WO2018095428A1 - Cd47抗体、其抗原结合片段及其医药用途 - Google Patents

Cd47抗体、其抗原结合片段及其医药用途 Download PDF

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WO2018095428A1
WO2018095428A1 PCT/CN2017/113081 CN2017113081W WO2018095428A1 WO 2018095428 A1 WO2018095428 A1 WO 2018095428A1 CN 2017113081 W CN2017113081 W CN 2017113081W WO 2018095428 A1 WO2018095428 A1 WO 2018095428A1
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seq
antibody
variable region
antigen
binding fragment
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PCT/CN2017/113081
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English (en)
French (fr)
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叶鑫
孙乐
徐韶瑜
胡齐悦
陶维康
张连山
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN201780016827.3A priority Critical patent/CN108779179B/zh
Publication of WO2018095428A1 publication Critical patent/WO2018095428A1/zh

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the present disclosure relates to the fields of biology and medicine; more specifically, the present disclosure relates to a CD47 antibody and antigen-binding fragment thereof, a chimeric antibody thereof, a humanized antibody, and a medicament comprising the CD47 antibody or antigen-binding fragment thereof A composition, and its use as an anticancer drug.
  • tumor immunotherapy has increasingly become a powerful weapon for humans to fight against cancer.
  • Tumor targeting monoclonal antibodies is one of the important means in the field of tumor immunotherapy.
  • the phagocytic effect of macrophages requires two signals to work simultaneously: one is the activation of the "eat me” signal that targets the cell surface, and the other is the inactivation of the "don't eat me” signal on the same cell surface. The absence of any one signal is not enough to trigger the phagocytic effect.
  • CD47 is a type of "don't eat me” signal that inhibits macrophage phagocytosis by binding to SIRP-alpha on the surface of macrophages.
  • CD47 is a member of the immunoglobulin Ig superfamily, which is widely expressed on the cell surface of different tissues, such as red blood cells, lymphocytes, platelets, and hepatocytes. CD47 is highly expressed on various tumor cells. Studies have shown that the expression of CD47 on the surface of various tumor cells is about 3 times higher than that on normal cells. In addition, patients with cancer cells expressing large amounts of CD47 have shorter survival times than patients with low levels of CD47 expression. At present, it has been found that blocking anti-CD47 monoclonal antibody has a very good effect on tumor treatment, but the mechanism of action of this process is still unclear.
  • CD47 as a target for cancer treatment has the following incomparable advantages:
  • CD47 patents such as WO2016065329, WO2016109415, WO2014087248 and WO2014093678. It has a good clinical prospect by effectively blocking the binding between CD47 and SIRP- ⁇ and promoting the phagocytosis of tumor cells by macrophages in vivo.
  • a first aspect of the present disclosure provides a CD47 antibody or antigen-binding fragment thereof comprising: a CDR region of an antibody heavy chain variable region (hereinafter referred to as an HCDR) and/or a CDR region of an antibody light chain variable region (hereinafter Called LCDR).
  • HCDR antibody heavy chain variable region
  • Called LCDR antibody light chain variable region
  • the HCDR is selected from any one or more of the following: SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, and a CDR having at least 85% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: .
  • the LCDR is selected from any one or more of the following CDRs: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19. CDR.
  • sequence identity means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 100% sequence identity.
  • a humanized antibody or antigen-binding fragment thereof comprising an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises SEQ ID NO: 8. HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 9 and SEQ ID NO: 10, or CDRs having at least 85% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. .
  • a humanized antibody or antigen-binding fragment thereof comprising an antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises SEQ ID NO: 14 HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 15 and SEQ ID NO: 16, or CDRs having at least 85% sequence identity to SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.
  • a humanized antibody or antigen-binding fragment thereof comprising an antibody light chain variable region, wherein the antibody light chain variable region comprises SEQ ID NO: 11. LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12 and SEQ ID NO: 13, or a CDR having at least 85% sequence identity to SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: .
  • a humanized antibody or antigen-binding fragment thereof comprising an antibody light chain variable region, wherein the antibody light chain variable region comprises SEQ ID NO: 17 LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18 and SEQ ID NO: 19, or CDRs having at least 85% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.
  • a CD47 antibody or antigen-binding fragment thereof which is a murine antibody or antigen-binding fragment thereof, is provided.
  • a CD47 antibody or antigen-binding fragment thereof wherein the light chain variable region of the antibody comprises a murine light chain FR region or a mutated sequence thereof;
  • the variable region comprises a murine heavy chain FR region or a mutated sequence thereof.
  • a CD47 antibody or antigen-binding fragment thereof comprising the heavy chain variable region of SEQ ID NO: 4 and the light chain of SEQ ID NO: 5 is provided Variable zone.
  • a CD47 antibody or antigen-binding fragment thereof comprising the heavy chain variable region set forth in SEQ ID NO: 6 and the light chain variable set forth in SEQ ID NO: 7 is provided Area.
  • a CD47 antibody or antigen-binding fragment thereof which is a chimeric antibody or antigen-binding fragment thereof, is provided.
  • a CD47 humanized antibody or antigen-binding fragment thereof wherein the sequence of the heavy chain FR region of the heavy chain variable region is selected from the human germline heavy chain IGHV1-3*01 Combination with hjh6.1 and its mutated sequence, or a combination selected from human germline heavy chains IGHV1-2*02 and hjh6.1 and its mutated sequences.
  • a CD47 humanized antibody or antigen-binding fragment thereof wherein the sequence of the light chain FR region of the light chain variable region is selected from the human germline light chain IGKV4-1*01 and The combination of hjk2.1 and its mutated sequence, or a combination selected from the human germline light chain IGKV1-39*01 and hjk4.1 and its mutated sequence.
  • a CD47 humanized antibody or antigen-binding fragment thereof wherein the FR region of the variable region of the humanized antibody or antigen-binding fragment thereof comprises 0 to 10 amino acids
  • Back mutations include, but are not limited to, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid back mutations.
  • a CD47 humanized antibody or antigen-binding fragment thereof comprising the heavy chain variable region set forth in SEQ ID NO: 20 or a mutant thereof and SEQ ID NO: 21 The light chain variable region shown or its mutated sequence.
  • a CD47 humanized antibody or antigen-binding fragment thereof comprising the heavy chain variable region set forth in SEQ ID NO: 22 or a mutant sequence thereof and SEQ ID NO: The light chain variable region shown in 23 or a mutant sequence thereof.
  • a CD47 humanized antibody or antigen-binding fragment thereof comprising a light chain variable region mutated sequence, the SEQ ID NO: Based on the light chain variable region shown by 21, the 66th amino acid residue D is replaced by E (expressed as D66E, the same applies hereinafter).
  • a CD47 humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable region mutated sequence, the SEQ ID NO:
  • the heavy chain variable region shown by 20 comprises a mutation selected from the group consisting of R72A, M48I, E46D, V68A, I70L, R38K, R67K, A97S, and combinations thereof.
  • the heavy chain variable region mutated sequence comprises a mutation selected from the group consisting of: R72A, M48I, E46D, V68A, I70L, based on the heavy chain variable region set forth in SEQ ID NO: And their combinations.
  • a CD47 humanized antibody or antigen-binding fragment thereof comprising a light chain variable region mutated sequence, the SEQ ID NO:
  • the light chain variable region shown by 23 comprises a mutation selected from the group consisting of V58I, I2V, M4I, Q38E, A43T, P44H, and combinations thereof.
  • the light chain variable region mutation sequence is based on the light chain variable region set forth in SEQ ID NO: 23, comprising a mutation I2V, M4I and combinations thereof selected from the group consisting of:
  • a CD47 humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable region mutated sequence, the SEQ ID NO:
  • the heavy chain variable region indicated by 22 includes a mutation selected from the group consisting of R72V, M48V, V68A, M70L, T74K, A40R, R38K, R67K and combinations thereof.
  • a CD47 antibody or antigen-binding fragment thereof wherein: the humanized antibody variable region comprises: the heavy chain variable region of SEQ ID NO: 26 or a mutant sequence, and the light chain variable region of SEQ ID NO: 27 or a mutant sequence thereof; or
  • the humanized antibody variable region comprises: the heavy chain variable region set forth in SEQ ID NO: 28 or a mutant sequence thereof, and the light chain variable region set forth in SEQ ID NO: 29 or a mutant sequence thereof.
  • a CD47 chimeric antibody or a CD47 humanized antibody is provided, the heavy chain comprising:
  • a CD47 chimeric antibody or a CD47 humanized antibody is provided, the heavy chain comprising:
  • a CD47 chimeric antibody or CD47 humanized antibody comprising: a human IgG4 heavy chain constant region comprising a F234A and L235A mutation (eg SEQ ID NO: 30) ).
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • a therapeutically effective amount of a CD47 antibody or antigen-binding fragment thereof according to the present disclosure is provided.
  • One or more pharmaceutically acceptable carriers, diluents or excipients • One or more pharmaceutically acceptable carriers, diluents or excipients.
  • the CD47 antibody or antigen-binding fragment thereof according to the present disclosure may be formulated in the composition as the sole pharmaceutically active ingredient in the pharmaceutical composition, or may be formulated in the composition in combination with other active ingredients.
  • carrier includes pharmaceutically acceptable carriers, diluents or excipients that are not toxic when contacted with a cell or mammal at the dosages and concentrations employed.
  • a pharmaceutically acceptable carrier generally refers to a buffered aqueous solution.
  • pharmaceutically acceptable carriers include, but are not limited to, buffers; antioxidants; polypeptides; proteins; hydrophilic polymers; amino acids; sugars; chelating agents; sugar alcohols; ions; surfactants.
  • a pharmaceutically acceptable carrier, diluent or excipient can also be in the form of a liposome, including liposomes targeted to cancerous tissue, and can also be used as a pharmaceutically acceptable carrier. These can all be prepared according to methods known to those skilled in the art.
  • a “therapeutically effective amount” can be determined by testing a CD47 antibody or antigen-binding fragment thereof of the present disclosure in known in vitro and in vivo systems, for example, based on an effective amount as demonstrated in an animal, and then inferred for use in humans. The amount.
