antibody (sdAb).
  • the Fab fragment is a monovalent fragment composed of VL, VH, CL and CH1 domains.
  • the Fab fragment can be obtained by digesting a complete antibody with papain.
  • pepsin digests the complete antibody under the disulfide bond in the hinge region to produce F(ab') 2 , which is a dimer of Fab' and a bivalent antibody fragment.
  • F(ab') 2 can be reduced by breaking the disulfide bond in the hinge region under neutral conditions, thereby converting F(ab') 2 dimers into Fab' monomers.
  • the Fab' monomer is basically a Fab fragment with a hinge region (for a more detailed description of other antibody fragments, please refer to: Fundamental Immunology, edited by WEPaul, Raven Press, NY (1993)).
  • the Fv fragment is composed of the VL and VH domains of one arm of the antibody.
  • the two domains VL and VH of the Fv fragment are encoded by independent genes, using recombination methods, they can be connected by a synthetic linking peptide that can produce these two domains as a single protein chain.
  • the VL and VH regions in a single protein chain are paired to form a single chain Fv.
  • the antibody fragments can be obtained by chemical methods, recombinant DNA methods or protease digestion methods.
  • monoclonal antibody refers to a preparation of antibody molecules having a single amino acid composition, and does not refer to the method by which it is produced.
  • Monoclonal antibodies or antigen-binding fragments thereof can be produced, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology such as CDR grafting, or a combination of such or other techniques known in the art.
  • humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • the humanized antibody comprises all or substantially all of the CDRs corresponding to those of the non-human antibody and all or substantially all of the FR regions corresponding to those of the human antibody.
  • the humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.
  • Human antibody “fully human antibody”, and “fully human antibody” are used interchangeably in this application and refer to an antibody having an amino acid sequence corresponding to the amino acid sequence of such an antibody. Produced from human or human cells or derived from non-human sources, which utilize human antibody libraries or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.
  • the term "specifically binds" or "bind” when referring to an antigen and an antibody means that the antibody forms a complex with an antigen that is relatively stable under physiological conditions.
  • Methods for determining whether an antibody specifically binds to an antigen include, for example, surface plasmon resonance assays, MSD assays (Estep, P. et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning, MAbs, 2013.5(2): p.270-278), ForteBio affinity assay (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs, 2013.5(2): p.270-8) and so on.
  • the anti-CD47 antibody that "specifically binds" to CD47 in the pharmaceutical combination of the present invention is measured in the ForteBio affinity assay to at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably The affinity constant of about 10 9 M -1 , 10 10 M -1 or stronger binds to CD47 on the surface of tumor cells, thereby blocking or inhibiting the binding of CD47 to SIRP ⁇ on the surface of macrophages, and promoting the macrophage of tumor tissue infiltration area. Phagocytosis of tumor cells by phages.
  • the anti-VEGF antibody that "specifically binds" VEGF in the pharmaceutical combination of the present invention is measured in the ForteBio affinity assay to at least about 10 7 M -1 , preferably about 10 8 M -1 and more.
  • an affinity constant of about 10 9 M -1 , 10 10 M -1 or greater binds to vascular endothelial cell growth factor (VEGF), thereby blocking or inhibiting the binding of VEGF to its receptor on the surface of vascular endothelial cells and subsequent Signal conduction.
  • anti-CD47 antibody refers to antibodies that can specifically bind to CD47 protein or its protein with sufficient affinity. Fragments and block or inhibit the binding of CD47 to SIRP ⁇ on the surface of macrophages, so that the antibody can be used as a preventive and/or therapeutic agent targeting CD47.
  • anti-VEGF antibody refers to antibodies that can specifically bind to VEGF protein or its protein with sufficient affinity. Fragments and block or inhibit the binding of VEGF to its receptor on the surface of vascular endothelial cells, so that the antibody can be used as a preventive and/or therapeutic agent targeting VEGF.
  • biosimilar is also called biosimilar or biosimilar. It is highly similar to the approved reference biological product, although there is a small difference in the inactive components; but it is clinically and the reference biological product. Compared with the products, there are no significant differences in safety, purity and efficacy.
  • non-fixed combination refers to non-fixed combination products or fixed combination products, including but not limited to kits and pharmaceutical compositions.
  • non-fixed combination means that the active ingredients (for example, (i) anti-CD47 antibodies and/or antigen-binding fragments thereof, and (ii) anti-VEGF antibodies and/or antigen-binding fragments thereof) are simultaneously and without Specific time limits or sequential administration to the subject at the same or different time intervals, wherein such administration provides prophylactic or therapeutically effective levels of the two antibodies in the subject.
  • the anti-CD47 antibody and/or antigen-binding fragment thereof and the anti-VEGF antibody and/or antigen-binding fragment thereof used in the pharmaceutical combination are administered at a level not exceeding the level when they are used alone.
  • the term "fixed combination" means that the two antibodies in the pharmaceutical combination of the present invention are simultaneously administered to a subject in the form of a single entity.
  • the dosage and/or time interval of the two antibodies in the drug combination of the present invention are selected so that the combined use of each antibody can produce an effect greater than that achieved by using any antibody alone in the treatment of diseases or disorders.
  • Each antibody may be in the form of a separate preparation, and the preparation form may be the same or different.
  • administration refers to the physical introduction of each active ingredient of the pharmaceutical combination of the present invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art.
  • the administration route of each antibody in the pharmaceutical combination of the present invention includes oral, intravenous (for example, infusion (also called drip) or injection), intramuscular, subcutaneous, intraperitoneal, spinal, topical or other parenteral administration routes.
  • parenteral administration refers to modes of administration other than gastrointestinal and local administration, including, without limitation, intramuscular, intraarterial, intravenous, intrathecal, intralymphatic, intralesional, intrasaccular, and intraorbital , Intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions, as well as in vivo electroporation.
  • each antibody in the pharmaceutical combination of the present invention can be formulated into capsules, tablets, injections (including infusions or injections), syrups, sprays, lozenges, liposomes or suppositories, etc.
  • a therapeutically effective amount refers to an amount that is effective to achieve the desired preventive and/or therapeutic results at a required dose and for a required period of time.
  • the preventive and/or therapeutically effective amount can be varied according to various factors such as disease state, age, sex, and weight of the subject.
  • a therapeutically effective amount is any amount whose toxic or harmful effects are not as good as the therapeutically beneficial effects.
  • a "therapeutically effective amount” preferably inhibits a measurable parameter (such as tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more. Preferably at least about 80%.
  • prophylactically effective amount refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since the prophylactic dose is used in the subject before or at the early stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.
  • dose is the amount of a drug that elicits a preventive and/or therapeutic effect. Unless otherwise stated, the dosage is related to the amount of the free form of the drug. If the drug is in the form of a pharmaceutically acceptable salt, the amount of the drug is increased in proportion to the amount of the drug in the free form. For example, the dosage will be stated on the product packaging or product information sheet.
  • inhibitor means that a given molecule (e.g. (i) anti-CD47 antibody and/or antigen-binding fragment thereof; (ii) anti-VEGF antibody and/or antigen-binding fragment thereof) enables certain parameters (e.g., CD47 activity; VEGFR activity) ) Reduction.
  • the term includes inhibition of activity of at least 5%, 10%, 20%, 30%, 40% or more. Therefore, the suppression does not have to be 100%.
  • treatment refers to alleviating the disease (ie, slowing down or preventing or reducing the development of the disease or at least one clinical symptom thereof), preventing or delaying the onset or development or progression of the disease.
  • prevention includes the suppression or delay of the occurrence or frequency of the occurrence or occurrence of a disease or condition or its symptoms, which usually refers to the administration of a drug before the occurrence or occurrence of the symptoms or symptoms, especially before the occurrence of the symptoms or symptoms in subjects at risk .
  • subject refers to mammals and non-mammals. Mammal refers to any member of the mammalian class, including but not limited to: humans; non-human primates, cows, horses, sheep, pigs, rabbits, dogs, cats, etc.
  • subject does not limit a specific age or gender. In some embodiments, the subject is a human.
  • the pharmaceutical combination of the present invention comprises (i) an anti-CD47 antibody and/or an antigen-binding fragment thereof; and (ii) an anti-VEGF antibody and/or an antigen-binding fragment thereof.
  • the anti-CD47 antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention is measured at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M as measured in the ForteBio affinity assay.
  • -1 , 10 10 M -1 or stronger affinity constants specifically bind to CD47 on the surface of tumor cells, thereby blocking or inhibiting the binding of CD47 to SIRP ⁇ on the surface of macrophages, and promoting the pairing of macrophages in the infiltrating area of tumor tissue Phagocytosis of tumor cells.
  • the anti-CD47 antibody in the drug combination of the present invention includes 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2 3 CDRs in the amino acid sequence.
  • the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 1 of the anti-CD47 antibody in the drug combination of the present invention are SYYWS (SEQ ID NO: 3) HCDR1, YIYYSGSTNYNPSLKS (SEQ ID NO: 4) shown in HCDR2 and GKTGSAA (SEQ ID NO: 5) shown in HCDR3; wherein the light chain variable region amino acid shown in SEQ ID NO: 2
  • the 3 CDRs in the sequence are LCDR1 shown in RASQGISRWLA (SEQ ID NO: 6) according to the Kabat numbering scheme, LCDR1 shown in AASSLQS (SEQ ID NO: 7) and LCDR3 shown in QQTVSFPIT (SEQ ID NO: 8) ; Or the anti-CD47 antibody in the drug combination of the present invention.
  • the three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO:1 are GSISSYYWS (SEQ ID NO :9) HCDR1, YIYYSGSTNYNPSLKS (SEQ ID NO: 4) shown in HCDR2 and ARGKTGSAA (SEQ ID NO: 10) shown in HCDR3; wherein the light chain variable region shown in SEQ ID NO: 2
  • the 3 CDRs in the amino acid sequence are the LCDR1 shown in RASQGISRWLA (SEQ ID NO: 6) determined according to the combination of Kabat, AbM, and IMGT numbering schemes, LCDR1 shown in AASSLQS (SEQ ID NO: 7), and LCDR2 and QQTVSFPIT (SEQ ID NO) :8) LCDR3 shown in Table A.
  • the anti-CD47 antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:1 or has at least 90%, 95%, 98% or A sequence with 99% identity, and the light chain variable region includes the sequence of SEQ ID NO: 2 or a sequence with at least 90%, 95%, 98% or 99% identity with it.
  • the anti-CD47 antibody comprises SEQ ID NO: 11 or a heavy chain sequence having at least 90%, 95%, 98% or 99% identity with SEQ ID NO: 12 or at least 90% with SEQ ID NO: 12, 95%, 98% or 99% identity of the light chain sequence.
  • the anti-CD47 antibody is the anti-CD47 antibody ADI-26630 disclosed in WO2019042285A1.
  • the anti-VEGF antibody in the pharmaceutical combination of the present invention is an antibody that can bind to VEGF and thereby block or inhibit the binding of VEGF to its receptor VEGFR.
  • the anti-VEGF antibody described in US Patent No. 6,884,879 and WO 2005/044853.
  • the anti-VEGF antibody is an anti-VEGF monoclonal antibody.
  • the anti-VEGF monoclonal antibody is selected from the group consisting of Ranibizumab, brolucizumab, varisacumab, Bevacizumab and their biological analogs.
  • the anti-VEGF monoclonal antibody is bevacizumab.
  • the anti-VEGF antibody and/or antigen-binding fragment thereof is measured at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 as measured in the ForteBio affinity assay.
  • the affinity constant of M -1 , 10 10 M -1 or stronger binds to vascular endothelial cell growth factor (VEGF), thereby blocking or inhibiting the binding of VEGF to its receptor VEGFR on the surface of vascular endothelial cells and subsequent signal transduction.
