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WO2019042119A1 - Anticorps dirigé contre cd47 humain et son utilisation - Google Patents

Anticorps dirigé contre cd47 humain et son utilisation Download PDF

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Publication number
WO2019042119A1
WO2019042119A1 PCT/CN2018/100262 CN2018100262W WO2019042119A1 WO 2019042119 A1 WO2019042119 A1 WO 2019042119A1 CN 2018100262 W CN2018100262 W CN 2018100262W WO 2019042119 A1 WO2019042119 A1 WO 2019042119A1
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Prior art keywords
antibody
sequence
seq
amino acid
variable region
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PCT/CN2018/100262
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English (en)
Chinese (zh)
Inventor
刘志刚
刘玉兰
郭晶晶
郝小勃
万姝南
胡俊杰
Original Assignee
北京智仁美博生物科技有限公司
智翔（上海）医药科技有限公司
重庆智翔金泰生物制药有限公司
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Publication of WO2019042119A1 publication Critical patent/WO2019042119A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates generally to the field of genetic engineering and antibody drugs; in particular, to the field of anti-human CD47 antibodies and uses thereof.
  • the present application develops a novel anti-human CD47 antibody and provides the use of the antibody in the treatment of CD47 mediated diseases.
  • CD47 belongs to the immunoglobulin superfamily and is a type I transmembrane glycoprotein, including an amino-terminal V-type immunoglobulin-like extracellular domain, a transmembrane domain composed of five highly hydrophobic transmembrane segments and a hydrophilic The carboxy-terminal intracellular domain has a molecular weight between 47 and 55 kD.
  • CD47 was recognized for its interaction with integrin ⁇ v ⁇ 3 , so it is also known as integrin-associated protein (IAP) 1 .
  • CD47 interacts with the ⁇ 3 subunit of integrin ⁇ v ⁇ 3 or ⁇ IIb ⁇ 3 via an extracellular domain, activating the corresponding integrin.
  • SIRP ⁇ Signal regulatory protein ⁇
  • SHPS-1 including Src homology 2 domain-containing protein tyrosine phosphatase substrate-1) /BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs)/CD172a, also a transmembrane glycoprotein with 3 immunoglobulins in the extracellular domain
  • ITIM immunoreceptor tyrosine inhibitory sequence
  • CD47 is a ligand for SIRP ⁇ , which interacts through the extracellular domain to form an intercellular communication complex.
  • CD47 binds to SIRP ⁇ , it induces intracellular ITIM phosphorylation of SIRP ⁇ , and phosphorylation site binds to activate tyrosine phosphatase SHP-1 and SHP-2 containing SH2 (Src homology 2) domain.
  • SHP-1 is mainly expressed in hematopoietic cells, which negatively regulates the function of these cells.
  • SHP-2 is widely expressed, regulating small G proteins Ras and Rho, and positively controlling cell growth and proliferation 3 .
  • CD47-SIRP ⁇ signaling system plays an important role in regulating macrophage phagocytosis of mature blood cells.
  • the CD47 molecule on the surface of normal healthy cells such as erythrocyte 5 or platelet 6 ) interacts with the receptor SIRP ⁇ on macrophages to produce an inhibitory signal, inhibiting its phagocytic activity, and regulating the life cycle of blood cells and their number in the blood. purpose.
  • SIRP ⁇ on monocytes interacts with CD47 on erythrocytes, and Fc ⁇ receptor-dependent phagocytosis is inhibited by myosin-IIA dephosphorylation 7 .
  • the CD47-SIRP ⁇ signaling system inhibits dendritic cell activation and participates in various physiological activities such as nervous system development, neutrophil chemotactic activation and stromal cell-supported hematopoietic cell formation, and induces T cell immune tolerance, activation, and apoptosis.
  • Other aspects also play a variety of regulatory roles 8 .