  • a CD47 antibody or antigen-binding fragment thereof of the present disclosure is mixed with a suitable pharmaceutical carrier to form a pharmaceutical composition.
  • concentration of the CD47 antibody or antigen-binding fragment thereof in the composition will depend on the absorption, inactivation, metabolic rate, dosage regimen, dosage, dosage form of the antibody or antigen-binding fragment thereof, as well as other factors known to those skilled in the art.
  • compositions of the present disclosure may be administered by any route known to those skilled in the art, such as, but not limited to, oral, topical, intracerebral, intraocular, intracardiac, intrathecal, Intravenous, intramuscular, intraperitoneal, intradermal, intratracheal, and combinations thereof.
  • the most suitable route of administration will vary depending on the intended use.
  • various cancers such as breast cancer, bladder cancer, and intestinal cancer
  • local administration may be employed, including administration to a site of tumor growth, since it is advantageous in the CD47 antibody or antigen thereof of the present disclosure.
  • the binding fragment can be administered at a higher concentration.
  • a solution or suspension for parenteral, intradermal, or subcutaneous administration including but not limited to: a sterile diluent; an antibacterial agent; an antioxidant; a buffer; and an agent for adjusting the osmotic pressure.
  • Formulations for parenteral administration can be placed in ampoules, syringes or single or multiple dose vials made of glass, plastic or other suitable materials.
  • the present disclosure further provides a bispecific antibody comprising a CD47 antibody or antigen-binding fragment thereof according to the present disclosure.
  • the disclosure further provides an isolated nucleic acid encoding a CD47 antibody or antigen-binding fragment thereof according to the present disclosure.
  • the disclosure further provides an expression vector that expresses a CD47 antibody or antigen-binding fragment thereof according to the present disclosure.
  • the disclosure further provides an expression vector comprising an isolated nucleic acid according to the present disclosure.
  • the disclosure further provides a host cell transformed with an expression vector according to the present disclosure.
  • the host cell is selected from the group consisting of a prokaryotic cell or a eukaryotic cell. In a specific embodiment, the host cell is a eukaryotic cell. In a specific embodiment, the host cell is a mammalian cell.
  • the present disclosure further provides a method for producing a CD47 antibody and antigen-binding fragment thereof, comprising:
  • the present disclosure further provides a method for inhibiting growth of tumor cells in a subject, comprising:
  • the CD47 antibody or antigen-binding fragment thereof of the present disclosure, or a pharmaceutical composition of the present disclosure, is administered to the subject.
  • the present disclosure further provides the use of a CD47 antibody or antigen-binding fragment thereof of the present disclosure in the manufacture of a medicament for treating cancer.
  • a pharmaceutical composition of the present disclosure in the manufacture of a medicament for the treatment of cancer.
  • a bispecific antibody of the present disclosure in the manufacture of a medicament for the treatment of cancer.
  • a CD47 antibody or antigen-binding fragment thereof of the present disclosure is capable of treating cancer, including, but not limited to, ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer , kidney cancer, pancreatic cancer, uterine cancer, liver cancer, bladder cancer, cervical cancer, oral cancer, brain Cancer, testicular cancer, skin cancer, thyroid cancer, and hematological malignancies.
  • the hematological malignancy is selected from the group consisting of myeloma, chronic leukemia, and acute leukemia.
  • Figure 1 is a CD47 antibody of the present disclosure enhancing antibody-dependent cellular phagocytosis (ADCP) of CCRF-CEM cells in vitro.
  • ADCP antibody-dependent cellular phagocytosis
  • Figure 2 is a erythrocyte agglutination assay of the CD47 antibody of the present disclosure.
  • Figure 3 is a red cell lysis assay of the CD47 antibody of the present disclosure.
  • Figure 4 is a pharmacokinetics of the CD47 antibody hu055-5 in vivo of the present disclosure.
  • Figure 5 is a pharmacokinetics of the CD47 antibody hu167-33 in vivo of the present disclosure.
  • CD47 is a widely expressed immunoglobulin on the surface of cell membranes, also known as integrin-related protein.
  • Singal regulatory protein (SIRP) is a member of the superfamily of inhibitory receptors and belongs to the immunoglobulin superfamily, which is mainly expressed on the surface of macrophages, dendritic cells and nerve cells. Contact regulates cell migration and phagocytic activity, immune homeostasis, and neural networks.
  • CD47 is an extracellular ligand of human signal-regulated protein alpha (SIRP ⁇ ), which reduces the phagocytic activity by binding to SIRP ⁇ on the surface of macrophages, thereby inhibiting the innate immune system. This signal is described as “ Don't eat me signal.
  • the antibody described in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain, respectively. , ⁇ chain, ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a ⁇ chain and a ⁇ chain by the difference of the constant region.
  • Each of the five types of Ig may have a kappa chain or a ⁇ chain.
  • the antibody light chain may further comprise a light chain constant region comprising a human or murine kappa, a constant region of a lambda chain or a mutant sequence thereof.
  • the antibody heavy chain may further comprise a heavy chain constant region comprising a constant region of IgG1, 2, 3, 4 of human or murine origin or a mutant sequence thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2. FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment thereof of the invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or in accordance with the numbering rules of kabat and chothia (HCDR1).
  • murine antibody is in this disclosure a monoclonal antibody to human CD47 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the CD47 antigen at the time of preparation, and then the antibody having the desired sequence or functional properties is isolated and expressed.
  • the murine CD47 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a mutated sequence thereof, or further comprising a murine IgG1, IgG2 The heavy chain constant region of IgG3 or its mutated sequence.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the mouse hybridoma cell, and the mouse variable region gene is cloned as needed, and the mouse variable region is cloned.
  • the gene is ligated into the vector after being ligated into a chimeric gene, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or a mutated sequence thereof.
  • the antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a mutated sequence thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or is significantly reduced using amino acid mutations ADCC (antibody-dependent cell-mediated cytotoxicity) toxic IgG4 constant region.
  • humanized antibody also referred to as a CDR-grafted antibody, refers to an antibody produced by engrafting a mouse CDR sequence into a human antibody variable region framework (FR). It is possible to overcome the side effects of chimeric antibodies on the human body induced by carrying a large amount of mouse protein components.
  • Human germline antibody variable region framework (FR) sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 014441351.
  • IMGT ImMunoGeneTics
  • the human antibody variable region can be reverse mutated (backmuted) to maintain activity.
  • the "antigen-binding fragment” described in the present specification means a Fab fragment having antigen-binding activity, a Fab' fragment, an F(ab')2 fragment, and an Fv fragment, scFv fragment which binds to human CD47.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the most antigen binding sites.
  • Small antibody fragment In general, Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (scFv).
  • binding to CD47 refers to the ability to interact with human CD47.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and that is recognized by an antibody or antigen-binding fragment of the present disclosure.
  • the "ADCC” described in the present disclosure ie, antibody-dependent cell-mediated cytotoxicity, is an antibody-dependent cell-mediated cytotoxic effect, in which a cell expressing an Fc receptor directly kills an antibody by coating the Fc portion of the antibody. Target cells.
  • the fusion protein described in the present disclosure is a protein product which is co-expressed by the two genes obtained by DNA recombination.
  • Recombinant CD47 extracellular domain Fc fusion protein A fusion protein that co-expresses the extracellular domain of CD47 and the human antibody Fc fragment by DNA recombination.
  • the CD47 extracellular region refers to a portion of the CD47 protein expressed outside the cell membrane, and the sequence is shown in SEQ ID NO: 1.
  • a mouse can be immunized with human CD47 or a fragment thereof, and the obtained antibody can be renatured, purified, and subjected to amino acid sequencing by a conventional method.
  • the antigen-binding fragment can also be prepared by a conventional method.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region. Human FR germline sequences are available on the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN 014441351.
  • the antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified by conventional methods.
  • a cDNA sequence encoding a heavy chain CDR and a light chain can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human CD47. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium secreted with the antibody can be purified by a conventional technique.
  • the column is passed through a Protein A or G Sepharose FF column with an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted with an acidic buffer, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -80 ° C, or lyophilized.
  • An antibody of the present disclosure refers to a monoclonal antibody.
  • the monoclonal antibody or mAb according to the present disclosure refers to an antibody obtained from a single clonal cell strain, and the cell strain is not limited to a eukaryotic, prokaryotic or phage clonal cell strain.
  • Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, for example, hybridoma technology, recombinant techniques, phage display technology, synthetic techniques (e.g., CDR-grafting), or other prior art techniques.
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Tester, Contact of cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluids that are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administration of a therapeutic agent for internal or external use to a patient, such as a composition comprising a CD47 antibody or antigen-binding fragment thereof, the patient having one or more symptoms of the disease.
  • a therapeutic agent is administered in a subject or population to be treated in an amount effective to alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any clinically measurable extent.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount” effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect on the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition.
  • embodiments of the present disclosure eg, methods of treatment or preparations
  • any statistical test methods known in the art such as Student's t test, chi-square test, Mann's and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • “Mutation” in “mutation sequence” as described in the present disclosure includes, but is not limited to, “backmutation”, “conservative modification” or “conservative substitution or substitution”.
  • “Conservatively modified” or “conservative substitution or substitution” as used in the present disclosure refers to other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.)
  • the amino acids allow frequent changes without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • the "mutation sequence" as used in the present invention means that the nucleotide sequence and the amino acid sequence of the present invention are different from the nucleotide sequence and the amino acid sequence of the present invention by appropriately modifying a nucleotide sequence and an amino acid sequence, such as substitution, insertion or deletion. Nucleotide sequence and amino acid sequence of percent sequence identity.
  • the sequence identity described in the present invention may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Sequence comparisons and percent identity determination between the two sequences can be performed by default settings of the BLASTN/BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • the effective amount for a particular patient or veterinary subject may be based on the following Mere changes: such as the condition to be treated, the overall health of the patient, the method of administration and dosage, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside of a living being, cell, or human, depending on the background.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the background.
  • the expression "cell”, “cell line” and “cell culture” are used interchangeably and all such names include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to intentional or unintentional mutations. Mutant progeny having the same function or biological activity as screened for in the originally transformed cell are included.