  • VEGF vascular endothelial cell growth factor
  • the anti-VEGF antibody in the drug combination of the present invention comprises 3 CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 13 and the light chain variable region shown in SEQ ID NO: 14 3 CDRs in the amino acid sequence.
  • the heavy chain variable region of an exemplary anti-VEGF antibody is the heavy chain variable region of an exemplary anti-VEGF antibody.
  • the three CDRs in the heavy chain variable region amino acid sequence shown in SEQ ID NO: 13 of the anti-VEGF antibody in the drug combination of the present invention are NYGMN (SEQ ID NO: 15) HCDR1, HCDR2 shown by WINTYTGEPTYAADFKR (SEQ ID NO: 16) and HCDR3 shown by YPHYYGSSHWYFDV (SEQ ID NO: 17); wherein the amino acid of the light chain variable region shown by SEQ ID NO: 14
  • the 3 CDRs in the sequence are LCDR1 shown in SASQDISNYLN (SEQ ID NO: 18) according to the Kabat numbering scheme, LCDR1 shown in FTSSLHS (SEQ ID NO: 19), and LCDR3 shown in QQYSTVPWT (SEQ ID NO: 20) Or wherein the three CDRs in the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 13 are HCDR1 and NTYTGE (SEQ ID NO: GYTFTNY (SEQ ID NO:
  • the anti-VEGF antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 13 or has at least 90%, 95%, 98% or 99% therewith.
  • the sequence of identity, and the variable region of the light chain includes the sequence of SEQ ID NO: 14 or a sequence that is at least 90%, 95%, 98% or 99% identical to it.
  • the anti-VEGF antibody comprises SEQ ID NO: 23 or a heavy chain sequence with at least 90%, 95%, 98% or 99% identity and SEQ ID NO: 24 or at least 90% therewith, 95%, 98% or 99% identity of the light chain sequence.
  • the present invention provides the aforementioned pharmaceutical combination of the present invention for preventing and/or treating the severity of at least one symptom or indication of cancer in a subject or inhibiting the growth of cancer cells.
  • the present invention provides a method of preventing or treating cancer, which comprises administering an effective amount of the pharmaceutical combination of the present invention to a subject in need.
  • the effective amount includes a preventive effective amount and a therapeutically effective amount.
  • the present invention provides the use of the aforementioned pharmaceutical combination of the present invention in the preparation of drugs for the prevention or treatment of cancer.
  • the cancer of the present invention includes solid tumors and hematological malignancies, for example, acute myeloid leukemia (AML), chronic myelogenous leukemia, acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple bone marrow Tumor (MM), lymphoma, breast cancer, stomach cancer, lung cancer, esophageal cancer, bowel cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, bladder cancer, glioma, melanoma, skin cancer.
  • the tumor and/or cancer is a solid tumor, and further is breast cancer or skin cancer.
  • the anti-CD47 antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention can be administered to a subject in need in one or more doses, where in the case of administering multiple doses, 3 days after the previous dose, The next dose is administered at 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 10 weeks.
  • a dose of the anti-CD47 antibody and/or antigen-binding fragment thereof can be selected from 0.1-10 mg/kg of the subject's body weight (e.g., 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg or 10mg/kg).
  • each dose contains 50-500 mg of anti-CD47 antibody and/or antigen-binding fragments thereof, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg or 500 mg of anti-CD47 antibody and / Or its antigen binding fragment.
  • the anti-VEGF antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention can be administered to a subject in need in one or more doses, wherein in the case of administering multiple doses, 3 days after the previous dose, The next dose is administered at 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, or 10 weeks.
  • a dose of the anti-VEGF antibody and/or antigen-binding fragment thereof can be selected from 0.1-10 mg/kg of the subject's body weight (e.g., 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg or 10mg/kg).
  • each dose contains 50-500 mg of anti-VEGF antibody and/or antigen-binding fragments thereof, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg of anti-VEGF antibody and / Or its antigen binding fragment.
  • the pharmaceutical combination of the present invention can be any dosage form known to those skilled in the art, such as tablets, capsules, granules, syrups, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions , Solutions, syrups, aerosols, ointments, creams and injections, etc.
  • the anti-CD47 antibody and/or its antigen-binding fragment and the anti-VEGF antibody and/or its antigen-binding fragment may each be in a separate dosage form, and their dosage forms may be different or the same.
  • the anti-CD47 antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention is an intravenous administration form, such as an intravenous injection.
  • the anti-CD47 antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention is an intraperitoneal administration dosage form, such as an intraperitoneal injection.
  • the anti-VEGF antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention is an intravenous administration dosage form, such as an intravenous injection.
  • the anti-VEGF antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention is an intraperitoneal administration dosage form, such as an intraperitoneal injection.
  • the anti-CD47 antibody and/or its antigen-binding fragment and the anti-VEGF antibody and/or its antigen-binding fragment in the pharmaceutical combination of the present invention can be administered separately, simultaneously or sequentially.
  • the anti-CD47 antibody and/or antigen-binding fragment thereof in the pharmaceutical combination of the present invention may be administered before, at the same time, or after the start of administration of the anti-VEGF antibody and/or antigen-binding fragment thereof.
  • the pharmaceutical combination of the present invention results in an increase, preferably a synergistic increase in the inhibition of tumor growth effect.
  • the drug combination of the present invention results in tumor growth being inhibited by at least About 50%, about 60%, about 70%, about 80%, about 90%, about 100%.
  • administration of the drug combination of the invention results in increased tumor regression, tumor shrinkage and/or disappearance.
  • the present invention compared with untreated subjects or with monotherapy with anti-CD47 antibodies and/or antigen-binding fragments thereof or monotherapy with anti-VEGF antibodies and/or antigen-binding fragments thereof, the present invention
  • the drug combination prevents tumor recurrence in the subject and/or increases the duration of survival, for example, the duration of survival is increased by more than 15 days, more than 1 month, more than 3 months, more than 6 months, more than 12 months Months, more than 18 months, more than 24 months, more than 36 months, or more than 48 months.
  • the drug combination of the present invention can increase progression-free survival or overall survival.
  • the drug combination of the present invention can reduce adverse events caused by the administration of each single agent, for example, hematological toxicity, non-hematological toxicity, or other toxic reactions.
  • Another object of the present invention is to provide a kit of medicines comprising the drug combination of the present invention, preferably the kit is in the form of a drug dosage unit, so that the drug dosage unit can be provided according to a dosing schedule.
  • kit of the present invention contains in the same package:
  • -A second container containing anti-VEGF antibodies and/or antigen-binding fragments thereof for parenteral administration.
  • MDA-MB-231 cells (human breast cancer cells) are ATCC products, the article number is HTB-26, and the batch number is 62235654.
  • Human PBMC human peripheral blood mononuclear cells
  • AllCells the batch number is 3024811.
  • SPF-grade NOG female mice (15-18g, 35-41 days old) were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and the certificate number was 1100111911046485.
  • h-IgG is an Equitech-Bio product, the article number is SLH56, and the batch number is 160308-02.
  • Anti-CD47 antibodies are antibodies that block or inhibit the interaction of CD47 ligand SIRP ⁇ with CD47 by binding to CD47, such as any anti-CD47 monoclonal antibody disclosed in Patent Publication No. WO2019042285A1.
  • This example illustrates the use of the anti-CD47 antibody ADI-26630 disclosed in WO2019042285A1. It should be noted that those skilled in the art can also use other anti-CD47 antibodies, including but not limited to other anti-CD47 antibodies disclosed in WO2019042285A1 for use in the pharmaceutical combination of the present invention.
  • amino acid sequences of the CDR region, the light chain variable region and the heavy chain variable region, the light chain and the heavy chain of the anti-CD47 antibody ADI-26630 are given in the "Detailed Description of the Invention" of the present application.
  • the anti-CD47 antibody in the pharmaceutical combination of the present invention can be produced by any antibody preparation technique known to those skilled in the art.
  • the expression and purification of anti-CD47 antibodies in yeast or CHO-S cells are exemplified.
  • Yeast-based antibody presentation library (Adimab) was amplified according to existing methods (WO2009036379; WO 2010105256; WO2012009568), and the diversity of each library reached 1 ⁇ 10 9 .
  • the first two rounds of screening used Miltenyi's MACS system for magnetically activated cell sorting.
  • FACS washing buffer phosphate buffer, containing 0.1% bovine serum protein
  • the buffer containing 100 nM biotin Labeled CD47 antigen (Acro Biosystems, catalog number CD7-H5227-1 mg).
  • LC-FITC FITC-labeled goat anti-human immunoglobulin F(ab') kappa chain antibody, Southern Biotech
  • SA-633 streptavidin-633, Molecular Probes
  • SA-PE streptavidin-phycoerythrin, Sigma
  • the yeast cells expressing the anti-CD47 antibody obtained by the screening were induced by shaking at 30° C. for 48 hours to express the anti-CD47 antibody. After the induction, the yeast cells were removed by centrifugation at 1300 rpm for 10 min, and the supernatant was harvested. Protein A was used to purify the anti-CD47 antibody in the supernatant, eluted with pH2.0 acetic acid solution, and the anti-CD47 antibody was harvested with a purity of >95%. Use papain digestion and purification with KappaSelect (GE Life Medical Group) to obtain the corresponding Fab fragments.
  • KappaSelect GE Life Medical Group
  • the kit produces a CHO-S cell line expressing antibodies.
  • the constructed pCHO1.0 plasmid was transformed into the CHO cell line by chemical transfection and electrotransfection. ForteBio was used to detect the antibody production to judge the transfection efficiency 48 hours after transfection.
  • the transfected cells undergo two rounds of pressure screening to obtain a pool of highly expressing antibodies. Then expand the cell pool to express a large amount of antibody, and collect the cell supernatant and purify the supernatant with Protein A to make the purity of the antibody>95%.
  • An anti-VEGF antibody is an antibody that can bind to VEGF and thereby block or inhibit the binding of VEGF to its receptor VEGFR, for example, the anti-VEGF antibody described in US Patent No. 6,884,879, WO 2005/044853, preferably bevacizumab (Bevacizumab).
  • the anti-VEGF antibody used in this example is the antibody IBI305 produced by Xinda Biopharmaceutical (Suzhou) Co., Ltd.
  • the amino acid sequence of its heavy chain and light chain is the same as that of bevacizumab, which is biologically similar to bevacizumab. Things.
  • those skilled in the art can also use other anti-VEGF antibodies, including but not limited to other anti-VEGF antibodies disclosed in US Patent No. 6,884,879 and WO 2005/044853 for use in the pharmaceutical combination of the present invention.
  • the preparation method of antibody IBI305 is as follows:
  • the kit produces a CHO-S cell line expressing antibodies.
  • the constructed pCHO1.0 plasmid was transferred into the CHO cell line by chemical transfection and electrotransfection. The transfected cells undergo two rounds of pressure screening to obtain a pool of highly expressing antibodies. Then expand the cell pool to express a large amount of antibody, and collect the cell supernatant and purify the supernatant with Protein A to make the purity of the antibody>95%.
  • an MDA-MB-231 tumor-bearing NOG mouse model was established, which is a humanized animal model.
  • mice After SPF grade NOG female mice (15-18g, 35-41 days old) arrived at the experimental center, the mice were domesticated and reared for 3 days. Then, the mice were used for experiments.
  • NOG mouse is a kind of mouse that lacks T cells, B cells and NK cells.
  • NOG mice were inoculated with 2 million peripheral blood mononuclear cells (PBMC) per mouse through the eye vein, so that the human PBMC can directly pass through the blood circulation of NOG mice, and the simulation was successfully reconstructed in a short period of time NOG mouse model of the human immune environment.