  • CD47-SIRP ⁇ signaling system has been paid attention to by regulating the phagocytosis of macrophages in tumor immune surveillance.
  • the expression level of CD47 is significantly up-regulated in many malignant tumors such as ovarian cancer 9 , acute myeloid leukemia (AML) 10 , B-cell lymphoma 11 and solid tumor 12 , and the average expression level is about 3.3 times that of normal cells, and This up-regulation is directly related to the poor prognosis of patients with malignant tumors 13 .
  • CD47 In human myeloid leukemia cells (expressing low levels of CD47 endogenous, Rag2-Il2rg- not transplanted mice) Mice of CD47 expression, can be suppressed macrophage phagocytosis of tumor cells and promote tumor cells successfully transplanted 14. It can be inferred that CD47 on tumor cells and SIRP ⁇ on macrophages inhibit the clearance of tumor cells by macrophages and promote the growth and metastasis of tumors in vivo. High expression of CD47 is a common mechanism for tumor cells to evade immune surveillance. Broken CD47-SIRP ⁇ may be a new tumor immunotherapy strategy.
  • high affinity SIRP [alpha] mutant CD172a
  • CD172a can also antagonize the CD47 thereby blocking the CD47-SIRP ⁇ signal pathway, in the AML model can significantly increase the phagocytosis of macrophages AML cells, and inhibition of tumor growth 16 .
  • the application provides an antibody that specifically binds to human CD47, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, wherein
  • the HCDR1 sequence is NYWMH
  • the HCDR2 sequence is VIAPSDNYTNYNQKFQG
  • the HCDR3 sequence is GGKYSMDY
  • the LCDR1 sequence is RSSQSIVHSNGNTYLE
  • the LCDR2 sequence is KVSNRFS
  • the LCDR3 sequence is FQGSHVPFT;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPSSGNTKYAQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSVLYSSNQKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is GQYYAYPIT;
  • HCDR and LCDR sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 23, 28, 30, 32 or 33.
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 24, 29, 31, 34 or 35.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:23, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:24;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 29;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 31;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 35;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:35.
  • the application provides an antibody that specifically binds to human CD47, wherein the amino acid sequence of the heavy chain variable region of the antibody has any one of SEQ ID NO: 23, 28, 30, 32 or 33 At least 90% identity, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity to any of SEQ ID NO: 24, 29, 31, 34 or 35.
  • the application provides an antibody that specifically binds to human CD47, wherein the binding epitope of the antibody to the human CD47 segment of SEQ ID NO: 1 is a discontinuous epitope, including amino acids Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103, E104, G105 and E106, the above residue numbers refer to SEQ ID NO: 1.
  • the antibody is a monoclonal antibody.
  • the antibody is a non-activated antibody.
  • the antibody binds to and neutralizes human CD47, thereby blocking the CD47-SIRP ⁇ signaling pathway.
  • the antibody promotes phagocytosis of tumor cells by macrophages.
  • the antibody inhibits in vivo growth of tumor cells.
  • the antibody does not increase phagocytosis of normal blood cells by macrophages.
  • the antibody has at least one of the following properties:
  • the antibody is a whole antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or comprises a selected from the kappa subtype or the lambda subtype Light chain constant region.
  • the present application provides a nucleic acid molecule encoding the antibody of the first aspect to the third aspect or an antigen binding portion thereof.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first to third aspects and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is for treating a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the application provides the use of the antibodies of the first to third aspects for the preparation of a medicament for the prevention or treatment of a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the present application provides a method of preventing or treating a CD47-mediated disease, comprising administering the antibody of the first aspect to the third aspect or the pharmaceutical composition of the seventh aspect to an individual in need thereof.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • Figure 1 shows the results of the anti-CD47 mAb of the present application competing with hCD172a for binding to hCD47.
  • Figure 3 shows the effect of anti-CD47 mAb of the present application on the phagocytosis of Daudi cells by macrophages by flow cytometry.