  • PCR polymerase chain reaction
  • RNA Ribonucleic acid
  • DNA Ribonucleic acid
  • RNA Ribonucleic acid
  • DNA DNA
  • PCR Ribonucleic acid
  • the 5' terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used herein is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which comprises using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or Produce a specific portion of the nucleic acid.
  • “Pharmaceutical composition” means a mixture comprising one or more of the CD47 antibodies or antigen-binding fragments thereof described herein and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • CD47 human CD47 protein, Uniprot number: Q08722
  • amino acid sequences of the antigens and detection proteins involved in the present disclosure were designed, and optionally, different tags such as his tags were fused on the basis of CD47 protein. Or Fc, etc.
  • CD47-ECD-His His-tagged CD47 protein extracellular domain
  • CD47 extracellular domain and human IgG1 Fc fusion protein (CD47-ECD-Fc) as immunogen and detection reagent:
  • SIRP ⁇ and human IgG1 Fc fusion protein (SIRP ⁇ -Fc) as binding and blocking detection reagents:
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, and the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM.
  • the nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes. The displaced supernatant sample was placed on an IMAC column. The column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline. The column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected.
  • the protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex 200 (GE, 28-9893-35), and the mobile phase was PBS.
  • the polymer peak was removed and the elution peak was collected.
  • the obtained protein was identified by electrophoresis, peptide mapping, and LC-MS, and then used.
  • CD47-ECD-His SEQ ID NO: 1
  • CD47-ECD-His can also be used as an immunogen to stimulate mouse immunization by in vitro chemical reaction with KLH.
  • the cell expression supernatant samples were centrifuged at high speed to remove impurities, and the supernatant was subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography.
  • the MabSelect Sure column was first regenerated with 0.1 M NaOH, rinsed with pure water, and the column was equilibrated with PBS. The supernatant was combined and washed with PBS until the A280 reading dropped to baseline.
  • the protein of interest was eluted with 0.1 M acetate buffer at pH 3.5 and neutralized with 1 M Tris-HCl. The eluted sample was appropriately concentrated and further purified by PBS-equilibrated gel chromatography Superdex 200 (GE, 28-9893-35), and the receiving tube in which the protein of interest was collected was concentrated to an appropriate concentration.
  • This method was used to purify the CD47-ECD-Fc (SEQ ID NO: 2) and SIRP ⁇ -Fc (SEQ ID NO: 3) fusion proteins, which can also be used to purify the humanized antibody proteins involved in the present disclosure.
  • Anti-human CD47 monoclonal antibodies are produced by immunizing mice.
  • Experimental SJL white mice female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. Mice that have adapted to the environment are immunized according to different protocols, 6-10 per group.
  • the immune antigen may be CD47-ECD-Fc, CD47-ECD-His6, CD47-ECD-His6-KLH, etc., and a single agent may be combined with different immunological adjuvants or different types of immunogens for cross-immunization.
  • the immune site can be subcutaneously or subcutaneously, or alternately immunized in two locations.
  • An exemplary immunization method is emulsified with Freund's adjuvant (sigma Lot Num: F5881/F5506): the first use of Freund's complete adjuvant (CFA), and the rest of the booster with Freund's incomplete adjuvant (IFA).
  • the ratio of antigen to adjuvant was 1:1, 100 ⁇ g/only (first aid), 50 ⁇ g/only (boost boost).
  • IP intraperitoneal
  • Titermax Titermax
  • the ratio of antigen to adjuvant (titermax) was 1:1, and the ratio of antigen to adjuvant (Alum) was 3:1, 10-20 ⁇ g/only (first exempt), and 5 ⁇ g/only (boosting).
  • Spleen cell fusion was performed in mice with high antibody titer in serum (see Test Example 1, ELISA method in combination with CD47) and titer-to-platform, and intraperitoneal injection of CD47-ECD-Fc was performed 72 hours before fusion.
  • Select mice Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • the fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI) and dispensed into 96-well cell culture plates (1 ⁇ 10 5 /150 ⁇ l).
  • HAT complete medium was added, 50 ⁇ l/well, and incubated at 37 ° C, 5% CO 2 .
  • the whole medium was changed, and the medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), 200 ⁇ l/well, Incubate at 37 ° C, 5% CO 2 .
  • ELISA method for binding to CD47 was performed according to cell growth density (see Test Example 1).
  • the positive well cells combined with ELISA were subjected to blocking ELISA for CD47/SIRP ⁇ binding (see Test Example 2), and the positive wells were exchanged for fluidization, and expanded into 24-well plates according to cell density.
  • the cell line transferred into the 24-well plate was subjected to retesting and then subjected to seed conservation and first subcloning.
  • the first subcloning screen (see Test Example 1) was positive for conservation and subjected to a second or third subcloning until a single cell clone was obtained. Multiple fusions were obtained to obtain hybridoma cells that blocked the binding effect of CD47 and SIRP ⁇ (see Test Example 2).
  • Hybridoma clones 055 and 167 were screened by blocking experiments and binding experiments, and antibodies were further prepared by serum-free cell culture, and the antibodies were purified according to the purification examples for use in the test examples.
  • the murine anti-variable region sequences of hybridoma clone 055 were determined as follows:
  • DIVMTQSPSYLTVTAGEKVTMSC KSSQSLLTSGKQKNYLT WYQQKPGQPPKLLIY WASTRES GVPERFRGSGSGTDFTLTISSVQAEDLAVYYC QNDYSYPLT FGAGTKLELN (SEQ ID NO: 5).
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the murine anti-variable region sequences of hybridoma clone 167 are as follows:
  • the two murine antibodies were selected by using the IMGT human antibody heavy light chain variable region germline gene database and MOE software to select the heavy and light chain variable region germline genes with high homology to 055 and 167 as templates.
  • the CDRs were each transplanted into the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acid residues are determined and annotated by the Kabat numbering system.
  • the humanized light chain template of murine antibody 055 is IGKV4-1*01 and hjk2.1, and the humanized heavy chain template is IGHV1-3*01 and hjh6.1.
  • the sequence of humanized variable region is as follows:
  • DIVMTQSPDSLAVSLGERATINC KSSQSLLTSGKQKNYLT WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QNDYSYPLT FGQGTKLEIK (SEQ ID NO: 21).
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • D66E indicates that 66 bits of D are mutated back to E according to the Kabat numbering system. Grafted represents the murine antibody CDRs inserted into the human germline FR region sequence.
  • the humanized template of hybridoma clone 055 was subjected to back mutation design, and different back mutations were combined into different humanized antibodies, as shown in Table 3.
  • Hu055_VL.1 hu055_VL.1A hu055_VH.1 Hu055-1 Hu055-8 hu055_VH.1A Hu055-2 Hu055-9 hu055_VH.1B Hu055-3 Hu055-10 hu055_VH.1C Hu055-4 Hu055-11 hu055_VH.1D Hu055-5 Hu055-12 hu055_VH.1E Hu055-6 Hu055-13 hu055_VH.1F Hu055-7 Hu055-14
  • the humanized light chain template of mouse antibody 167 is IGKV1-39*01 and hjk4.1, humanized heavy chain model
  • the plates are IGHV1-2*02 and hjh6.1, and the humanized variable region sequences are as follows:
  • V58I indicates that 58-bit V is mutated back to I according to the Kabat numbering system. Grafting representative mouse antibody The CDRs are inserted into the human germline FR region sequence.
  • the humanized template of hybridoma clone 167 was subjected to a back mutation design, and different back mutations were combined into different humanized antibodies, as shown in Table 5.
  • Hu167_VL.1 hu167_VL.1A hu167_VL.1B hu167_VL.1C hu167_VL.1D hu167_VH.1 Hu167-1 Hu167-2 Hu167-3 Hu167-4 Hu167-5 hu167_VH.1A Hu167-6 Hu167-7 Hu167-8 Hu167-9 Hu167-10 hu167_VH.1B Hu167-11 Hu167-12 Hu167-13 Hu167-14 Hu167-15 hu167_VH.1C Hu167-16 Hu167-17 Hu167-18 Hu167-19 Hu167-20 hu167_VH.1D Hu167-21 Hu167-22 Hu167-23 Hu167-24 Hu167-25 hu167_VH.1E Hu167-26 Hu167-27 Hu167-28 Hu167-29 Hu167-30
  • the ELISA method combined with CD47 was performed (see Test Example 1), and the positive hole cells combined with the ELISA were subjected to blocking ELISA detection of CD47/SIRP ⁇ binding (see Test Example 2, the results are shown in Table 6), and the maximum value according to the test results was preferred.
  • the degree of back mutation combination that retains antibody binding ability and blocking ability.
  • the different combinations of 055 humanized antibodies basically maintained good in vitro blocking activity, and the affinity of different humanized antibodies was compared by Biacore. Some data are shown in Table 8. The results show that the humanized antibody combinations are maintained for CD47 has a good binding ability, especially hu055-3, hu055-4, hu055-5, hu055-6, hu055-7 affinity and chimeric antibody ch-055 are substantially similar.
  • the primers were designed to construct the VH/VK gene fragment of each humanized antibody, and then homologously recombined with the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) to construct the full-length antibody expression vector VH- CH1-FC-pHr/VK-CL-pHr.
  • the IgG4-AA antibody form can be obtained by simple point mutation of the IgG4 antibody form, and IgG4-AA represents the F234A and L235A mutations, and it is shown that it further reduces the binding ability of IgG4-Fc to FcyR, further reducing ADCC/CDC.
  • IgG4-S228P represents a mutation in the amino acid S at position 228 of the wild-type IgG4 hinge region to P, which can avoid a Fab-exchange-induced mismatch in the native IgG4 antibody.