  • PBMC peripheral blood mononuclear cells
  • MDA-MB-231 cells are human breast cancer cell lines, and the preparation method is as follows. Disperse MDA-MB-231 breast cancer cells with sterile PBS (1 ⁇ ) solution to make the cell density 10 ⁇ 10 6 cells/mL, then mix with Matrigel glue at a volume ratio of 1:1 to prepare a cell density of 5 ⁇ 10 6 Cells/mL of cell suspension. 6 million MDA-MB-231 cells were inoculated subcutaneously on the back of each mouse. On the day of subcutaneous inoculation of MDA-MB-231 cells on the back, the mice were randomly divided into groups. A total of 63 mice were randomly divided into 9 groups with 7 NOG mice in each group.
  • h-IgG group Dissolve h-IgG with PBS to 1.05mg/ml, and intraperitoneally inject each mouse at a dose volume of 10ml/kg, intraperitoneally once every 3 days for a total of 4 times.
  • Anti-CD47 antibody-1mg/kg group The anti-CD47 antibody ADI-26630 prepared in Example 1 was dissolved in PBS to 0.1mg/ml, and the mice were injected intraperitoneally according to the administration volume of 10ml/kg, every 3 days The medicine is administered once, a total of 4 times, and the first administration is the day after the tumor cells are inoculated.
  • Anti-CD47 antibody-2mg/kg group The anti-CD47 antibody ADI-26630 prepared in Example 1 was dissolved in PBS to 0.2mg/ml, and the mice were intraperitoneally injected according to the administration volume of 10ml/kg, every 3 days The medicine is administered once, a total of 4 times, and the first administration is the day after the tumor cells are inoculated.
  • Anti-VEGF antibody-0.25mg/kg group Dissolve the anti-VEGF antibody IBI305 prepared in Example 1 with PBS to 0.025mg/ml, and intraperitoneally inject it into each mouse according to the administration volume of 10ml/kg, intraperitoneally every 3 days Once, a total of 4 times, the first dose is the day after tumor cell inoculation.
  • Anti-VEGF antibody-0.5mg/kg group Dissolve the anti-VEGF antibody IBI305 prepared in Example 1 with PBS to 0.05mg/ml, and intraperitoneally inject it into each mouse according to the administration volume of 10ml/kg, intraperitoneally every 3 days Once, a total of 4 times, the first dose is the day after tumor cell inoculation.
  • Anti-CD47 antibody + anti-VEGF antibody-1 mg/kg+0.25 mg/kg group the anti-CD47 antibody ADI-26630 prepared in Example 1 and the anti-VEGF antibody IBI305 prepared in Example 1 were dissolved in PBS to prepare the preparation to contain 0.1 mg/kg/kg. ml of anti-CD47 antibody and 0.025mg/ml of anti-VEGF antibody solution were injected intraperitoneally into each mouse according to the administration volume of 10ml/kg, intraperitoneally once every 3 days, a total of 4 times, the first administration was inoculation of tumor cells Dosing on the following day.
  • Anti-CD47 antibody + anti-VEGF antibody-1mg/kg+0.5mg/kg group The anti-CD47 antibody ADI-26630 prepared in Example 1 and the anti-VEGF antibody IBI305 prepared in Example 1 were dissolved in PBS to prepare the preparation to contain 0.1 mg/kg. ml anti-CD47 antibody and 0.05 mg/ml anti-VEGF antibody solution were injected intraperitoneally into each mouse at a dose volume of 10ml/kg, intraperitoneally once every 3 days, a total of 4 times, the first dose was inoculation of tumor cells Dosing on the following day.
  • Anti-CD47 antibody + anti-VEGF antibody-2 mg/kg + 0.5 mg/kg group the anti-CD47 antibody ADI-26630 prepared in Example 1 and the anti-VEGF antibody IBI305 prepared in Example 1 were dissolved in PBS to prepare a concentration of 0.2 mg/kg.
  • ml anti-CD47 antibody and 0.05 mg/ml anti-VEGF antibody solution were injected intraperitoneally into each mouse at a dose volume of 10ml/kg, intraperitoneally once every 3 days, a total of 4 times, the first dose was inoculation of tumor cells Dosing on the following day.
  • Anti-CD47 antibody + anti-VEGF antibody-2 mg/kg + 0.5 mg/kg group the anti-CD47 antibody ADI-26630 prepared in Example 1 and the anti-VEGF antibody IBI305 prepared in Example 1 were dissolved in PBS to prepare a concentration of 0.2 mg/kg.
  • ml anti-CD47 antibody and 0.05 mg/ml anti-VEGF antibody solution were injected intraperitoneally into each mouse at a dose volume of 10ml/kg, intraperitoneally once every 3 days, a total of 4 times, the first dose was inoculation of tumor cells Dosing on the following day.
  • the body weight, the largest long axis (L) and the largest wide axis (W) of the tumor tissues in each group were monitored twice a week for 24 consecutive days. After the experiment, the relative tumor inhibition rate of each group of mice was calculated.
  • a vernier caliper (purchased from China Baogong, model PD-051) was used to determine the maximum long axis (L) and maximum wide axis (W) of the tumor in tumor-bearing mice.
  • the relative tumor inhibition rate is calculated as follows:
  • Tumor inhibition rate TGI (%) 100% ⁇ (end-of-tumor volume of the control group after administration-end-of-tumor volume after administration of the administration group)/(end-of-tumor volume after administration of the control group-before administration of the control group Tumor volume)
  • control group is the h-IgG group.
  • the tumor inhibition rate of each group is shown in Table 1.
  • TGI Tumor inhibition rate
  • the body weight change rate of each group of tumor-bearing mice is shown in Figure 1.
  • the effect of each administration group on the tumor volume of tumor-bearing mice over time is shown in Figure 2.
  • the effect of anti-CD47 antibody low-dose group on tumor volume of tumor-bearing mice over time is shown in Figure 3A and Figure 3B
  • the effect of anti-CD47 antibody high-dose group on tumor volume of tumor-bearing mice over time is shown in Figure 4A and Figure 4B. Show.
  • anti-CD47 antibody -1mg/kg single agent The tumor suppression rate of anti-CD47 antibody is 32.59%; the tumor suppression rate of anti-CD47 antibody-2mg/kg single drug is 40.20%; the tumor suppression rate of anti-VEGF antibody-0.25mg/kg single drug is 27.69%; anti-VEGF antibody-0.5mg/kg Tumor inhibition rate of single drug is -22.44%; anti-CD47 antibody+anti-VEGF antibody-1mg/kg+0.25mg/kg group combined tumor inhibition rate of 100.17%; anti-CD47 antibody+anti-VEGF antibody-1mg/kg+0.5mg/ The combined tumor inhibition rate of the kg group was 110.86%; the combined tumor inhibition rate of the anti-CD47 antibody + anti-VEGF antibody-2mg/kg+0.25mg/kg group was 8
  • the anti-CD47 antibody and anti-VEGF antibody combined tumor inhibition rate was significantly higher than the anti-CD47 antibody single-agent group and anti-VEGF antibody single-agent group, which has a good tumor suppression effect and statistical differences (the statistical method uses two-way variance Analysis-Tukey multiple comparison, in Figure 3A, Figure 3B, Figure 4A and Figure 4B, ns represents no significant difference, * represents the statistical P value is less than 0.1, ** represents the statistical P value is less than 0.01, *** represents the statistical P value Less than 0.001, **** means that the statistical P value is less than 0.0001), indicating that anti-CD47 antibody and anti-VEGF antibody have a synergistic effect on anti-tumor and have a good anti-tumor effect.
  • the anti-CD47 antibody has a certain anti-tumor efficacy, and its combination with anti-VEGF antibody significantly enhances its anti-tumor effect, indicating that the two drugs The combination has a synergistic effect.
  • the anti-CD47 antibody and anti-VEGF antibody combination drug has a significantly better tumor inhibition effect on MDA-MB-231 tumor-bearing mice than the anti-CD47 antibody single agent and the anti-VEGF antibody single agent.
  • an A431 tumor-bearing NOG mouse model was established.
  • NOG female mice 14-17 g, 35-41 days old were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. Grade: SPF grade, the quantity is 36, the certificate number is 110011201109345813. After arrival, the experiment was carried out after acclimation and breeding for 3 days.
  • A431 cells were inoculated 5 days later.
  • the amount of PBMC inoculated was 2 million cells per mouse, and the amount of subcutaneous inoculation of A431 cells (cutaneous squamous cell carcinoma, obtained from ATCC) was 1.5 million cells per mouse. mouse.
  • the cells were randomly grouped the next day after subcutaneous inoculation. A total of 36 mice were randomly divided into 6 groups with 6 mice in each group.
  • h-IgG group Dissolve h-IgG with PBS to 1.03 mg/ml, and intraperitoneally inject each mouse at a dose volume of 10 ml/kg, intraperitoneally once every 3 to 4 days for a total of 4 times.
  • Anti-CD47 antibody-0.1mg/kg group Dissolve the anti-CD47 antibody ADI-26630 prepared in Example 1 with PBS to 0.01mg/ml, and inject it into each mouse intraperitoneally according to the administration volume of 10ml/kg, every 3 ⁇ 4 Intraperitoneal administration once a day, a total of 4 times, the first administration is the next day after tumor cell inoculation.
  • Anti-CD47 antibody-0.3mg/kg group Dissolve the anti-CD47 antibody ADI-26630 prepared in Example 1 with PBS to 0.03mg/ml, and inject it into each mouse intraperitoneally according to the administration volume of 10ml/kg, every 3 ⁇ 4 Intraperitoneal administration once a day, a total of 4 times, the first administration is the next day after tumor cell inoculation.
  • Anti-VEGF antibody-1mg/kg Dissolve the anti-VEGF antibody IBI305 prepared in Example 2 with PBS to 0.1mg/ml, and inject it into each mouse intraperitoneally according to the administration volume of 10ml/kg, intraperitoneally every 3 to 4 days One time, a total of 4 times, the first dose was the next day after tumor cell inoculation.
  • Anti-CD47 antibody + anti-VEGF antibody-0.1 mg/kg + 1 mg/kg group the anti-CD47 antibody ADI-26630 prepared in Example 1 and the anti-VEGF antibody IBI305 prepared in Example 1 were dissolved in PBS to prepare to contain 0.01 mg/kg. ml anti-CD47 antibody and 0.1 mg/ml anti-VEGF antibody solution were injected into each mouse intraperitoneally according to the administration volume of 10ml/kg, intraperitoneally once every 3 to 4 days, a total of 4 times, the first administration is vaccination Tumor cells were administered the next day.
  • Anti-CD47 antibody + anti-VEGF antibody-0.3 mg/kg + 1 mg/kg group The anti-CD47 antibody ADI-26630 prepared in Example 1 and the anti-VEGF antibody IBI305 prepared in Example 1 were dissolved in PBS to prepare a concentration of 0.03 mg/kg. ml anti-CD47 antibody and 0.1 mg/ml anti-VEGF antibody solution were injected into each mouse intraperitoneally according to the administration volume of 10ml/kg, intraperitoneally once every 3 to 4 days, a total of 4 times, the first administration is vaccination Tumor cells were administered the next day.
  • the body weight, the largest long axis (L) and the largest wide axis (W) of the tumor tissues of each group were monitored twice a week for 22 consecutive days. After the experiment, the relative tumor inhibition rate of each group of mice was calculated.
  • a vernier caliper (purchased from China Baogong, model PD-051) was used to determine the maximum long axis (L) and maximum wide axis (W) of the tumor in tumor-bearing mice.
  • the relative tumor inhibition rate is calculated as follows:
  • Tumor inhibition rate TGI (%) 100% ⁇ (end-of-tumor volume of the control group after administration-end-of-tumor volume after administration of the administration group)/(end-of-tumor volume after administration of the control group-before administration of the control group Tumor volume)
  • control group is the h-IgG group.
  • the tumor inhibition rate of each group is shown in Table 2.
  • TGI Tumor inhibition rate
  • the weight change rate of each group of tumor-bearing mice is shown in Figure 5.