  • Figure 4 shows that the anti-CD47 mAb of the present application promotes the phagocytosis of macrophages to Daudi cells.
  • Figure 5 shows the effect of the humanized anti-CD47 mAb of the present application inhibiting the binding of hCD47 to hCD172a.
  • Figure 6 shows the effect of hS2C3 and its variants on inhibition of hCD47 and hCD172a-D1M1 binding.
  • Figure 7 shows the effect of the anti-CD47 mAb of the present application on the degree of red blood cell agglutination.
  • Figure 8 shows that the anti-CD47 mAb of the present application promotes the phagocytosis of macrophages to Daudi cells.
  • Figure 9 shows the effect of the anti-CD47 mAb of the present application on inhibiting tumor growth in mice in vivo.
  • Figure 10 shows the effect of anti-CD47 mAb of the present application on tumor growth of xenograft human myeloma RPMI 8226 in NOD-SCID mice.
  • Figure 11 shows the structure of the CD47-Fab complex obtained by the present application, wherein Panel A shows the overall structure of the CD47-Fab complex; Panel B shows the binding surface of the CD47-Fab complex, and the amino acid residues involved in protein interaction are modeled with a stick. It is indicated that the two main binding areas are marked with dashed lines, the C picture shows the details of the interaction of the area I in the B picture, and the D picture shows the details of the area II interaction in the B picture.
  • SEQ ID NO: 1 shows the amino acid sequence of human (homo sapiens) CD47 extracellular domain D1 (hCD47).
  • SEQ ID NO: 2 shows the amino acid sequence of mouse (mus musculus) CD47 extracellular domain D1 (mCD47).
  • SEQ ID NO: 3 shows the amino acid sequence of the macadam (Macaca mulatta) CD47 extracellular domain D1 (mmCD47).
  • SEQ ID NO: 4 shows the amino acid sequence of the high affinity mutant (hCD172a-D1M1) of the immunoglobulin-like domain of the human CD172a extracellular domain.
  • SEQ ID NO: 5 shows the amino acid sequence of the His tag (His).
  • SEQ ID NO: 6 shows the amino acid sequence of the Fc fragment (Fc) of human antibody IgG1.
  • SEQ ID NO: 8 shows the amino acid sequence of the human IgGl subtype heavy chain constant region.
  • SEQ ID NO: 9 shows the amino acid sequence of the human IgG2 subtype heavy chain constant region.
  • SEQ ID NO: 10 shows the amino acid sequence of the human IgG4 subtype heavy chain constant region.
  • SEQ ID NO: 11 shows the amino acid sequence of the heavy chain constant region of the murine IgGl subtype.
  • SEQ ID NO: 12 shows the amino acid sequence of the heavy chain constant region of the murine IgG2a subtype.
  • SEQ ID NO: 13 shows the amino acid sequence of the human kappa subtype light chain constant region.
  • SEQ ID NO: 14 shows the amino acid sequence of the human lambda subtype light chain constant region.
  • SEQ ID NO: 15 shows the amino acid sequence of the murine kappa subtype light chain constant region.
  • SEQ ID NO: 17 and SEQ ID NO: 18 show the amino acid sequences of the VH and VK sequences of the humanized anti-CD47 mAb Hu5F9-G4, respectively.
  • SEQ ID NO: 19 shows the full-length amino acid sequence of the murine single-chain antibody S4D12
  • SEQ ID NOS: 20 and 21 show the amino acid sequences of its VH and VK sequences, respectively.
  • SEQ ID NO: 22 shows the full-length amino acid sequence of the murine single-chain antibody S2H2
  • SEQ ID NOS: 23 and 24 show the amino acid sequences of the VH and VK sequences, respectively.
  • SEQ ID NO: 25 shows the full length amino acid sequence of the murine single-chain antibody S2C3
  • SEQ ID NOS: 26 and 27 show the amino acid sequences of its VH and VK sequences, respectively.