  • the heavy chain constant region sequences of IgG4-S228P are as follows:
  • IgG4-AA represents the heavy chain constant region sequence obtained by mutation of F234A and L235A in the underlined portion of the above sequence, and specifically the heavy chain constant region sequence of IgG4-AA (including S228P) in the present disclosure is as follows:
  • the light chain (Kappa chain) constant region sequence of the antibody is as follows:
  • Hu167-33 antibody form IgG4AA
  • Hu055-5 antibody form IgG4AA
  • Hu055-5 heavy chain sequence
  • Test Example 1 ELISA assay of CD47 antibody binding to CD47 protein
  • CD47-ECD-His SEQ ID NO: 1
  • PBS PBS
  • 96-well ELISA plate Costar, CAT #3590
  • H+L horseradish peroxidase-goat anti-human antibody
  • Jackson, CAT#109-035-088 horseradish peroxidase-goat anti-human antibody
  • the ability of anti-CD47 antibodies to block the binding of SIRP ⁇ and CD47 was determined by measuring the amount of binding of CD47 to SIRP ⁇ in the presence of antibodies.
  • the CD47-ECD-Fc (SEQ ID NO. 2) was diluted with phosphate buffer to 1 ⁇ g/ml and coated on a 96-well ELISA plate (Costar, CAT #3590). After washing the plate was blocked, biotin-labeled SIRP ⁇ -Fc was added.
  • CCRF-CEM cells (ATCC, CAT#CRM-CCL-119) were cultured in RPMI medium (Hyclone, CAT#SH30809.01B) (containing 10% fetal bovine serum), 1 ⁇ 10 6 cells/ml CCRF-CEM cells. After blocking with 5% BSA, add CD47 antibody sample to 1 ⁇ g/ml, wash twice, then add Alexa Fluor 488-goat anti-human (H+L) antibody (Invitrogen, CAT#A11013), wash twice, flow The cytometer reads the fluorescent signal value.
  • the FACS test results showed that the humanized antibody involved in the present disclosure has strong binding ability to the natural CD47 on the cell surface, and is superior to the binding ability of the control antibody B6H12 (US9017675B). The results are shown in Table 7.
  • Test Example 4 CD47 antibody enhances antibody-dependent cell-mediated phagocytosis (ADCP) in CCRF-CEM cells in vitro
  • Monocytes were isolated from human peripheral blood mononuclear cells and cultured in six-well plates in RPMI medium (Hyclone, CAT #SH30809.01B) (containing 10% fetal bovine serum) for 7 days. 1 ⁇ 10 6 cells/ml CCRF-CEM cells were added to CFSE (abcam, ab113853) to a final concentration of 0.02 ⁇ M, and incubated at 37 ° C for 5-10 minutes. After washing twice, a six-well plate containing differentiated monocytes was added and added. Different concentrations of anti-CD47 antibody samples were incubated at 37 ° C for 4 hours.
  • test results are shown in Figure 1, which shows that the addition of the humanized antibody of the present disclosure can effectively promote ADCP with a certain dose effect.
  • Fresh human blood was diluted 1 time with phosphate buffer to a 96-well plate, and anti-CD47 antibody samples diluted at different concentrations were added and incubated at 37 ° C for 4 hours after shaking. The supernatant was aspirated, and the cell sedimentation in different wells of the 96-well plate was observed. The different concentrations of each antibody in each well were used to cause red blood cell (RBC) agglutination, and the results were shown in the manner shown in Fig. 2, and the results were statistically performed. Comparison.
  • RBC red blood cell
  • Antibody-induced agglutination of red blood cells is a common phenomenon in the field of CD47.
  • the patented antibody 5F9 US9017675B, see patent vh2, SEQ ID NO 37 and vl2, SEQ ID NO 42
  • the erythrocyte agglutination phenomenon under the same conditions, the hu167-33 of the present disclosure does not cause erythrocyte agglutination at different concentrations tested, and another antibody molecule hu055-5 can cause erythrocyte agglutination, but the concentration range of red blood cell agglutination is significantly smaller than 5F9.
  • the results are shown in Figure 2.
  • the potential advantages of the antibodies of the present disclosure in terms of safety are suggested.
  • Fresh human blood was diluted 1 times with phosphate buffer to 96-well plate, 0.2mg/ml anti-CD47 antibody sample or Triton X-100 diluted at different concentrations, shaken at 37 ° C for 4 hours after shaking, centrifuged to extract supernatant A 96-well plate was used to measure OD540 on a microplate reader. The results are shown in Figure 3, which shows that in vitro addition of the anti-CD47 antibody shown in the present disclosure does not cause lysis of red blood cells.
  • Human Fab capture molecules were covalently coupled to a CM5 biosensor chip (Cat. #BR-1000-12, according to the method described in the Human Fab Capture Kit (Cat. #28-9583-25, GE) instructions. GE), thereby affinity capture of the antibody to be tested. Then, the surface of the chip was passed through human CD47-his (Yiqiao Shenzhou, CAT#12283-HCCH-50) antigen, and the reaction signal was detected by Biacore instrument in real time to obtain the binding and dissociation curves. The affinity values were obtained by fitting, see the following table. 8, Table 9. After each cycle of dissociation was completed in the experiment, the biochip was washed and regenerated using the regeneration solution disposed in the human Fab capture kit.
  • the corresponding humanized antibody of the disclosed antibody was detected by Biacore and found to have a higher affinity with the antigen, and further selected to optimize the humanized antibody with high affinity and less back mutation.
  • Animals were administered serum at different time points after administration of different concentrations of anti-CD47 antibody samples. According to the method of ELISA test of test case 1 anti-CD47 antibody binding to CD47 protein, the standard curve of different samples was used, and serum sample 1:1000 instead of anti-CD47 antibody. After adding the reaction system, the concentration of anti-CD47 antibody in serum was converted according to OD450 at different time points, and the obtained data were analyzed by Phoenix WinNonlin software to calculate the pharmacokinetic parameters.