  • the effect of each administration group on the tumor volume of tumor-bearing mice over time is shown in Figure 6.
  • the effect of the low-dose anti-CD47 antibody group on the tumor volume of tumor-bearing mice over time is shown in Figure 7, and the effect of the high-dose anti-CD47 antibody group on the tumor volume of tumor-bearing mice over time is shown in Figure 8.

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Abstract

提供了抗CD47抗体或其抗原结合片段和抗VEGF抗体或其抗原结合片段的药物组合、包含所述药物组合的药物组合物或成套药盒、以及使用所述药物组合、药物组合物或成套药盒预防和/或治疗肿瘤和/或癌症的方法。

Description

抗CD47抗体和抗VEGF抗体的组合及其用途 技术领域
本发明涉及医药学领域。具体地，本发明涉及一种抗肿瘤药物组合及其用途，尤其涉及一种包含抗CD47抗体和抗VEGF抗体的药物组合及其在预防和/或治疗肿瘤和/或癌症中的用途。
背景技术
分化抗原簇47(CD47)，也被称为整联蛋白相关蛋白(IAP)，是免疫球蛋白超家族成员。研究表明，几乎所有的肿瘤细胞和组织都高表达CD47。CD47通过与主要由巨噬细胞和树突细胞表达的一种作为其配体的细胞表面免疫球蛋白SIRPα相互作用，产生一系列的级联反应，并由此抑制巨噬细胞和树突细胞对表达CD47的肿瘤细胞的摄取和吞噬作用。
目前已经报道了多个抗CD47抗体，它们被开发用于各种肿瘤和/或癌症的治疗中。例如，在WO2019042285A1中，公开了一系列重组全人单克隆抗CD47抗体。对使用人伯基特氏淋巴瘤Raji细胞接种NOD-SCID小鼠产生的荷瘤小鼠施用所述抗CD47抗体，结果表明，所述抗CD47抗体能够改善巨噬细胞的吞噬作用，并具有显著的抗肿瘤活性，能够显著抑制肿瘤的生长，甚至能使得肿瘤完全消失。
由于在肿瘤的治疗中，仅针对一种靶点的单特异性抗体在临床应用上存在一些局限性。受试者在接受单特异性抗体治疗后可能会产生耐药性或无应答。为了克服抗CD47抗体可能会带来的耐药性和追求更大的临床获益，本领域存在将抗CD47抗体与其它抗肿瘤药物通过进行合理的联合用药来提高临床获益、减轻不良反应的需要。
VEGF是一种血管生成过程中重要的因子，在多数人类肿瘤的内皮细胞中过度病理表达。VEGF通过与其受体VEGFR结合极大地促进了血管内皮细胞的分裂增殖和迁移、提高了血管通透性、抑制肿瘤细胞凋亡，为肿瘤的生长和转移提供了良好的微环境。
抗VEGF抗体可以特异性结合VEGF，通过阻断VEGF与其血管内皮细胞表面上的受体结合，阻断PI3K-AKt/PKB和Ras-Raf-MEK-ERK等信号通路的传导，从而抑制血管内皮细胞的生长、增殖和迁移以及血管新生，降低血管通透性，阻断肿瘤组织的血液供应，抑制肿瘤细胞的增殖和转移，诱导肿瘤细胞凋亡，从而达到抗肿瘤的治疗效果。目前，已批准上市了基因泰克(Genentech)公司的人鼠嵌合抗VEGF抗体贝伐珠单抗(Bevacizumab，商品名Avastin)。
尚未报道在肿瘤的预防和/或治疗中将抗CD47抗体与抗VEGF抗体联用的临床研究。另外，抗CD47抗体的临床前实验结果显示，当单独使用该抗体时，在血液肿瘤上的药效不错，但是在实体肿瘤模型上药效一般。因此，也存在在实体瘤中开发比抗CD47抗体单药效果更好的联合治疗的需要，以便有效地治疗和/或预防癌症。
发明概述
本发明通过提供抗CD47抗体和抗VEGF抗体的药物组合来满足上述需求。
因此，在一个方面，本发明提供了一种药物组合，其包含(i)抗CD47抗体和/或其抗原结合片段；和(ii)抗VEGF抗体和/或其抗原结合片段。
本发明的药物组合中的抗CD47抗体可以是任何抗CD47抗体，只要是能够特异性结合CD47，并由此改善或增强巨噬细胞的吞噬作用的抗体即可。
在一个实施方案中，所述抗CD47抗体为全人单克隆抗CD47抗体。在一个实施方案中，所述抗CD47抗体包含SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR。
又在一个实施方案中，本发明的药物组合中的抗CD47抗体的SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SYYWS(SEQ ID NO:3)所示的HCDR1、YIYYSGSTNYNPSLKS(SEQ ID NO:4)所示的HCDR2和GKTGSAA(SEQ ID NO:5)所示的HCDR3；其中所述SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的RASQGISRWLA(SEQ ID NO:6)所示的LCDR1、AASSLQS(SEQ ID NO:7)所示的LCDR2和QQTVSFPIT(SEQ ID NO:8)所示的LCDR3；或者本发明的药物组合中的抗CD47抗体的SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR是根据组合Kabat、AbM、IMGT编号方案确定的GSISSYYWS(SEQ ID NO:9)所示的HCDR1、YIYYSGSTNYNPSLKS(SEQ ID NO:4)所示的HCDR2和ARGKTGSAA(SEQ ID NO:10)所示的HCDR3；其中所述SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR是根据组合Kabat、AbM、IMGT编号方案确定的RASQGISRWLA(SEQ ID NO:6)所示的LCDR1、AASSLQS(SEQ ID NO:7)所示的LCDR2和QQTVSFPIT(SEQ ID NO:8)所示的LCDR3，见下表A。
表A
Figure PCTCN2021078075-appb-000001
在一个实施方案中，本发明的药物组合中的抗CD47抗体包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:1的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:2的序列或与其具有至少90％,95％,98％或99％同一性的序列。优选地，所述抗CD47抗体包含SEQ ID NO:11或与之具有至少90％,95％,98％或99％同一性的重链序列以及SEQ ID NO：12或与之具有至少90％,95％,98％或99％同一性的轻链序列。
本发明的药物组合中的抗VEGF抗体可以是任何抗VEGF抗体，只要是能够特异性结合VEGF，并由此阻断或抑制VEGF与其受体VEGFR结合的抗体即可。例如，所述抗VEGF单克隆抗体为雷珠单抗(Ranibizumab)、brolucizumab、varisacumab或贝伐珠单抗(Bevacizumab)及其生物类似物。优选地，所述抗VEGF单克隆抗体为贝伐珠单抗。
在一个实施方案中，本发明的药物组合中的抗VEGF抗体包含SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个 CDR。
又在一个实施方案中，本发明的药物组合中的抗VEGF抗体的SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的NYGMN(SEQ ID NO:15)所示的HCDR1、WINTYTGEPTYAADFKR(SEQ ID NO:16)所示的HCDR2和YPHYYGSSHWYFDV(SEQ ID NO:17)所示的HCDR3；其中所述SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SASQDISNYLN(SEQ ID NO:18)所示的LCDR1、FTSSLHS(SEQ ID NO:19)所示的LCDR2和QQYSTVPWT(SEQ ID NO:20)所示的LCDR3；或者其中所述SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR是根据Chothia编号方案的GYTFTNY(SEQ ID NO:21)所示的HCDR1、NTYTGE(SEQ ID NO:22)所示的HCDR2和YPHYYGSSHWYFDV(SEQ ID NO:17)所示的HCDR3；其中所述SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR是根据Chothia编号方案的SASQDISNYLN(SEQ ID NO:18)所示的LCDR1、FTSSLHS(SEQ ID NO:19)所示的LCDR2和QQYSTVPWT(SEQ ID NO:20)所示的LCDR3。
在一个实施方案中，本发明的药物组合中的抗VEGF抗体包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:13的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:14的序列或与其具有至少90％,95％,98％或99％同一性的序列；
又在一个实施方案中，本发明的药物组合中的抗VEGF抗体包含SEQ ID NO:23或与之具有至少90％,95％,98％或99％同一性的重链序列以及SEQ ID NO：24或与之具有至少90％,95％,98％或99％同一性的轻链序列。
在第二方面，本发明提供了一种药物组合物，其包含抗CD47抗体和抗VEGF抗体的药物组合，以及可药用载体。
在第三方面，本发明提供了一种成套药盒，其包含抗CD47抗体和抗VEGF抗体的药物组合，优选地，所述药盒为药物剂量单元形式，由此可以依据给药方案提供药物剂量单元。
在一个实施方案中，本发明的成套药盒在同一包装内包含：
-含有用于胃肠外施用的抗CD47抗体和/或其抗原结合片段的第一容器；
-含有用于胃肠外施用的抗VEGF抗体和/或其抗原结合片段的第二容器。
在第四方面，本发明提供了抗CD47抗体和抗VEGF抗体的药物组合的用途，用于制备预防和/或治疗肿瘤和/或癌症的药物。不特别地限定所述肿瘤和/或癌症。所述肿瘤和/或癌症可以是各种血液癌和实体瘤，例如，急性骨髓性白血病(AML)，慢性骨髓性白血病，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)，多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤、皮肤癌等。优选地，所述肿瘤和/或癌症为实体瘤，进一步地为乳腺癌或皮肤癌。
在第五方面，本发明提供了抗CD47抗体和/或其抗原结合片段的用途，用于与抗VEGF抗体和/或其抗原结合片段组合来制备预防和/或治疗肿瘤和/或癌症的药物组合物。在一个实施方案中，所述抗CD47抗体为本发明第一方面中所述的抗CD47抗体。又在一个实施方案中，所述抗VEGF抗体为本发明第一方面中所述的抗VEGF抗体。不特别地限定所述肿瘤和/或癌症。所述肿瘤和/或癌症可以是各种血液癌和实体瘤，例如，急性骨髓性白血病(AML)，慢性骨髓性白血病，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)，多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、 膀胱癌、神经胶质瘤、黑素瘤、皮肤癌等。优选地，所述肿瘤和/或癌症为实体瘤，进一步地为乳腺癌或皮肤癌。
在第六方面，本发明提供了一种预防和/或治疗肿瘤和/或癌症的方法，所述方法包括向有需要的受试者施用有效量的本发明的药物组合、药物组合物或成套药盒。
本发明的抗CD47抗体与抗VEGF抗体的药物组合能够提供更好的抗肿瘤药效。在一个实施方案中，药物组合中的抗CD47抗体是WO2019042285A1中公开的全人单克隆抗CD47抗体。在一个实施方案中，将WO2019042285A1中公开的全人单克隆抗CD47抗体与美国专利号6,884,879或WO 2005/044853中记载的抗VEGF抗体组合，优选地与贝伐珠单抗(Bevacizumab)组合。本发明的药物组合取得的抗肿瘤药效显著优于抗CD47抗体或抗VEGF抗体以单药施用取得的抗肿瘤药效。
附图简述
结合以下附图一起阅读时，将更好地理解以下详细描述的本发明的优选实施方案。出于说明本发明的目的，图中显示了目前优选的实施方案。然而，应当理解本发明不限于图中所示实施方案的精确安排和手段。
图1：显示了各给药组中，MDA-MB-231荷瘤小鼠的体重变化率。
图2：显示了各给药组中，MDA-MB-231荷瘤小鼠的肿瘤体积变化。
图3A：显示了抗CD47抗体1mg/kg联合抗VEGF抗体0.25mg/kg施用时，与单药施用相比，对荷瘤小鼠的肿瘤体积变化的影响。
图3B：显示了抗CD47抗体1mg/kg联合抗VEGF抗体0.5mg/kg施用时，与单药施用相比，对荷瘤小鼠的肿瘤体积变化的影响。
图4A：显示了抗CD47抗体2mg/kg联合抗VEGF抗体0.25mg/kg施用时，与单药施用相比，对荷瘤小鼠的肿瘤体积变化的影响。
图4B：显示了抗CD47抗体2mg/kg联合抗VEGF抗体0.5mg/kg施用时，与单药施用相比，对荷瘤小鼠的肿瘤体积变化的影响。
图5：显示了各给药组中，A-431荷瘤小鼠的体重变化率。
图6：显示了各给药组中，A-431荷瘤小鼠的肿瘤体积变化。
图7：显示了抗CD47抗体0.1mg/kg联合抗VEGF抗体1mg/kg施用时，与单药施用相比，对荷瘤小鼠的肿瘤体积变化的影响。
图8：显示了抗CD47抗体0.3mg/kg联合抗VEGF抗体1mg/kg施用时，与单药施用相比，对荷瘤小鼠的肿瘤体积变化的影响。
发明详述
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
I.定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小10％的下限和比指定数字数值大10％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重链的结构域或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个可变结构域包含四个保守的构架区(FR)和三个互补决定区。(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。参见，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。
“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883，Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest,第4版,U.S.Department of Health and Human Services,National Institutes of Health(1987)),AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。
然而，应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。例如，使用Kabat和Chothia编号的CDR区域的不同定义下的残基范围如下表B所示。
表B
Figure PCTCN2021078075-appb-000002
Figure PCTCN2021078075-appb-000003
因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统规则或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
本发明抗体的CDR可以根据本领域的任何方案或其组合人工地评估确定边界。除非另有说明，否则在本发明中，术语“CDR”或“CDR序列”涵盖以上述任一种方式确定的CDR序列。
具有不同特异性(即，针对不同抗原的不同结合位点)的抗体具有不同的CDR。然而，尽管CDR在抗体与抗体之间是不同的，但是CDR内只有有限数量的氨基酸位置直接参与抗原结合。使用Kabat,Chothia,AbM、Contact和North方法中的至少两种，可以确定最小重叠区域，从而提供用于抗原结合的“最小结合单位”。最小结合单位可以是CDR的一个子部分。正如本领域技术人员明了，通过抗体的结构和蛋白折叠，可以确定CDR序列其余部分的残基。因此，本发明也考虑本文所给出的任何CDR的变体。例如，在一个CDR的变体中，最小结合单位的氨基酸残基可以保持不变，而根据Kabat或Chothia等定义的其余CDR残基可以被保守氨基酸残基替代。
除非另有说明，否则在本发明中，当提及抗体可变区中的残基位置(包括重链可变区残基和轻链可变区残基)时，是指根据Kabat编号系统(Kabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.(1991))的编号位置。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
术语“抗原结合片段”是比完整或完全抗体的氨基酸残基数要少的完整或完全抗体的一部分或一段，其能结合抗原或与完整抗体(即与抗原结合片段所来源的完整抗体)竞争结合抗原。可以通过重组DNA技术、或通过酶或化学切割完整的抗体制备抗原结合片段。抗原结合片段包括但不限于Fab、Fab’、F(ab’) 2、Fv、单链Fv、双体抗体(diabody)、单结构域抗体(sdAb)。所述Fab片段是一种由VL、VH、CL和CH1结构域组成的单价片段，例如，通过木瓜蛋白酶消化完全抗体能够获得Fab片段。此外，通过胃蛋白酶在铰链区的二硫键下面消化完全抗体产生F(ab') 2，其为Fab’的二聚体，是二价的抗体片段。F(ab') 2可以在中性条件下通过破坏铰链区中的二硫键而被还原，由此将F(ab') 2二聚体转化为Fab'单体。Fab'单体基本上是具有铰链区的Fab片段(其它抗体片段的更详细的描述请参见：基础免疫学(Fundamental Immunology),W.E.Paul编辑,Raven Press,N.Y.(1993))。所述Fv片段由抗体单臂的VL和VH结构域组成。另外，虽然Fv片段的两个结构域VL和VH由独立的基因编码，但是使用重组方法，可以将它们通过能够使这两个结构域作为单条蛋白链产生的合成性连接肽连接，在所述单条蛋白链中VL区和VH区配对以形成单链Fv。可以通过化学方法、重组DNA方法或蛋白酶消化法获得所述抗体片段。
术语“单克隆抗体”指具有单一氨基酸组成的抗体分子的制备物，而不指其产生的方法。单克隆抗体或其抗原结合片段可以例如通过杂交瘤技术、重组技术、噬菌体展示技术、合成技术例如CDR嫁接、或此类或其它本领域已知的技术的组合来产生。
术语“人源化”抗体指包含来自非人类CDR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体包含全部或基本上全部的CDR与非人抗体的那些CDR对应并且全部或基本上全部的FR区与人抗体的那些FR对应。人源化抗体任选地可以包含从人抗体衍生的抗体恒定区的至少一部分。抗体(例如非人抗体)的“人源化形式”指已经历过人源化的抗体。
“人抗体”、“全人抗体”、“完全人抗体”在本申请中可互换地使用，指具有这样的氨基酸序列的抗体，所述氨基酸序列对应于这样抗体的氨基酸序列，所述抗体由人或人细胞生成或来源于非人来源，其利用人抗体库或其它人抗体编码序列。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。
当谈及抗原和抗体时使用的术语“特异性结合”或“结合”意指抗体与生理条件下相对稳定的抗原形成复合物。用于确定抗体是否与抗原特异性结合的方法是本领域熟知的并且例如包括表面等离振子共振测定法、MSD测定法(Estep,P.等人,High throughput solution-based measurement of antibody-antigen affinity and epitope binning,MAbs,2013.5(2):p.270-278)、ForteBio亲和力测定法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):p.270-8)等。在一个实施方案中，本发明药物组合中的“特异性结合”CD47的抗CD47抗体如ForteBio亲和力测定法中测量，以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1、10 10M -1或更强的亲和力常数与肿瘤细胞表面的CD47结合，由此阻断或抑制CD47与巨噬细胞表面SIRPα的结合，促进肿瘤组织浸润区的巨噬细胞对肿瘤细胞的吞噬作用。又在一个实施方案中，本发明药物组合中的“特异性结合”VEGF的抗VEGF抗体如ForteBio亲和力测定法中测量，以至少约10 7M -1、 优选地约10 8M -1和更优选地约10 9M -1、10 10M -1或更强的亲和力常数与血管内皮细胞生长因子(VEGF)结合，由此阻断或抑制VEGF与其血管内皮细胞表面上的受体结合以及随后的信号传导。
术语“抗CD47抗体”、“抗CD47”、“CD47抗体”或“结合CD47的抗体”可互换地使用，是指这样的抗体，所述抗体能够以足够的亲和力特异性结合CD47蛋白或其片段，并阻断或抑制CD47与巨噬细胞表面SIRPα的结合，以致所述抗体可以用作靶向CD47的预防剂和/或治疗剂。
术语“抗VEGF抗体”、“抗VEGF”、“VEGF抗体”或“结合VEGF的抗体”可互换地使用，是指这样的抗体，所述抗体能够以足够的亲和力特异性结合VEGF蛋白或其片段，并阻断或抑制VEGF与其血管内皮细胞表面上的受体结合，以致所述抗体可以用作靶向VEGF的预防剂和/或治疗剂。
术语“生物类似物(biosimilar)”也称为生物仿制物、生物仿制品，是与已批准的参比生物产品高度相似，尽管无活性组分有小的差异；但在临床上和参比生物产品相比在安全性、纯度与效力方面没有显著差异。
术语“药物组合”是指非固定组合产品或固定组合产品，包括但不限于药盒、药物组合物。术语“非固定组合”意指活性成分(例如，(i)抗CD47抗体和/或其抗原结合片段、以及(ii)抗VEGF抗体和/或其抗原结合片段)以分开的实体被同时、无特定时间限制或以相同或不同的时间间隔、依次地施用于受试者，其中这类施用在受试者体内提供预防或治疗有效水平的所述两种抗体。在一些实施方案中，药物组合中使用的抗CD47抗体和/或其抗原结合片段和抗VEGF抗体和/或其抗原结合片段以不超过它们单独使用时的水平施用。术语“固定组合”意指本发明药物组合中的两种抗体以单个实体的形式被同时施用于受试者。优选对本发明药物组合中的两种抗体的施用剂量和/或时间间隔进行选择，从而使各抗体的联合使用能够在治疗疾病或病症时产生大于单独使用任何一种抗体所能达到的效果。各抗体可以各自呈单独的制剂形式，其制剂形式可以相同也可以不同。
术语“施用”指用本领域技术人员已知的多种方法和递送系统中的任一种将本发明的药物组合中的各活性成分物理导入至受试者。本发明的药物组合中的各抗体的施用途径包括口服、静脉内(例如输注(又称滴注)或注射)、肌内、皮下、腹膜内、脊髓、局部或其他胃肠外施用途径。本文所用的短语“胃肠外施用”指胃肠和局部施用之外的施用方式，非限制性地包括肌内、动脉内、静脉内，鞘内、淋巴内、病灶内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注，以及体内电穿孔。相应地，本发明的药物组合中的各抗体可以被配制成胶囊剂、片剂、注射剂(包括输液或注射液)、糖浆、喷雾剂、锭剂、脂质体或栓剂等。
术语“有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防和/或治疗结果的量。可以根据多种因素如疾病状态、受试者的年龄、性别和重量等变动预防和/或治疗有效量。治疗有效量是任何有毒或有害作用不及治疗有益作用的量。相对于未治疗的受试者，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约60％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价本发明的药物组合抑制可度量参数(例如，肿瘤体积)的能力。“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在受试者中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量小于治疗有效量。
术语“剂量”是引发预防和/或治疗效果的药物的量。除非另有说明，否则剂量与游离形式的药物的量有关。如果药物是可药用盐形式，药物的量与游离形式的药物的量相比成比例地增加。例如，剂量将在产品包装或产品信息单中声明。
术语“抑制”是指给定分子(例如(i)抗CD47抗体和/或其抗原结合片段；(ii)抗VEGF抗体和/或其抗原结合片段)使得某些参数(例如CD47活性；VEGFR活性)的降低。例如，该术语包括抑制至少5％、10％、20％、30％、40％或更多的活性。因此，抑制不必是100％。
在谈及疾病时，术语“治疗”是指减轻所述疾病(即，减缓或阻止或减少所述疾病或其至少一个临床症状的发展)、防止或延迟所述疾病的发作或发展或进展。
术语“预防”包括对疾病或病症或其症状的发生或发生频率的抑制或推迟，其通常是指在病征或症状发生前，特别是在具有风险受试者的病征或症状发生前的药物施用。
术语“受试者”指哺乳动物和非哺乳动物。哺乳动物指哺乳类的任何成员，其包括但不限于：人；非人灵长类动物，牛、马、羊、猪、兔、狗和猫等。术语“受试者”并不限定特定的年龄或性别。在一些实施方案中，受试者是人。