  • the drug metabolism parameters of hu055-5 at 3mpk dose were determined in rats. The results showed that hu055-5 had good drug metabolism in rats, and the average drug half-life t1/2 was about 285 hours, as shown in Figure 4, suggesting that the antibody It is stable in rats.
  • the drug of the present disclosure hu167-33 was administered in mice. Metabolic test, the drug half-life t1/2 was found to be about 192 hours at a dose of 10 mpk, as shown in Figure 5, showing that the antibody was stable in mice.

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Abstract

本发明公开了CD47抗体、其抗原结合片段及其医药用途。进一步地，本发明公开了包含所述CD47抗体CDR区的嵌合抗体、人源化抗体，包含所述CD47抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。

Description

CD47抗体、其抗原结合片段及其医药用途 技术领域
本公开涉及生物学和医学领域；更具体而言，本公开涉及一种CD47抗体及其抗原结合片段、其嵌合抗体、人源化抗体，以及包含所述CD47抗体或其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。
背景技术
随着人类对免疫***和肿瘤发生机制上认识的深入，肿瘤免疫疗法已经日益成为人类对抗肿瘤的有力武器。
肿瘤靶向单克隆抗体是肿瘤免疫治疗领域的重要手段之一。巨噬细胞发挥吞噬效应需要两个信号同时起作用：一个是靶向细胞表面的“eat me”信号的激活，另一个是同一细胞表面“don't eat me”信号的失活。任何一个信号的缺少都不足以引发吞噬效应的发生。越来越多的证据表明，CD47是一类“don't eat me”信号，它通过与巨噬细胞表面的SIRP-α相互结合而抑制巨噬细胞的吞噬功能。
CD47是免疫球蛋白Ig超家族成员，其广泛表达于不同组织的细胞表面，比如红细胞、淋巴细胞、血小板、肝细胞。CD47在各种肿瘤细胞上有较高表达，有研究显示多种肿瘤细胞表面的CD47表达量高于正常细胞平均约3倍多。此外，癌细胞表达大量CD47的患者相比于CD47表达低水平的患者具有更短的生存期。目前研究已经发现，阻断性抗CD47单克隆抗体在肿瘤治疗方面有着非常良好的效果，但是这一过程的作用机制尚不清楚。
CD47作为癌症治疗的靶点具有以下不可比拟的优势：
1.它广泛地表达于各类癌细胞表面，因此可以用于治疗各种类型的癌症；
2.正常细胞由于缺乏“eat me”信号，因此单单阻断CD47并不能引发巨噬细胞对正常细胞的吞噬效应。因此，CD47阻断剂的副作用十分小。前期的很多基础实验数据已经支持了这一观点。研究者通过小鼠异体肿瘤移植模型说明了CD47阻断的有效性与安全性。
目前已有相关的CD47专利，如WO2016065329、WO2016109415、WO2014087248和WO2014093678。有效阻断CD47与SIRP-α之间的结合并促进体内巨噬细胞对于肿瘤细胞的吞噬，具有良好的临床前景。
发明内容
本公开的第一个方面提供一种CD47抗体或其抗原结合片段，其包含：抗体重链可变区的CDR区(以下称为HCDR)和/或抗体轻链可变区的CDR区(以下称为LCDR)。
在一些实施方案中，HCDR选自以下的任一个或多个CDR：SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、以及与SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16具有至少85％序列同一性的CDR。
在另一些实施方案中，LCDR选自以下的任一个或多个CDR：SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、以及与SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19具有至少85％序列同一性的CDR。
在本公开的上下文中，表述“至少85％序列同一性”是指至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％的序列同一性。
在本公开一个具体的实施方案中，提供了一种CD47的人源化抗体或其抗原结合片段，其包含抗体重链可变区；其中所述的抗体重链可变区包含SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10所示的HCDR1、HCDR2和HCDR3，或与SEQ ID NO:8、SEQ ID NO:9或SEQ ID NO:10具有至少85％序列同一性的CDR。
在另一个具体的实施方案中，提供了一种CD47的人源化抗体或其抗原结合片段，其包含抗体重链可变区；其中所述的抗体重链可变区包含SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的HCDR1、HCDR2和HCDR3，或与SEQ ID NO:14、SEQ ID NO:15或SEQ ID NO:16具有至少85％序列同一性的CDR。
在本公开一个具体的实施方案中，提供了一种CD47的人源化抗体或其抗原结合片段，其包含抗体轻链可变区；其中所述的抗体轻链可变区包含SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的LCDR1、LCDR2和LCDR3，或与SEQ ID NO:11、SEQ ID NO:12或SEQ ID NO:13具有至少85％序列同一性的CDR。
在另一个具体的实施方案中，提供了一种CD47的人源化抗体或其抗原结合片段，其包含抗体轻链可变区；其中所述的抗体轻链可变区包含SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示的LCDR1、LCDR2和LCDR3，或与SEQ ID NO:17、SEQ ID NO:18或SEQ ID NO:19具有至少85％序列同一性的CDR。
在本公开一个具体的实施方案中，提供了一种CD47抗体或其抗原结合片段，其为鼠源抗体或其抗原结合片段。
在本公开一个具体的实施方案中，提供了一种CD47抗体或其抗原结合片段，所述抗体的轻链可变区包含鼠源轻链FR区或其突变序列；所述抗体的重链可变区包含鼠源重链FR区或其突变序列。
在本公开一个具体的实施方案中，提供了一种CD47抗体或其抗原结合片段，所述抗体包含SEQ ID NO:4所示的重链可变区和SEQ ID NO:5所示的轻链可变区。在另一个实施方案中，提供了一种CD47抗体或其抗原结合片段，所述CD47抗体包含SEQ ID NO:6所示的重链可变区和SEQ ID NO:7所示的轻链可变区。
在本公开一个具体的实施方案中，提供了一种CD47抗体或其抗原结合片段，其为嵌合抗体或其抗原结合片段。
在本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其重链可变区上重链FR区的序列选自人种系重链IGHV1-3*01和hjh6.1的组合及其突变序列，或选自人种系重链IGHV1-2*02和hjh6.1的组合及其突变序列。
本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其轻链可变区上轻链FR区的序列选自人种系轻链IGKV4-1*01和hjk2.1的组合及其突变序列，或选自人种系轻链IGKV1-39*01和hjk4.1的组合及其突变序列。
在本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，所述人源化抗体或其抗原结合片段的可变区上FR区包含0至10个氨基酸的回复突变，包括但不限于0、1、2、3、4、5、6、7、8、9或10个氨基酸的回复突变。
在本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其包含SEQ ID NO:20所示的重链可变区或其突变序列和SEQ ID NO:21所示的轻链可变区或其突变序列。
在本公开另一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其包含SEQ ID NO:22所示的重链可变区或其突变序列和SEQ ID NO:23所示的轻链可变区或其突变序列。
在本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其包含轻链可变区突变序列，所述轻链可变区突变序列是在SEQ ID NO:21所示的轻链可变区基础上，将第66位氨基酸残基D替换为E(以D66E表示，以下同)。
在本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其包含重链可变区突变序列，所述重链可变区突变序列是在SEQ ID NO:20所示的重链可变区基础上，包含选自以下的突变：R72A、M48I、E46D、V68A、I70L、R38K、R67K、A97S及其组合。在一个具体的实施方案中，所述重链可变区突变序列是在SEQ ID NO:20所示的重链可变区基础上包含选自以下的突变：R72A、M48I、E46D、V68A、I70L及其组合。
在本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其包含轻链可变区突变序列，所述轻链可变区突变序列是在SEQ ID NO:23所示的轻链可变区基础上，包含选自以下的突变：V58I、I2V、M4I、Q38E、A43T、P44H及其组合。在一个具体的实施方案中，所述轻链可变区突变序列是在SEQ ID NO:23所示的轻链可变区基础上，包含选自以下的突变I2V、M4I及其组合。
在本公开一个具体的实施方案中，提供了一种CD47人源化抗体或其抗原结合片段，其包含重链可变区突变序列，所述重链可变区突变序列是在SEQ ID NO:22所示的重链可变区基础上，包含选自以下的突变：R72V、M48V、V68A、M70L、 T74K、A40R、R38K、R67K及其组合。
在本公开一个具体的实施方案中，提供了一种CD47抗体或其抗原结合片段，其中：所述人源化抗体可变区包含：SEQ ID NO:26所示的重链可变区或其突变序列，和SEQ ID NO:27所示的轻链可变区或其突变序列；或
所述人源化抗体可变区包含：SEQ ID NO:28所示的重链可变区或其突变序列，和SEQ ID NO:29所示的轻链可变区或其突变序列。
在本公开一个具体的实施方案中，提供了一种CD47嵌合抗体或CD47人源化抗体，其重链包含：
·人源IgG1、IgG2、IgG3或IgG4的重链恒定区或其突变序列；和
·人源κ链、λ链的轻链恒定区或其突变序列。
在本公开一个具体的实施方案中，提供了一种CD47嵌合抗体或CD47人源化抗体，其重链包含：
·人源IgG4的重链恒定区或其突变序列，和
·人源κ链的轻链恒定区或其突变序列。
在本公开一个具体的实施方案中，提供了一种CD47嵌合抗体或CD47人源化抗体，其重链包含：含有F234A和L235A突变的人源IgG4重链恒定区(如SEQ ID NO:30)。
本公开进一步提供一种药物组合物，其含有：
·治疗有效量的根据本公开的CD47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。
根据本公开的CD47抗体或其抗原结合片段可以作为药物组合物中唯一的药物活性成分而配制于组合物中，或者可以与其它活性组分组合配制在组合物中。
本文所用的“载体”包括当其以所用剂量和浓度与细胞或哺乳动物接触时没有毒性的药学上可接受的载体、稀释剂或赋形剂。药学上可接受的载体通常是指缓冲水溶液。药学上可接受的载体的例子包括但不限于缓冲液；抗氧化剂；多肽；蛋白质；亲水性聚合物；氨基酸；糖；螯合剂；糖醇；离子；表面活性剂。再比如，药学上可接受的载体、稀释剂或赋形剂还可以是脂质体的形式，包括以癌组织为靶标的脂质体也可以用作药物学上可接受的载体。这些都可以依据本领域技术人员已知的方法来制备。
“治疗有效量”可以通过在已知的体外和体内***中测试本公开的CD47抗体或其抗原结合片段而确定，例如根据在动物体上所证实的有效量，然后由此推断出用于人的量。
为了全身性的、外部的或局部的给药，将本公开的CD47抗体或其抗原结合片段与合适的药物载体混合，从而形成药物组合物。组合物中CD47抗体或其抗原结合片段的浓度取决于抗体或其抗原结合片段的吸收、失活、代谢率、给药方案、给药量、剂型以及本领域技术人员已知的其它因素。
本公开的药物组合物可以通过本领域技术人员已知的任何途径来施用，例如但不限于口服、局部给药、脑内给药、眼内给药、心内给药、鞘内给药、静脉内给药、肌内给药、腹腔内给药、皮内给药、气管内给药及其组合。
最合适的给药途径将依据预期用途而变化。例如，对于各种癌症的治疗，例如乳腺癌、膀胱癌和肠胃癌的治疗，可以采用局部给药，包括施用至肿瘤生长的位点是优选的，因为优点在于本公开的CD47抗体或其抗原结合片段能够以较高的浓度被施用。
用于肠胃外、皮内、皮下的溶液或悬浮液，包括但不限于：无菌稀释剂；抗菌剂；抗氧化剂；缓冲液；以及用于调整渗透压的试剂。肠胃外给药的制剂可以装入玻璃、塑料或其他合适材料制成的安瓿、注射器或者单剂量或多次剂量的小瓶中。
本公开进一步提供一种双特异性抗体，其包含根据本公开的CD47抗体或其抗原结合片段。
本公开进一步提供一种分离的核酸，其编码根据本公开的CD47抗体或其抗原结合片段。
本公开进一步提供一种表达载体，其表达根据本公开的CD47抗体或其抗原结合片段。
本公开进一步提供一种表达载体，其包含根据本公开的分离的核酸。
本公开进一步提供一种宿主细胞，其转化有根据本公开的表达载体。
在具体的实施方案中，所述宿主细胞选自原核细胞或真核细胞。在具体的实施方案中，所述宿主细胞是真核细胞。在具体的实施方案中，所述宿主细胞是哺乳动物细胞。