II.本发明的药物组合
本发明的药物组合包含(i)抗CD47抗体和/或其抗原结合片段；和(ii)抗VEGF抗体和/或其抗原结合片段。
本发明的药物组合中的抗CD47抗体和/或其抗原结合片段如ForteBio亲和力测定法中测量，以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1、10 10M -1或更强的亲和力常数与肿瘤细胞表面的CD47特异性结合，由此阻断或抑制CD47与巨噬细胞表面SIRPα的结合，促进肿瘤组织浸润区的巨噬细胞对肿瘤细胞的吞噬作用。
在一个实施方案中，本发明药物组合中的抗CD47抗体包含SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR。
例示的抗CD47抗体的重链可变区：
Figure PCTCN2021078075-appb-000004
例示的抗CD47抗体的轻链可变区：
Figure PCTCN2021078075-appb-000005
又在一个实施方案中，本发明的药物组合中的抗CD47抗体的SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SYYWS(SEQ ID NO:3)所示的HCDR1、YIYYSGSTNYNPSLKS(SEQ ID NO:4)所示的HCDR2和GKTGSAA(SEQ ID NO:5)所示的HCDR3；其中所述SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的RASQGISRWLA(SEQ ID NO:6)所示的LCDR1、AASSLQS(SEQ ID NO:7)所示的LCDR2和QQTVSFPIT(SEQ ID NO:8)所示的LCDR3；或者本发明的药物组合中的抗CD47抗体的SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR是根据组合Kabat、AbM、IMGT编号方案确定的GSISSYYWS(SEQ ID NO:9)所示的HCDR1、YIYYSGSTNYNPSLKS(SEQ ID NO:4)所示的HCDR2和ARGKTGSAA(SEQ ID NO:10)所示的HCDR3；其中所述SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR是根据组合Kabat、AbM、IMGT编号方案确定的RASQGISRWLA(SEQ ID NO:6)所示的LCDR1、AASSLQS(SEQ ID NO:7)所示的LCDR2和QQTVSFPIT(SEQ ID NO:8)所示的LCDR3， 同见表A。
优选地，所述抗CD47抗体包含重链可变区VH和轻链可变区VL，其中重链可变区包含SEQ ID NO:1的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:2的序列或与其具有至少90％,95％,98％或99％同一性的序列。
优选地，所述抗CD47抗体包含SEQ ID NO:11或与之具有至少90％,95％,98％或99％同一性的重链序列以及SEQ ID NO：12或与之具有至少90％,95％,98％或99％同一性的轻链序列。
例示的抗CD47抗体的重链：
Figure PCTCN2021078075-appb-000006
例示的抗CD47抗体的轻链：
Figure PCTCN2021078075-appb-000007
优选地，所述抗CD47抗体是WO2019042285A1中公开的抗CD47抗体ADI-26630。
本发明药物组合中的抗VEGF抗体是能够结合VEGF，并由此阻断或抑制VEGF与其受体VEGFR结合的抗体。例如，美国专利号6,884,879、WO 2005/044853中记载的抗VEGF抗体。在一些实施方案中，所述抗VEGF抗体为抗VEGF单克隆抗体。优选地，所述抗VEGF单克隆抗体选自雷珠单抗(Ranibizumab)、brolucizumab、varisacumab、贝伐珠单抗(Bevacizumab)和它们的生物类似物。在一个实施方案中，所述抗VEGF单克隆抗体为贝伐珠单抗。
在一个实施方案中，所述抗VEGF抗体和/或其抗原结合片段如ForteBio亲和力测定法中测量，以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1、10 10M -1或更强的亲和力常数与血管内皮细胞生长因子(VEGF)结合，由此阻断或抑制VEGF与其血管内皮细胞表面上的受体VEGFR结合以及随后的信号传导。
在一个实施方案中，本发明药物组合中的抗VEGF抗体包含SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR。
例示的抗VEGF抗体的重链可变区：
Figure PCTCN2021078075-appb-000008
例示的抗VEGF抗体的轻链可变区：
Figure PCTCN2021078075-appb-000009
又在一个实施方案中，本发明的药物组合中的抗VEGF抗体的SEQ ID NO:13所示的重 链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的NYGMN(SEQ ID NO:15)所示的HCDR1、WINTYTGEPTYAADFKR(SEQ ID NO:16)所示的HCDR2和YPHYYGSSHWYFDV(SEQ ID NO:17)所示的HCDR3；其中所述SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SASQDISNYLN(SEQ ID NO:18)所示的LCDR1、FTSSLHS(SEQ ID NO:19)所示的LCDR2和QQYSTVPWT(SEQ ID NO:20)所示的LCDR3；或者其中所述SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR是根据Chothia编号方案的GYTFTNY(SEQ ID NO:21)所示的HCDR1、NTYTGE(SEQ ID NO:22)所示的HCDR2和YPHYYGSSHWYFDV(SEQ ID NO:17)所示的HCDR3；其中所述SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR是根据Chothia编号方案的SASQDISNYLN(SEQ ID NO:18)所示的LCDR1、FTSSLHS(SEQ ID NO:19)所示的LCDR2和QQYSTVPWT(SEQ ID NO:20)所示的LCDR3。
优选地，所述抗VEGF抗体包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:13的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:14的序列或与其具有至少90％,95％,98％或99％同一性的序列。
优选地，所述抗VEGF抗体包含SEQ ID NO:23或与之具有至少90％,95％,98％或99％同一性的重链序列以及SEQ ID NO：24或与之具有至少90％,95％,98％或99％同一性的轻链序列。
例示的抗VEGF抗体的重链：
Figure PCTCN2021078075-appb-000010
例示的抗VEGF抗体的轻链：
Figure PCTCN2021078075-appb-000011
III.本发明的药物组合的用途和使用本发明的药物组合的治疗方法
本发明提供了前述本发明的药物组合，其用于预防和/或治疗受试者中癌症的至少一种症状或指征的严重性或抑制癌细胞生长。
本发明提供了预防或治疗癌症的方法，其包括向有需要的受试者施用有效量的本发明的药物组合。所述有效量包括预防有效量和治疗有效量。
本发明提供了前述本发明的药物组合在制备用于预防或治疗癌症的药物中的用途。
本发明所述癌症包括实体瘤和血液学恶性肿瘤，例如，急性骨髓性白血病(AML)，慢性骨髓性白血病，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)，多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤、皮肤癌。优选地，所述肿瘤和/或癌症为实体瘤，进一步地为乳腺癌、皮肤癌。
本发明药物组合中的抗CD47抗体和/或其抗原结合片段可以以一个或多个剂量施用于有需要的受试者，其中在施用多个剂量的情况下，在前一剂量之后3天、1周、2周、4周、6周、8周、或10周施用下一个剂量。抗CD47抗体和/或其抗原结合片段的一个剂量可以选自0.1-10mg/kg受试者体重(例如，0.2mg/kg、0.5mg/kg、1mg/kg、2mg/kg、5mg/kg或10mg/kg)。在一些其他实施方案中，每个剂量包含50-500mg的抗CD47抗体和/或其抗原结合片段，例如50mg、100mg、150mg、200mg、250mg、300mg、350mg、400mg、450mg或500mg抗CD47抗体和/或其抗原结合片段。
本发明药物组合中的抗VEGF抗体和/或其抗原结合片段可以以一个或多个剂量施用于有需要的受试者，其中在施用多个剂量的情况下，在前一剂量之后3天、1周、2周、4周、6周、8周、或10周施用下一个剂量。抗VEGF抗体和/或其抗原结合片段的一个剂量可以选自0.1-10mg/kg受试者体重(例如，0.2mg/kg、0.5mg/kg、1mg/kg、2mg/kg、5mg/kg或10mg/kg)。在一些其他实施方案中，每个剂量包含50-500mg的抗VEGF抗体和/或其抗原结合片段，例如50mg、100mg、150mg、200mg、250mg、300mg、350mg、400mg、450mg或500mg抗VEGF抗体和/或其抗原结合片段。
本发明的药物组合可以是本领域技术人员已知的任何剂型，例如片剂、胶囊剂、颗粒剂、糖浆剂、粉末、锭剂、药囊、扁囊剂、酏剂、混悬剂、乳剂、溶液、糖浆剂、气雾剂、软膏剂、乳膏剂和注射剂等。其中，抗CD47抗体和/或其抗原结合片段与抗VEGF抗体和/或其抗原结合片段可以各自呈单独的剂型，其剂型可以是不同或相同的。
在一个实施方案中，本发明的药物组合中的抗CD47抗体和/或其抗原结合片段是静脉内施用剂型，例如静脉内注射剂。在一个实施方案中，本发明的药物组合中的抗CD47抗体和/或其抗原结合片段是腹膜内施用剂型，例如腹膜内注射剂。
在一个实施方案中，本发明的药物组合中的抗VEGF抗体和/或其抗原结合片段是静脉内施用剂型，例如静脉内注射剂。在一个实施方案中，本发明的药物组合中的抗VEGF抗体和/或其抗原结合片段是腹膜内施用剂型，例如腹膜内注射剂。
本发明的药物组合中的抗CD47抗体和/或其抗原结合片段和抗VEGF抗体和/或其抗原结合片段可以分开、同时或依次施用。
本发明的药物组合中的抗CD47抗体和/或其抗原结合片段可以在开始施用抗VEGF抗体和/或其抗原结合片段之前、同时、或者之后施用。
与施用抗CD47抗体和/或其抗原结合片段的单一疗法或施用抗VEGF抗体和/或其抗原结合片段的单一疗法相比，本发明的药物组合导致增加、优选协同地增加对肿瘤生长的抑制作用。在一些实施方案中，与施用抗CD47抗体和/或其抗原结合片段的单一疗法或施用抗VEGF抗体和/或其抗原结合片段的单一疗法相比，本发明的药物组合导致肿瘤生长被抑制至少约50％、约60％、约70％、约80％、约90％、约100％。在一些实施方案中，本发明的药物组合的施用导致增加肿瘤消退、肿瘤缩小和/或消失。在一些实施方案中，与未治疗的受试者或与使用抗CD47抗体和/或其抗原结合片段的单一疗法或使用抗VEGF抗体和/或其抗原结合片段的单一疗法相比，本发明的药物组合预防受试者的肿瘤复发和/或增加生存持续时间，例如，将生存持续时间增加多于15天、多于1个月、多于3个月、多于6个月、多于12个月、多于18个月、多于24个月、多于36个月、或多于48个月。在一些实施方案中，本发明的药物组合可增加无进展生存或总体生存。
在一些实施方案中，本发明的药物组合可以减少由施用各单药导致的不良事件，例如，血液学毒性反应、非血液学毒性反应或其他毒性反应。
IV.本发明的药盒
本发明的另一个目的是提供一种成套药盒，其包含本发明的药物组合，优选地所述药盒为药物剂量单元形式，由此可以依据给药方案提供药物剂量单元。
在一个实施方案中，本发明的成套药盒在同一包装内包含：
-含有用于胃肠外施用的抗CD47抗体和/或其抗原结合片段的第一容器；
-含有用于胃肠外施用的抗VEGF抗体和/或其抗原结合片段的第二容器。
本发明所述的各个实施方案/技术方案以及各个实施方案/技术方案中的特征应当被理解为可以任意进行相互组合，这些相互组合得到的各个方案均包括在本发明的范围内，就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。
实施例
通过以下实施例向本领域普通技术人员提供如何制备和使用本发明的方法和组合物的完整公开和描述，并且不旨在限制本发明所涵盖的范围。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
MDA-MB-231细胞(人乳腺癌细胞)为ATCC产品，货号为HTB-26，批号为62235654。
人PBMC(人外周血单个核细胞)为AllCells产品，批号为3024811。
SPF级NOG雌性小鼠(15-18g，35-41日龄)购自北京维通利华实验动物技术有限公司，合格证号为1100111911046485。
h-IgG为Equitech-Bio产品，货号为SLH56，批号为160308-02。
实施例1.抗CD47抗体和抗VEGF抗体的生产和纯化
1、抗CD47抗体
抗CD47抗体是通过结合CD47，从而阻断或抑制CD47配体SIRPα与CD47相互作用的抗体，例如专利公开号WO2019042285A1中公开的任一抗CD47单克隆抗体。为了本申请的目的，该PCT申请的全部内容特此并入本文作为参考。本实施例例示使用的是WO2019042285A1中公开的抗CD47抗体ADI-26630。需要说明的是，本领域技术人员也可以使用其他抗CD47抗体，包括但不限于WO2019042285A1中公开的其他抗CD47抗体用于本发明的药物组合中。
抗CD47抗体ADI-26630的CDR区、轻链可变区和重链可变区、轻链和重链的氨基酸序列在本申请的“发明详述”中给出。
本发明的药物组合中的抗CD47抗体可以通过本领域技术人员公知的任一抗体制备技术来生成。在下文中，例示了通过在酵母或CHO-S细胞中表达并且纯化抗CD47抗体。
酵母中的表达和纯化：
基于酵母的抗体展示(yeast-based antibody presentation)文库(Adimab)，按照现有的方法(WO2009036379；WO 2010105256；W02012009568)进行扩增，其中每个库的多样性达到1×10 9。简言之，前两轮的筛选使用Miltenyi公司的MACS系统进行磁性激活细胞分选。首先，将文库的酵母细胞(～1×10 10个细胞/文库)分别在FACS洗涤缓冲液中(磷酸盐缓冲液，含有0.1％牛血清蛋白)室温孵化15分钟，缓冲液中含有100nM生物素标记的CD47抗原(Acro Biosystems，目录号CD7-H5227-1mg)。使用50ml预冷的FACS洗涤缓冲液洗一次，再用40ml相同洗涤缓冲液重悬细胞，并加入500μl链霉素微珠(Miltenyi LS)于4℃孵化15分钟。1000rpm离心5min弃去上清后用5ml FACS洗涤缓冲液重悬细胞，将细胞溶液加到Miltenyi LS柱中。加样完成后，用FACS洗涤缓冲液洗柱3次，每次3ml。从磁性区域取下Miltenyi LS柱，用5ml生长培养基洗脱，收集洗脱的酵母细胞并在37℃过夜生长。
使用流式细胞仪进行下一轮的分选：将经过MACS系统筛选获得的大约1×10 8个酵母细胞用FACS缓冲液洗三次，于含有低浓度生物素(100-1nM)标记的CD47抗原中室温下培养。弃去培养液，细胞用FACS洗涤缓冲液洗两次之后，将细胞与LC-FITC(FITC标记山羊抗人免疫球蛋白F(ab’)kappa链抗体，Southern Biotech)(1:100稀释)混合，并与SA-633(链霉亲和素-633,Molecular Probes)(1:500稀释)或SA-PE(链霉亲和素-藻红蛋白,Sigma)(1:50稀释)试剂混合，4℃下培养15分钟。用预冷的FACS洗涤缓冲液洗脱两次，并重悬于0.4ml缓冲液中，将细胞转移到带滤器的分离管中。使用FACS ARIA(BD Biosciences)分选细胞。
将通过筛选获得的表达抗CD47抗体的酵母细胞在30℃下震荡诱导48小时以表达抗CD47的抗体。诱导结束之后，1300rpm离心10min去除酵母细胞，收获上清液。使用Protein A对上清液中的抗CD47抗体进行纯化，pH2.0醋酸溶液洗脱，收获抗CD47抗体，抗体纯度＞95％。使用木瓜蛋白酶消化并用KappaSelect(GE生命医疗集团)进行纯化可获得相应的Fab片段。
CHO-S细胞中的表达和纯化：
根据制造商的说明书，使用
Figure PCTCN2021078075-appb-000012
试剂盒(Invitrogen)产生表达抗体的CHO-S细胞系。首先将抗体分子重链和轻链的DNA序列插入到同一个pCHO1.0质粒中，其中重链DNA序列在轻链DNA序列的上游。之后采用化学转染法和电转染法将构建的pCHO1.0质粒转入CHO细胞系，转染48小时之后利用ForteBio检测抗体产量以判断转染效率。转染后的细胞经过两轮加压筛选得到高表达抗体的细胞池(pool)。之后扩增细胞池，大量表达抗体，并收集细胞上清用Protein A纯化上清液，使抗体的纯度＞95％。
2、抗VEGF抗体
抗VEGF抗体是能够结合VEGF，并由此阻断或抑制VEGF与其受体VEGFR结合的抗体，例如，美国专利号6,884,879、WO 2005/044853中记载的抗VEGF抗体，优选地为贝伐珠单抗(Bevacizumab)。本实施例例示使用的抗VEGF抗体是信达生物制药(苏州)有限公司生产的抗体IBI305，其重链和轻链的氨基酸序列和贝伐珠单抗相同，是贝伐珠单抗的生物类似物。需要说明的是，本领域技术人员也可以使用其他抗VEGF抗体，包括但不限于美国专利号6,884,879、WO 2005/044853中公开的其他抗VEGF抗体用于本发明的药物组合中。
抗体IBI305的制备方法如下：
根据制造商的说明书，使用
Figure PCTCN2021078075-appb-000013
试剂盒(Invitrogen)产生表达抗体的CHO-S细胞系。首先将编码抗体IBI305的如SEQ ID NO:23所示的重链氨基酸序列的DNA序列和 编码抗体IBI305的如SEQ ID NO:24所示的轻链氨基酸序列的DNA序列插入到同一个pCHO1.0质粒中，其中重链DNA序列在轻链DNA序列的上游。之后采用化学转染法和电转染法将构建的pCHO1.0质粒转入CHO细胞系。转染后的细胞经过两轮加压筛选得到高表达抗体的细胞池(pool)。之后扩增细胞池，大量表达抗体，并收集细胞上清用Protein A纯化上清液，使抗体的纯度＞95％。
实施例2、MDA-MB-231荷瘤NOG小鼠模型的建立和本发明的药物组合的抗肿瘤作用
在本实施例中，建立了MDA-MB-231荷瘤NOG小鼠模型，这是一种人源化动物模型。通过在这种临床前模型中研究抗CD47抗体与抗VEGF抗体联合的抗肿瘤功效，对将来临床开展所述药物组合具有重要意义。
1.实验操作过程：
SPF级NOG雌性小鼠(15-18g，35-41日龄)到达实验中心后，驯化饲养小鼠3天。然后，将所述小鼠用于实验。
NOG小鼠是T细胞、B细胞和NK细胞均缺失的一种小鼠。以2百万个外周血单个核细胞(PBMC)每只小鼠的量通过眼静脉接种NOG小鼠，由此使得所述人PBMC能够直接通过NOG小鼠的血液循环，在短期内重建成功模拟人类免疫环境的NOG小鼠模型。使用模拟人类免疫环境的NOG小鼠模型进行的研究更贴近临床研究。
人PBMC眼静脉接种NOG小鼠5天后，对NOG小鼠背部皮下接种MDA-MB-231细胞，所述MDA-MB-231细胞为人乳腺癌细胞系，其制备方法如下。以无菌PBS(1×)溶液分散MDA-MB-231乳腺癌细胞使得细胞密度为10×10 6个/mL，再与Matrigel胶以体积比1:1混合制备成细胞密度为5×10 6个/mL的细胞悬液。以6百万个MDA-MB-231细胞每只小鼠进行背部皮下接种。MDA-MB-231细胞背部皮下接种当天对小鼠随机分组。共63只小鼠，随机分为9组，每组7只NOG小鼠。
2.各组小鼠的给药方案：
h-IgG组：将h-IgG用PBS溶解为1.05mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次。
抗CD47抗体-1mg/kg组：将实施例1制备的抗CD47抗体ADI-26630用PBS溶解为0.1mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。抗CD47抗体-2mg/kg组：将实施例1制备的抗CD47抗体ADI-26630用PBS溶解为0.2mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。
抗VEGF抗体-0.25mg/kg组：将实施例1制备的抗VEGF抗体IBI305用PBS溶解为0.025mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。
抗VEGF抗体-0.5mg/kg组：将实施例1制备的抗VEGF抗体IBI305用PBS溶解为0.05mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。
抗CD47抗体+抗VEGF抗体-1mg/kg+0.25mg/kg组：将实施例1制备的抗CD47抗体ADI-26630和实施例1制备的抗VEGF抗体IBI305分别用PBS溶解制备为含有0.1mg/ml抗CD47抗体、0.025mg/ml抗VEGF抗体的溶液，分别按照10ml/kg的给药体积腹腔注射给各 小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。
抗CD47抗体+抗VEGF抗体-1mg/kg+0.5mg/kg组：将实施例1制备的抗CD47抗体ADI-26630和实施例1制备的抗VEGF抗体IBI305分别用PBS溶解制备为含有0.1mg/ml抗CD47抗体、0.05mg/ml抗VEGF抗体的溶液，分别按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。
抗CD47抗体+抗VEGF抗体-2mg/kg+0.5mg/kg组：将实施例1制备的抗CD47抗体ADI-26630和实施例1制备的抗VEGF抗体IBI305分别用PBS溶解制备为含有0.2mg/ml抗CD47抗体、0.05mg/ml抗VEGF抗体的溶液，分别按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。
抗CD47抗体+抗VEGF抗体-2mg/kg+0.5mg/kg组：将实施例1制备的抗CD47抗体ADI-26630和实施例1制备的抗VEGF抗体IBI305分别用PBS溶解制备为含有0.2mg/ml抗CD47抗体、0.05mg/ml抗VEGF抗体的溶液，分别按照10ml/kg的给药体积腹腔注射给各小鼠，每3天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后当天给药。
每周监测各组小鼠体重、瘤组织最大长轴(L)和最大宽轴(W)两次，连续监测24天。实验结束后，计算各组小鼠相对肿瘤抑制率。
3.实验结果和结论：
(i)对荷瘤小鼠的肿瘤体积的影响：
采用游标卡尺(购自中国宝工，型号PD-051)测定荷瘤小鼠中肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。
如下计算相对肿瘤抑制率：
肿瘤抑制率TGI(％)＝100％×(对照组给药后肿瘤终末体积–给药组给药后肿瘤终末体积)/(对照组给药后肿瘤终末体积–对照组给药前肿瘤体积)
其中，对照组为h-IgG组。
各组的肿瘤抑制率见表1。
表1.NOG小鼠接种MDA-MB-231细胞第24天的肿瘤抑制率(TGI)％
Figure PCTCN2021078075-appb-000014
各组荷瘤小鼠的体重变化率如图1所示。各给药组对荷瘤小鼠肿瘤体积随时间的影响如图2所示。抗CD47抗体低剂量组对荷瘤小鼠肿瘤体积随时间的影响如图3A和图3B所示，抗CD47抗体高剂量组对荷瘤小鼠肿瘤体积随时间的影响如图4A和图4B所示。
由表1可知，给药4次后，相对于h-IgG组，在MDA-MB-231接种后第24天，PBMC 接种29天后，与h-IgG对比，抗CD47抗体-1mg/kg单药的肿瘤抑制率为32.59％；抗CD47抗体-2mg/kg单药的肿瘤抑制率为40.20％；抗VEGF抗体-0.25mg/kg单药的肿瘤抑制率27.69％；抗VEGF抗体-0.5mg/kg单药的肿瘤抑制率-22.44％；抗CD47抗体+抗VEGF抗体-1mg/kg+0.25mg/kg组联合的肿瘤抑制率100.17％；抗CD47抗体+抗VEGF抗体-1mg/kg+0.5mg/kg组联合的肿瘤抑制率110.86％；抗CD47抗体+抗VEGF抗体-2mg/kg+0.25mg/kg组联合的肿瘤抑制率88.14％；抗CD47抗体+抗VEGF抗体-2mg/kg+0.5mg/kg组联合的肿瘤抑制率106..44％。抗CD47抗体与抗VEGF抗体联合时的肿瘤抑制率明显高于抗CD47抗体单药组以及抗VEGF抗体单药组，具有很好的抑瘤效果，且具有统计学差异(统计方法采用双因素方差分析-Tukey多重比较，在图3A、图3B、图4A和图4B中，n.s代表无明显差异，*代表统计P值小于0.1，**代表统计P值小于0.01，***代表统计P值小于0.001，****代表统计P值小于0.0001)，表明抗CD47抗体和抗VEGF抗体在抗肿瘤方面具有协同作用，具有很好的抑瘤效果。