本公开进一步提供一种用于制备CD47抗体及其抗原结合片段的方法，包括：
·在适合表达本公开的CD47抗体或其抗原结合片段的条件下，培养如上所述的宿主细胞，以及
·自该宿主细胞中分离本公开的CD47抗体或其抗原结合片段。
本公开进一步提供一种用于抑制受试者中的肿瘤细胞的生长的方法，包括：
向所述受试者施用本公开的CD47抗体或其抗原结合片段、或本公开的药物组合物。
本公开进一步提供了本公开的CD47抗体或其抗原结合片段在制备用于治疗癌症的药物中的用途。根据另一方面，提供了本公开的药物组合物在制备用于治疗癌症的药物中的用途。根据又一方面，提供了本公开的双特异性抗体在制备用于治疗癌症的药物中的用途。
在一些实施方案中，本公开的CD47抗体或其抗原结合片段能够治疗癌症，所述癌症包括，但不限于：卵巢癌、黑色素瘤、***癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子***、口腔癌、脑 癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤。在一些实施方案中，血液学恶性肿瘤选自骨髓瘤、慢性白血病和急性白血病。
附图说明
图1为本公开CD47抗体在体外增强CCRF-CEM细胞的抗体依赖性细胞的吞噬作用(ADCP)。
Figure PCTCN2017113081-appb-000001
表示hu055-5；
Figure PCTCN2017113081-appb-000002
表示hu167-33；
Figure PCTCN2017113081-appb-000003
表示5F9。
图2为本公开CD47抗体红细胞凝集实验。
图3为本公开CD47抗体红细胞裂解实验。
图4为本公开CD47抗体hu055-5大鼠体内药代动力学。
图5为本公开CD47抗体hu167-33大鼠体内药代动力学。
具体实施方式
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
CD47是一种广泛表达的细胞膜表面的免疫球蛋白，又称整合素相关蛋白。信号调节蛋白(singal regulatory protein，SIRP)是抑制性受体超家族的一个成员，属于免疫球蛋白超家族，主要表达于巨噬细胞、树突状细胞和神经细胞表面，通过细胞表面受配体接触调节细胞的迁移和吞噬活性、免疫自稳及神经元网络。CD47是人信号调节蛋白α(SIRPα)的胞外配体，通过与巨噬细胞表面SIRPα结合传导抑制性信号而降低吞噬活性，进而对固有免疫***产生抑制作用，该信号被形象的描述为“don't eat me(别吃我)”信号。
本公开所述的抗体指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链、λ链。五类Ig中每类Ig都可以有κ链、λ链。
在本公开中，所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链的恒定区或其突变序列。
在本公开中，所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1,2,3,4的恒定区或其突变序列。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
发明所述的抗体或其抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3、HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人CD47的单克隆抗体。制备时用CD47抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体。在本公开一个优选的实施方案中，所述的鼠源CD47抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其突变序列的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其突变序列的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。在本发明优选的实施方案中，所述的嵌合抗体的抗体轻链进一步包含人源κ、λ链或其突变序列的轻链恒定区。所述的嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3或IgG4或其突变序列的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后显著降低ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4恒定区。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架(FR)中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的对人体的免疫副反应。人种系抗体可变区框架(FR)序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区进行反向突变(回复突变)，以保持活性。
本公开中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab’片段、F(ab’)2片段、以及与人CD47结合的Fv片段、scFv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，并具有全部抗原结合位点的最 小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(scFv)。
本公开的术语“与CD47结合”，指能与人CD47相互作用。
本公开的术语“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
本公开中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。
本公开中所述的融合蛋白是一种通过DNA重组，将得到的两个基因共表达的蛋白产物。重组CD47胞外区Fc融合蛋白通过DNA重组，把CD47胞外区和人抗体Fc片段共表达的融合蛋白。所述的CD47胞外区，是指CD47蛋白表达在细胞膜以外的部分，序列见SEQID NO:1。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，老鼠可以用人CD47或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链CDR和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的技术，哺乳动物表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人CD47特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌有抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的Protein A或G Sepharose FF柱进行过柱。洗去非特异性结合的组分。再用酸性缓冲液洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-80℃，或者冻干。
本公开的抗体指单克隆抗体。本公开所述的单克隆抗体或mAb，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、 细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施、研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，诸如包含CD47抗体或其抗原结合片段的组合物，所述患者具有一种或多种疾病症状。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物对患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解某个患者的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
本公开中所述的“突变序列”中的“突变”包括但不限于“回复突变”、“保守修饰”或“保守置换或取代”。本公开中所述的“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
本发明所述的“突变序列”是指对本发明的核苷酸序列和氨基酸序列进行适当的替换、***或缺失等突变修饰情况下，得到的与本发明的核苷酸序列和氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和氨基酸序列。本发明中所述的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因 素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据背景在生物、细胞或人体外产生的物质。“内源性”指根据背景在细胞、生物或人体内产生的物质。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
本文使用的“聚合酶链式反应”或“PCR”是指微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述CD47抗体或其抗原结合片段与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
实施例
以下结合实施例进一步描述本公开，但这些实施例并非限制本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；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。
实施例1.CD47抗原及检测用蛋白的制备
以UniProt Leukocyte surface antigen CD47(人CD47蛋白，Uniprot号：Q08722)作为CD47的模板，设计本公开涉及的抗原及检测用蛋白的氨基酸序列，可选的在CD47蛋白基础上融合不同的标签如his标签或Fc等。
1.带His标签的CD47蛋白胞外域(CD47-ECD-His)：
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNEHHHHHH(SEQ ID NO：1)。注释：划线部分为6×his标签。
2.CD47胞外域与人IgG1Fc融合蛋白(CD47-ECD-Fc)作为免疫原及检测试剂：
QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNEEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：2)。注释：划线部分为人IgG1-Fc部分。
3.人SIRPα与人IgG1Fc融合蛋白(SIRPα-Fc)作为结合及阻断检测试剂：
EEELQIIQPDKSVSVAAGESAILHCTITSLFPVGPIQWFRGAGPARVLIYNQRQGPFPRVTTVSETTKRENMDFSISISNITPADAGTYYCIKFRKGSPDTEFKSGAGTELSVRAKPSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO：3)。注释：划线部分为huamn-IgG1-Fc部分。
实施例2.CD47、SIRPα相关重组蛋白的纯化
1.带His标签的重组蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，并将缓冲液置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的上清样品上IMAC柱。用含有5mM咪唑的PBS溶液冲洗柱子，至A280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用凝胶层析Superdex200(GE，28-9893-35)进一步纯化，流动相为PBS。去除聚体峰，收集洗脱峰。所得到的蛋白经电泳、肽图、LC-MS鉴定为正确后，分装备用。
得到带His标签的CD47-ECD-His(SEQ ID NO：1)用于本公开抗体的免疫原或检测试剂。CD47-ECD-His也可以通过体外化学法与KLH进行偶联反应后作为免疫原刺激小鼠免疫。
2.CD47-ECD-Fc及SIRPα-Fc融合蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，上清进行MabSelect Sure(GE，17-5438-01)亲和层析。MabSelect Sure层析柱先用0.1M NaOH再生，利用纯水冲洗后用PBS平衡柱子，将上清结合后，利用PBS进行洗涤至A280读数降至基线。用0.1M醋酸缓冲液在pH3.5条件下洗脱目的蛋白，用1MTris-HCl中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE，28-9893-35)进一步纯化，合并收集目的蛋白所在接收管浓缩至适当浓度。
此方法用来纯化CD47-ECD-Fc(SEQ ID NO：2)及SIRPα-Fc(SEQ ID NO：3)融合蛋白，该方法也可以用来纯化本公开中涉及的人源化抗体蛋白。
实施例3.抗人CD47杂交瘤单克隆抗体的获得和制备
1.免疫
抗人CD47单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。
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％。将已适应环境的小鼠按不同方案免疫，每组6-10只。
免疫抗原可以是CD47-ECD-Fc、CD47-ECD-His6、CD47-ECD-His6-KLH等，可以单独一种试剂配合不同的免疫佐剂或者不同类型免疫原交叉免疫。免疫部位可以是腹腔或者背部皮下，或者两种位置交替免疫。示例性免疫方法如用弗氏佐剂(sigma Lot Num：F5881/F5506)乳化：首免用弗氏完全佐剂(CFA)，其余加强免疫用弗氏不完全佐剂(IFA)。抗原与佐剂比例为1:1，100μg/只(首免)，50μg/只(加强免疫)。第0天腹膜内(IP)注射100μg/只的乳化后抗原，首免后每两周一次，共6-8周。或用Titermax(sigma Lot Num：T2684)与Alum(Thremo Lot Num：77161)交叉免疫。抗原与佐剂(titermax)比例为1:1，抗原与佐剂(Alum)比例为3:1，10-20μg/只(首免)，5μg/只(加强免疫)。第0天腹膜内注射20/10μg/只的乳化后抗原，首免后每周一次，Titermax和Alum交替使用，共6至11周。免疫四周后，根据背部结块和腹部肿胀情况，选择背部或腹膜内注射抗原。
2.细胞融合
选择血清中抗体滴度高(见后面的测试例1，结合CD47的ELISA方法)并且滴度趋于平台的小鼠进行脾细胞融合，融合前72小时利用CD47-ECD-Fc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细胞Sp2/0细胞(
Figure PCTCN2017113081-appb-000004
CRL-8287TM)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RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×105/150μl/孔)，37℃，5％CO2孵育。 融合后的第5天加入HAT完全培养基，50μl/孔，37℃，5％CO2孵育。融合后第7天至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RPMI-1640培养基)，200μl/孔，37℃，5％CO2孵育。
3.杂交瘤细胞筛选
融合后第7-9天，根据细胞生长密度，进行结合CD47的ELISA方法检测(见测试例1)。并将结合ELISA检测的阳性孔细胞进行CD47/SIRPα结合的阻断ELISA检测(见测试例2)，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选(见测试例1)为阳性的进行保种，并进行第二次或第三次亚克隆，直至获得单细胞克隆。多次融合获得有阻断CD47和SIRPα结合效果(见测试例2)的杂交瘤细胞。
通过阻断实验和结合实验筛选得到杂交瘤克隆055和167，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
测得杂交瘤克隆055的鼠抗可变区序列如下：
克隆055的鼠源重链可变区序列：
Figure PCTCN2017113081-appb-000005
克隆055的鼠源轻链可变区序列：
DIVMTQSPSYLTVTAGEKVTMSCKSSQSLLTSGKQKNYLTWYQQKPGQPPKLLIYWASTRESGVPERFRGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLELN(SEQ ID NO：5)。注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
杂交瘤克隆167的鼠抗可变区序列如下：
克隆167的鼠源重链可变区序列：
Figure PCTCN2017113081-appb-000006
克隆167的鼠源轻链可变区序列：
DVQITQSPSYLAASPGETITINCRTSKSISKFLAWYQEKPGKTHKLLIYSGSTLQ SGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNEYPWTFGGGTKLEIK(SEQID NO：7)。注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
表1.各重链及轻链CDR区序列
Figure PCTCN2017113081-appb-000007
Figure PCTCN2017113081-appb-000008
实施例4.抗人CD47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，分别挑选与055和167同源性高的重轻链可变区种系基因作为模板，将这两个鼠源抗体的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。氨基酸残基由Kabat编号***确定并注释。
1.杂交瘤克隆055人源化
1.1杂交瘤克隆055人源化构架选择
鼠源抗体055的人源化轻链模板为IGKV4-1*01和hjk2.1，人源化重链模板为IGHV1-3*01和hjh6.1，人源化可变区序列如下：
hu055VH-CDR嫁接：
Figure PCTCN2017113081-appb-000009
hu055VL CDR嫁接：
DIVMTQSPDSLAVSLGERATINCKSSQSLLTSGKQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGQGTKLEIK(SEQ ID NO：21)。注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
1.2杂交瘤克隆055的人源化模板选择和回复突变设计。
表2.杂交瘤克隆055的人源化模板选择和回复突变设计
Figure PCTCN2017113081-appb-000010
注：如D66E表示依照Kabat编号***，将66位D突变回E。嫁接(Grafted)代表鼠抗体CDR植入人种系FR区序列。
1.3杂交瘤克隆055的人源化
杂交瘤克隆055的人源化模板进行回复突变设计，不同的回复突变组合成不同的人源化抗体，具体见表3。
表3：鼠抗055人源化序列组合
  hu055_VL.1 hu055_VL.1A
hu055_VH.1 hu055-1 hu055-8
hu055_VH.1A hu055-2 hu055-9
hu055_VH.1B hu055-3 hu055-10
hu055_VH.1C hu055-4 hu055-11
hu055_VH.1D hu055-5 hu055-12
hu055_VH.1E hu055-6 hu055-13
hu055_VH.1F hu055-7 hu055-14
2.杂交瘤克隆167人源化
2.1鼠源抗体167的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模
板为IGHV1-2*02和hjh6.1，人源化可变区序列如下：
Hu167VH-CDR嫁接
Figure PCTCN2017113081-appb-000011
Hu167VL-CDR嫁接
Figure PCTCN2017113081-appb-000012
2.2杂交瘤克隆167的人源化模板选择和回复突变设计，见下表4：
表4.鼠抗167人源化序列组合
Figure PCTCN2017113081-appb-000013
注：如V58I表示依照Kabat编号***，将58位V突变回I。嫁接代表鼠抗体 CDR植入人种系FR区序列。
2.3杂交瘤克隆167的人源化
杂交瘤克隆167的人源化模板进行回复突变设计，不同的回复突变组合成不同的人源化抗体，具体见表5。
表5.鼠抗167人源化序列组合
  hu167_VL.1 hu167_VL.1A hu167_VL.1B hu167_VL.1C hu167_VL.1D
hu167_VH.1 hu167-1 hu167-2 hu167-3 hu167-4 hu167-5
hu167_VH.1A hu167-6 hu167-7 hu167-8 hu167-9 hu167-10
hu167_VH.1B hu167-11 hu167-12 hu167-13 hu167-14 hu167-15
hu167_VH.1C hu167-16 hu167-17 hu167-18 hu167-19 hu167-20
hu167_VH.1D hu167-21 hu167-22 hu167-23 hu167-24 hu167-25
hu167_VH.1E hu167-26 hu167-27 hu167-28 hu167-29 hu167-30
3.人源化抗体筛选
进行结合CD47的ELISA方法检测(见测试例1)，并将结合ELISA检测的阳性孔细胞进行CD47/SIRPα结合的阻断ELISA检测(见测试例2，结果见表6)，根据测试结果优选最大程度保留抗体结合能力及阻断能力的回复突变组合。
055人源化抗体的不同组合基本都保持了良好的体外阻断活性，通过Biacore进行不同人源化抗体的亲和力比较，部分数据如表8所示，结果显示人源化抗体组合均保持了对于CD47良好的结合能力，尤其是hu055-3、hu055-4、hu055-5、hu055-6、hu055-7亲和力和嵌合抗体ch-055相比基本接近。
同样对于167人源化抗体的不同组合进行结合CD47的ELISA方法检测(见测试例1)，并将结合ELISA检测的阳性孔细胞进行CD47/SIRPα结合的阻断ELISA检测(见测试例2)，根据测试结果优选最大程度保留抗体结合能力及阻断能力的回复突变组合。体外阻断实验结果显示大部分抗体均保持了良好的阻断能力，相互之间无明显差异，从中选择了部分进行Biacore亲合力比较，结果如表9所示，大部分都保持了很好的对于CD47的结合能力，尤其是hu167-4、hu167-6、hu-167-9、hu167-14。
根据亲和力及阻断活性比较不同人源化组合形式，发现回复突变中的V58I对于亲和力的维持影响很小，因此在原设计基础上增加三条轻链(分别为：hu167_VL.1E(I2V)、hu167_VL.1F(M4I)、hu167_VL.1G(I2V，M4I))和hu167_VH.1组合产生的hu167-31、hu167-32、hu167-33，体外Biacore检测及阻断活性显示三者均保持了相当的CD47结合能力及阻断活性。通过Biacore检测比较hu167-31、hu167-32、hu167-33及hu167-4的亲和力(KD)，结果显示相比hu167-4的KD比值分别为1.3、1.2、0.98，提示hu167-31、hu167-32、hu167-33很好的保持了抗体的结合能力。
实施例5.构建和表达CD47人源化抗体的IgG4-S228P及IgG4-AA形式
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pHr(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-pHr/VK-CL-pHr。IgG4-AA抗体形式可以通过IgG4抗体形式简单点突变获得，IgG4-AA代表F234A和L235A突变，结果显示其进一步降低IgG4-Fc与FcyR的结合能力，进一步降低ADCC/CDC。IgG4-S228P代表野生型IgG4铰链区第228位的氨基酸S突变成P，该位点突变能够避免天然IgG4抗体在体内发生Fab-交换(exchange)导致的错配。
IgG4-S228P的重链恒定区序列如下：
Figure PCTCN2017113081-appb-000014
IgG4-AA代表上述序列下划线部分发生F234A和L235A突变所得的重链恒定区序列，具体本公开中IgG4-AA(包含S228P)的重链恒定区序列如下：
Figure PCTCN2017113081-appb-000015
抗体的轻链(Kappa链)恒定区序列如下：
Figure PCTCN2017113081-appb-000016
构建后的抗体序列列举如下：
hu167-33：抗体形式IgG4AA
hu167-33重链序列：
Figure PCTCN2017113081-appb-000017
Figure PCTCN2017113081-appb-000018
hu167-33轻链序列:
Figure PCTCN2017113081-appb-000019
hu055-5：抗体形式IgG4AA
hu055-5重链序列：
Figure PCTCN2017113081-appb-000020
hu055-5轻链序列：
Figure PCTCN2017113081-appb-000021
测试例
测试例1.CD47抗体结合CD47蛋白的ELISA实验
抗CD47抗体的结合力通过抗体与固定在ELISA板上CD47的结合的量来检测。用PBS稀释CD47-ECD-His(SEQ ID NO：1)至1μg/ml包被在96孔ELISA板(Costar，CAT#3590)上，洗板封闭后，加入不同浓度稀释的抗CD47抗体样品，再洗板后加入辣根过氧化物酶-羊抗人(H+L)抗体(Jackson，CAT#109-035-088)，再洗板加入四甲基联苯胺溶液显色，最后加入终止液，在酶标仪上测量OD450并计算其EC50值。
测试例2.CD47抗体对SIRPα/CD47结合的阻断
抗CD47抗体对SIRPα和CD47结合的阻断能力，通过检测在抗体存在的条件下CD47与SIRPα结合的量来确定。用磷酸缓冲液稀释CD47-ECD-Fc(SEQ ID NO.2)至1μg/ml包被在96孔ELISA板(Costar，CAT#3590)上，洗板封闭后，加入生物素标记的SIRPα-Fc(SEQ ID NO：3)(用磷酸缓冲液稀释至终浓度0.5μg/ml)和抗体样品(用磷酸缓冲液稀释)的混合液100μl/孔，洗板后加入辣根过氧化物酶-链霉亲和素(sigma，CAT#S2438)，再洗板加入四甲基联苯胺溶液显色，最后加入终止液，在酶标仪上测量OD450并计算其IC50值，结果见表6。
表6. 055人源化抗体阻断活性
Figure PCTCN2017113081-appb-000022
测试例3.CD47抗体结合CCRF-CEM细胞的实验
CCRF-CEM细胞(ATCC，CAT#CRM-CCL-119)培养在RPMI培养基中(Hyclone，CAT#SH30809.01B)(含10％胎牛血清)，1×106细胞/ml CCRF-CEM细胞用5％BSA封闭后，加入CD47抗体样品至1μg/ml，洗两次后，再加入Alexa Fluor 488-羊抗人(H+L)抗体(Invitrogen，CAT#A11013)，洗两次后，流式细胞仪读取荧光信号值。