总之，由图1可知，抗CD47抗体单药、抗VEGF抗体单药或者抗CD47抗体与抗VEGF抗体联合用药在该临床前小鼠模型中均未见到明显毒副作用，小鼠体重正常，未出现下降。
由图2、图3A、图3B、图4A、图4B和表1可以看出，抗CD47抗体具有一定的抗肿瘤药效，与抗VEGF抗体联用显著增强了其抗肿瘤作用，说明两药联用具有协同效应。并且，与相同剂量的各单药相比较，抗CD47抗体和抗VEGF抗体联合用药对MDA-MB-231荷瘤小鼠肿瘤抑制效果明显优于抗CD47抗体单药和抗VEGF抗体单药。
实施例3、A431荷瘤NOG小鼠模型的建立和本发明的药物组合的抗肿瘤作用
在本实施例中，建立了A431荷瘤NOG小鼠模型。通过在这种临床前模型中研究抗CD47抗体与抗VEGF抗体联合的抗肿瘤功效，对将来临床开展所述药物组合具有重要意义。
1.实验操作过程：
NOG雌性小鼠(14-17g，35-41日龄)购自北京维通利华实验动物技术有限公司。等级：SPF级，数量为36只，合格证编号为110011201109345813。到达后驯化饲养3天后进行实验。
人PBMC眼静脉接种后，5天后接种A431细胞，PBMC接种量为200万个细胞每只小鼠，A431细胞(皮肤鳞状细胞癌，获自ATCC)皮下接种量为150万个细胞每只小鼠。细胞皮下接种后第二天随机分组。共36只小鼠，随机分组，共分为6组，每组6只。
2.各组小鼠的给药方案：
h-IgG组：将h-IgG用PBS溶解为1.03mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3～4天腹腔给药一次，一共4次。
抗CD47抗体-0.1mg/kg组：将实施例1制备的抗CD47抗体ADI-26630用PBS溶解为0.01mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3～4天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后第二天给药。
抗CD47抗体-0.3mg/kg组：将实施例1制备的抗CD47抗体ADI-26630用PBS溶解为0.03mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3～4天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后第二天给药。
抗VEGF抗体-1mg/kg：将实施例2制备的抗VEGF抗体IBI305用PBS溶解为0.1mg/ml，按照10ml/kg的给药体积腹腔注射给各小鼠，每3～4天腹腔给药一次，一共4次，首次给药 为接种肿瘤细胞后第二天给药。
抗CD47抗体+抗VEGF抗体-0.1mg/kg+1mg/kg组：将实施例1制备的抗CD47抗体ADI-26630和实施例1制备的抗VEGF抗体IBI305分别用PBS溶解制备为含有0.01mg/ml抗CD47抗体、0.1mg/ml抗VEGF抗体的溶液，分别按照10ml/kg的给药体积腹腔注射给各小鼠，每3～4天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后第二天给药。
抗CD47抗体+抗VEGF抗体-0.3mg/kg+1mg/kg组：将实施例1制备的抗CD47抗体ADI-26630和实施例1制备的抗VEGF抗体IBI305分别用PBS溶解制备为含有0.03mg/ml抗CD47抗体、0.1mg/ml抗VEGF抗体的溶液，分别按照10ml/kg的给药体积腹腔注射给各小鼠，每3～4天腹腔给药一次，一共4次，首次给药为接种肿瘤细胞后第二天给药。
每周监测各组小鼠体重、瘤组织最大长轴(L)和最大宽轴(W)两次，连续监测22天。实验结束后，计算各组小鼠相对肿瘤抑制率。
3.实验结果和结论：
(i)对荷瘤小鼠的肿瘤体积的影响：
采用游标卡尺(购自中国宝工，型号PD-051)测定荷瘤小鼠中肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。
如下计算相对肿瘤抑制率：
肿瘤抑制率TGI(％)＝100％×(对照组给药后肿瘤终末体积–给药组给药后肿瘤终末体积)/(对照组给药后肿瘤终末体积–对照组给药前肿瘤体积)
其中，对照组为h-IgG组。
各组的肿瘤抑制率见表2。
表2.NOG小鼠接种A431细胞第22天的肿瘤抑制率(TGI)％
Figure PCTCN2021078075-appb-000015
各组荷瘤小鼠的体重变化率如图5所示。各给药组对荷瘤小鼠肿瘤体积随时间的影响如图6所示。抗CD47抗体低剂量组对荷瘤小鼠肿瘤体积随时间的影响如图7所示，抗CD47抗体高剂量组对荷瘤小鼠肿瘤体积随时间的影响如图8所示。
由表2可知，在A431细胞接种后第22天，PBMC接种27天后，与h-IgG组对比，抗CD47抗体-0.1mg/kg单药组抑制率为为23.84％；抗CD47抗体-0.3mg/kg单药组抑制率为8.79％；抗VEGF抗体-1mg/kg单药组抑制率为14.66％；抗CD47抗体+抗VEGF抗体-0.1mg/kg+1mg/kg联合组抑制率为38.59％；抗CD47抗体+抗VEGF抗体-0.3mg/kg+1mg/kg联合组抑制率为48.13％；综上所述，抗CD47抗体与抗VEGF抗体联合时的肿瘤抑制率明显高于抗CD47抗体单药组以及抗VEGF抗体单药组，具有很好的抑瘤效果，说明抗CD47抗体和抗VEGF抗体在抗肿瘤中具有协同作用。
由图5可知，实验期间，各组小鼠体重均无明显下降。由图6、图7、图8和表2可以看出，抗CD47抗体具有一定的抗肿瘤药效，与抗VEGF抗体联用显著增强了其抗肿瘤作用，说明两药联用具有协同效应。并且，与相同剂量的各单药相比较，抗CD47抗体和抗VEGF抗体联合用药对A431荷瘤小鼠肿瘤抑制效果明显优于抗CD47抗体单药和抗VEGF抗体单药。
尽管已经出于说明本发明的目的显示了某些代表性实施方案和细节，但是本领域技术人员显而易见的是可以对它们进行多种变化和修改而不脱离主题发明的范围。在这个方面，本发明范围仅由以下权利要求限定。

Claims (14)

  1. 药物组合，其包含(i)抗CD47抗体和/或其抗原结合片段；和(ii)抗VEGF抗体和/或其抗原结合片段，
    优选地，所述抗CD47抗体包含SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR。
  2. 根据权利要求1所述的药物组合，其中所述SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2和SEQ ID NO:5所示的HCDR3；其中所述SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:8所示的LCDR3；或者
    其中所述SEQ ID NO:1所示的重链可变区氨基酸序列中的3个CDR是根据组合Kabat、AbM、IMGT编号方案确定的SEQ ID NO:9所示的HCDR1、SEQ ID NO:4所示的HCDR2和SEQ ID NO:10所示的HCDR3；其中所述SEQ ID NO:2所示的轻链可变区氨基酸序列中的3个CDR是根据组合Kabat、AbM、IMGT编号方案确定的SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:8所示的LCDR3。
  3. 根据权利要求1或2所述的药物组合，其中所述抗CD47抗体包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:1的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:2的序列或与其具有至少90％,95％,98％或99％同一性的序列；
    优选地，所述抗CD47抗体包含SEQ ID NO:11或与之具有至少90％,95％,98％或99％同一性的重链序列以及SEQ ID NO：12或与之具有至少90％,95％,98％或99％同一性的轻链序列；
    优选地，所述抗CD47抗体为全人单克隆抗CD47抗体。
  4. 根据权利要求1至3中任一项所述的药物组合，其中所述抗VEGF抗体为抗VEGF单克隆抗体；
    优选地，所述抗VEGF单克隆抗体选自雷珠单抗(Ranibizumab)、brolucizumab、varisacumab、贝伐珠单抗(Bevacizumab)和它们的生物类似物；
    优选地，所述抗VEGF单克隆抗体为贝伐珠单抗。
  5. 根据权利要求1至4中任一项所述的药物组合，其中所述抗VEGF抗体包含SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR和SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR。
  6. 根据权利要求5所述的药物组合，其中所述SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SEQ ID NO:15所示的HCDR1、SEQ ID NO:16所示的HCDR2和SEQ ID NO:17所示的HCDR3；其中所述SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR是根据Kabat编号方案的SEQ ID NO:18所示的LCDR1、SEQ ID NO:19所示的LCDR2和SEQ ID NO:20所示的LCDR3；或者
    其中所述SEQ ID NO:13所示的重链可变区氨基酸序列中的3个CDR是根据Chothia编号方案的SEQ ID NO:21所示的HCDR1、SEQ ID NO:22所示的HCDR2和SEQ ID NO:17所示的HCDR3；其中所述SEQ ID NO:14所示的轻链可变区氨基酸序列中的3个CDR是根据Chothia编号方案的SEQ ID NO:18所示的LCDR1、SEQ ID NO:19所示的LCDR2和SEQ ID NO:20所示的LCDR3。
  7. 根据权利要求1至6中任一项所述的药物组合，其中所述抗VEGF抗体包含重链可变区和轻链可变区，其中重链可变区包含SEQ ID NO:13的序列或与其具有至少90％,95％,98％或99％同一性的序列，且轻链可变区包含SEQ ID NO:14的序列或与其具有至少90％,95％,98％或99％同一性的序列；
    优选地，所述抗VEGF抗体包含SEQ ID NO:23或与之具有至少90％,95％,98％或99％同一性的重链序列以及SEQ ID NO：24或与之具有至少90％,95％,98％或99％同一性的轻链序列。
  8. 药物组合物，其包含有效量的权利要求1至7中任一项所述的药物组合和可药用载体。
  9. 成套药盒，其包含权利要求1至7中任一项所述的药物组合，优选地所述药盒为药物剂量单元形式；优选地，所述药盒在同一包装内包含：
    -含有用于胃肠外施用的抗CD47抗体和/或其抗原结合片段的第一容器；
    -含有用于胃肠外施用的抗VEGF抗体和/或其抗原结合片段的第二容器。
  10. 根据权利要求1至7中任一项所述的药物组合、根据权利要求8所述的药物组合物、根据权利要求9所述的成套药盒的用途，用于制备预防和/或治疗肿瘤和/或癌症的药物。
  11. 根据权利要求10所述的用途，其中所述肿瘤和/或癌症是各种血液癌和实体瘤，例如，急性骨髓性白血病(AML)，慢性骨髓性白血病，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)，多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤、皮肤癌；
    优选地，所述肿瘤和/或癌症为实体瘤，进一步地为乳腺癌或皮肤癌。
  12. 抗CD47抗体和/或其抗原结合片段的用途，用于与抗VEGF抗体和/或其抗原结合片段组合来制备预防和/或治疗肿瘤和/或癌症的药物组合物，其中所述抗CD47抗体为权利要求1至3中任一项所述的抗CD47抗体，所述肿瘤和/或癌症是各种血液癌和实体瘤，例如，急性骨髓性白血病(AML)，慢性骨髓性白血病，急性淋巴细胞白血病(ALL)，非霍奇金淋巴瘤(NHL)，多发性骨髓瘤(MM)、淋巴瘤、乳腺癌、胃癌、肺癌、食管癌、肠癌、卵巢癌、宫颈癌、肾癌、胰腺癌、膀胱癌、神经胶质瘤、黑素瘤、皮肤癌；
    优选地，所述肿瘤和/或癌症为实体瘤，进一步地为乳腺癌或皮肤癌。
  13. 根据权利要求12所述的用途，其中所述抗VEGF抗体为权利要求4至7中任一项所述的抗VEGF抗体。
  14. 一种预防和/或治疗肿瘤和/或癌症的方法，所述方法包括向有需要的受试者施用有效量的权利要求1至7中任一项所述的药物组合、权利要求8所述的药物组合物、或权利要求9所述的成套药盒。
PCT/CN2021/078075 2020-02-27 2021-02-26 抗cd47抗体和抗vegf抗体的组合及其用途 WO2021170077A1 (zh)

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