FACS检测结果显示，本公开涉及的人源化抗体对细胞表面的天然CD47具有很强的结合能力，明显优于对照抗体B6H12(US9017675B)的结合能力，结果见表7。
表7.本公开人源化抗体与细胞表面的天然CD47的结合能力测试
序号 样品名称 流式结合信号
1 hu167-33 2.78E4
2 hu055-5 3.41E4
3 B6H12 2713
4 IgG4-Fc对照 65.1
5 空白对照 30.0
测试例4.CD47抗体增强CCRF-CEM细胞体外抗体依赖的细胞介导的细胞吞噬作用(ADCP)的实验
单核细胞分离于人外周血单核细胞，培养在RPMI培养基中(Hyclone，CAT#SH30809.01B)(含10％胎牛血清)的六孔板中7天。1×106细胞/ml  CCRF-CEM细胞加入CFSE(abcam，ab113853)至终浓度0.02μM，37℃孵育5-10分钟，洗两次后，加入含分化后单核细胞的六孔板，加入不同浓度稀释的抗CD47抗体样品，37℃孵育4小时，洗两次后，刮下分化后单核细胞，加入APC-抗人CD14抗体(BioLegend，CAT#325608)，4℃孵育0.5小时，洗两次后，流式细胞仪读取荧光信号值。
测试结果见图1，显示加入本公开的人源化抗体可以有效促进ADCP，且有一定的剂量效应。
测试例5.CD47抗体红细胞凝集实验
新鲜人血用磷酸缓冲液稀释1倍加入96孔板，加入不同浓度稀释的抗CD47抗体样品，震荡后37℃孵育4小时。吸去上清，观察96孔板上不同孔中的细胞沉淀情况，记录各孔中各抗体不同浓度引起红细胞(RBC)凝集的情况，以图2中所示的方式进行标记，并统计结果进行比较。
抗体引起红细胞发生凝集是CD47领域内抗体较为普遍的现象，比如专利抗体5F9(US9017675B，见专利中vh2，SEQ ID NO 37和vl2，SEQ ID NO 42)经检测能够在很大浓度范围内引起体外红细胞发生凝集现象，而同样条件下本公开的hu167-33在测试的不同浓度下均不会引起红细胞凝集，另外一个抗体分子hu055-5能够引起红细胞凝集，但其引起红细胞凝集的浓度范围明显小于5F9。结果见图2。提示本公开的抗体在安全性方面的潜在优势。
测试例6.CD47抗体红细胞裂解实验
新鲜人血用磷酸缓冲液稀释1倍加入96孔板，加入0.2mg/ml的抗CD47抗体样品或不同浓度稀释的Triton X-100，震荡后37℃孵育4小时，离心后吸取上清至另一块96孔板，在酶标仪上测量OD540。结果见图3，显示体外加入本公开所示的抗CD47抗体不会引起红细胞的裂解。
测试例7.BIAcore检测CD47抗体亲和力实验
用Biacore仪器测试本公开嵌合抗体及相应的人源化抗体对人CD47-his抗原的亲和力。
按照人Fab捕获试剂盒(Cat.#28-9583-25，GE)说明书中所述的方法，将人Fab捕获分子共价偶联于CM5生物传感芯片(Cat.#BR-1000-12，GE)上，从而亲和捕获待测抗体。然后于芯片表面流经人CD47-his(义翘神州，CAT#12283-HCCH-50)抗原，利用Biacore仪器实时检测反应信号，从而获得结合和解离曲线，通过拟合得到亲和力数值，见下表8、表9。在实验中每个循环解离完成后，用人Fab捕获试剂盒里配置的再生溶液将生物芯片洗净再生。
表8.本公开的hu055系列抗体对人CD47-his抗原的亲和力
配体 ka(1/Ms) kd(1/s) KD(M) KD的倍数差异
ch-055 2.32E6 3.89E-4 1.67E-10 1
hu055-1 1.67E6 9.76E-4 5.85E-10 3.5
hu055-2 1.81E6 7.76E-4 4.29E-10 2.6
hu055-3 1.98E6 7.32E-4 3.70E-10 2.2
hu055-4 1.87E6 6.99E-4 3.73E-10 2.2
hu055-5 2.10E6 3.82E-4 1.82E-10 1.1
hu055-6 2.00E6 3.27E-4 1.64E-10 1.0
hu055-7 2.11E6 4.33E-4 2.05E-10 1.2
hu055-8 1.60E6 9.87E-4 6.17E-10 3.7
表9.本公开167系列抗体对人CD47-his抗原的亲和力
配体 ka(1/Ms) kd(1/s) KD(M) KD的倍数差异
ch-167 1.72E6 5.58E-4 3.25E-10 1
hu167-1 1.49E6 9.91E-4 6.64E-10 2.04
hu167-2 1.50E6 9.85E-4 6.55E-10 2.01
hu167-3 1.21E6 8.76E-4 7.22E-10 2.22
hu167-4 1.60E6 7.01E-4 4.37E-10 1.34
hu167-6 1.61E6 9.46E-4 5.89E-10 1.81
hu167-9 1.38E6 6.10E-4 4.43E-10 1.36
hu167-14 1.37E6 5.48E-4 4.00E-10 1.23
本公开抗体相应的人源化抗体经Biacore检测，发现其与抗原有较高的亲和力，并进一步筛选到具有高亲和力且回复突变少的优化人源化抗体。
测试例8.CD47抗体的体内药代动力学实验
动物在给药不同浓度抗CD47抗体样品后于不同时间点采取血清，根据测试例1抗CD47抗体结合CD47蛋白的ELISA实验中的方法作不同样品的标准曲线，血清样品1：1000代替抗CD47抗体加入反应体系后根据OD450换算不同时间点抗CD47抗体于血清中的浓度，所得数据由Phoenix WinNonlin软件分析计算药代动力学相关参数。
在大鼠中测定了hu055-5在3mpk剂量下的药物代谢参数，结果显示hu055-5具有良好的大鼠体内药物代谢表现，平均药物半衰期t1/2约285小时，见图4，提示该抗体在大鼠体内稳定性良好。在小鼠体内对本公开抗体hu167-33，进行了药 物代谢检测，在10mpk剂量下发现其药物半衰期t1/2约192小时，见图5，显示该抗体在小鼠体内稳定性良好。

Claims (20)

  1. 一种CD47抗体或其抗原结合片段，其包含选自以下的任意一个或多个CDR区：
    抗体重链可变区的HCDR区：SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、以及与SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16具有至少85％序列同一性的CDR；和
    抗体轻链可变区的LCDR区：SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、以及与SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19具有至少85％序列同一性的CDR。
  2. 根据权利要求1所述的CD47抗体或其抗原结合片段，其中：
    所述的抗体重链可变区包含SEQ ID NO:8、SEQ ID NO:9和SEQ ID NO:10所示的HCDR1、HCDR2和HCDR3，或与SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10具有至少85％序列同一性的CDR；和/或
    所述的抗体轻链可变区包含SEQ ID NO:11、SEQ ID NO:12和SEQ ID NO:13所示的LCDR1、LCDR2和LCDR3，或与SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13具有至少85％序列同一性的CDR。
  3. 根据权利要求1所述的CD47抗体或其抗原结合片段，其中：
    所述的抗体重链可变区包含SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示的HCDR1、HCDR2和HCDR3，或与SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16具有至少85％序列同一性的CDR；和/或
    所述的抗体轻链可变区包含SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示的LCDR1、LCDR2和LCDR3，或与SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19具有至少85％序列同一性的CDR。
  4. 根据权利要求1所述的CD47抗体或其抗原结合片段，所述的抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段，嵌合抗体或其片段，和/或人源化抗体或其片段。
  5. 根据权利要求4所述的CD47抗体或其抗原结合片段，其中所述鼠源抗体或其抗原结合片段的轻链可变区包含鼠源的轻链FR区或其突变序列；所述鼠源抗体或其抗原结合片段的重链可变区包含鼠源的重链FR区或其突变序列。
  6. 根据权利要求5所述的CD47抗体或其抗原结合片段，其中
    所述CD47抗体包含SEQ ID NO:4所示的重链可变区和SEQ ID NO:5所示的轻链可变区，或
    所述CD47抗体包含SEQ ID NO:6所示的重链可变区和SEQ ID NO:7所示的轻链可变区。
  7. 根据权利要求4所述的CD47抗体或其抗原结合片段，其中
    所述人源化抗体的重链可变区上的重链FR区序列选自人种系重链IGHV1-3*01和hjh6.1的组合及其突变序列，或选自人种系重链IGHV1-2*02和hjh6.1的组合序列及其突变序列；
    所述人源化抗体的轻链可变区上的轻链FR区序列选自人种系轻链IGKV4-1*01和hjk2.1的组合及其突变序列，或选自人种系轻链IGKV1-39*01和hjk4.1的组合序列及其突变序列。
  8. 根据权利要求7所述的CD47抗体或其抗原结合片段，所述人源化抗体的可变区上的FR区包含0至10个氨基酸的回复突变。
  9. 根据权利要求8所述的CD47抗体或其抗原结合片段，其中
    所述人源化抗体可变区包含：SEQ ID NO:20所示的重链可变区或其突变序列，和SEQ ID NO:21所示的轻链可变区或其突变序列；或
    所述人源化抗体可变区包含：SEQ ID NO:22所示的重链可变区或其突变序列，和SEQ ID NO:23所示的轻链可变区或其突变序列。
  10. 根据权利要求9所述的CD47抗体或其抗原结合片段，其中：
    SEQ ID NO:21所示的轻链可变区的突变序列包含突变D66E；
    SEQ ID NO:20所示的重链可变区的突变序列包含选自以下的突变：R72A、M48I、E46D、V68A、I70L、R38K、R67K、A97S及其组合；优选R72A、M48I、E46D、V68A、I70L及其组合；
    SEQ ID NO:23所示的轻链可变区的突变序列包含选自以下的突变：V58I、I2V、M4I、Q38E、A43T、P44H及其组合；优选I2V、M4I及其组合；
    SEQ ID NO:22所示的重链可变区的突变序列包含选自以下的突变：R72V、M48V、V68A、M70L、T74K、A40R、R38K、R67K及其组合。
  11. 根据权利要求7-9任一所述的CD47抗体或其抗原结合片段，其中：
    所述人源化抗体可变区包含：SEQ ID NO:26所示的重链可变区或其突变序列，和SEQ ID NO:27所示的轻链可变区或其突变序列；或
    所述人源化抗体可变区包含：SEQ ID NO:28所示的重链可变区或其突变序列， 和SEQ ID NO:29所示的轻链可变区或其突变序列。
  12. 根据权利要求4，7-10任一项所述的CD47抗体或其抗原结合片段，
    所述嵌合抗体或人源化抗体的重链包含：
    人源IgG1、IgG2、IgG3或IgG4的重链恒定区或其突变序列，和
    人源κ、λ链的轻链恒定区或其突变序列；
    优选地，所述嵌合抗体或人源化抗体的重链包含：
    人源IgG4的重链恒定区或其突变序列，和
    人源κ链轻链恒定区或其突变序列；
    更优选，所述嵌合抗体或人源化抗体的重链包含：
    含有F234A突变和L235A突变的人源IgG4重链恒定区SEQ ID NO:30。
  13. 一种药物组合物，其含有：
    治疗有效量的权利要求1至12任一项所述的CD47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。
  14. 一种双特异性抗体，其包含：
    权利要求1至12任一项所述的CD47抗体或其抗原结合片段。
  15. 一种分离的核酸，其编码权利要求1-12任一项所述的CD47抗体或其抗原结合片段。
  16. 一种表达载体，其含有根据权利要求15所述的分离的核酸。
  17. 一种宿主细胞，其转化有权利要求16所述的表达载体，所述宿主细胞选自原核细胞和真核细胞；所述真核细胞优选为哺乳动物细胞。
  18. 一种用于制备权利要求1至12任一项所述CD47抗体或其抗原结合片段的方法，包括步骤：
    在权利要求17所述的宿主细胞中表达抗体，以及从宿主细胞中分离所述抗体。
  19. 一种用于抑制受试者中的肿瘤细胞生长的方法，包括步骤：
    向受试者施用权利要求1至12任一项所述的CD47抗体或其抗原结合片段、或权利要求13所述的药物组合物。
  20. 选自以下的任一项在制备用于治疗癌症的药物中的用途：权利要求1至12任一项所述的CD47抗体或其抗原结合片段、权利要求13所述的药物组合物以 及权利要求14所述的双特异性抗体，
    所述癌症选自卵巢癌、黑色素瘤、***癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子***、口腔癌、脑癌、睾丸癌、皮肤癌、甲状腺癌以及血液学恶性肿瘤；
    优选地，所述血液学恶性肿瘤选自骨髓瘤、慢性白血病和急性白血病。
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