  • SEQ ID NOS: 28 and 29 show the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of humanized S4D12 (hS4D12), respectively.
  • SEQ ID NOS: 30 and 31 show the heavy chain variable region amino acid sequence and the light chain variable region amino acid sequence of humanized S2C3 (hS2C3), respectively.
  • SEQ ID NO:32 shows the amino acid sequence of the heavy chain variable region mutant H10C7.
  • SEQ ID NO:33 shows the amino acid sequence of the heavy chain variable region mutant H11E5.
  • SEQ ID NO:34 shows the amino acid sequence of the light chain variable region mutant L25B8.
  • SEQ ID NO: 35 shows the amino acid sequence of the light chain variable region mutant L26A6.
  • a novel anti-human CD47 antibody or antigen-binding fragment thereof obtained novel anti-human CD47 antibodies by antibody engineering techniques.
  • a novel anti-human CD47 antibody or antigen-binding fragment thereof a polynucleotide encoding the antibody or antigen-binding fragment thereof, a vector comprising the polynucleotide, comprising the polynucleotide Or a host cell of a vector, a method of making and purifying the antibody, and a medical and biological application of the antibody or antigen-binding fragment thereof.
  • a full-length antibody molecule can be constructed as a medicament for the treatment of a clinically mediated CD47 disease.
  • antibody refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of an immunoglobulin molecule.
  • Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, and the like.
  • antibody includes not only intact (ie, full-length) antibodies, but also antigen-binding fragments thereof (eg, Fab, Fab', F(ab') 2 , Fv), variants thereof, and antibody-containing portions thereof.
  • Fusion proteins humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (eg bispecific antibodies) and any other immunoglobulin containing an antigen recognition site of the desired specificity Modified configurations of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • a full or full length antibody comprises two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variant region (VH) and first, second and third constant regions (CH1, CH2 and CH3).
  • Each light chain contains a light chain variant region (VL) and a constant region (CL).
  • the full length antibody can be any kind of antibody, such as IgD, IgE, IgG, IgA or IgM (or a subclass of the above), but the antibody does not need to belong to any particular class.
  • Immunoglobulins can be assigned to different classes depending on the antibody amino acid sequence of the constant domain of the heavy chain.
  • immunoglobulins there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant domains corresponding to different immunoglobulin classes are referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • Subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • antigen binding domain refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variant region (VH), a light chain variant region (VL), or both.
  • VH and VL typically contains three complementarity determining regions, CDR1, CDR2 and CDR3.
  • CDRs complementarity determining regions
  • VH or VL There are two common definitions for the CDR sequences of VH or VL, namely the kabat definition and the Chothia definition.
  • CDR sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In an embodiment of the present application, CDR sequences are defined using Kabat.
  • variable region sequences of a given antibody the CDR region sequences in the variable region sequences can be analyzed in a variety of ways, for example, using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab') 2 fragment, which may have two a bivalent fragment of a Fab' fragment joined by a disulfide bridge of the hinge region (ie, a dimer of Fab'); (3) an Fv fragment of the VL and VH domains of the one arm of the antibody; 4) a single-chain Fv (scFv), which may be a single multi-peptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) 2 , which may comprise two linked by a peptide A coupled VH domain and two VL domains are combined with the two VH domains via a disulfide bridge.
  • a Fab fragment which may be a monovalent fragment having a VL-CL chain and a VH-
  • the term "specifically binds" as used herein refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population are identical except that there may be naturally occurring mutations in a small number of individuals.
  • Monoclonal antibodies as used herein specifically include “chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence derived from a particular species or antibody belonging to a particular antibody or subclass, but The remainder of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies as long as they exhibit desired Biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • tumor refers to a neoplasm or solid lesion formed by abnormal cell growth.
  • the tumor can be benign, malignant or malignant.
  • a “primary tumor” is a tumor that exists at an initial site in an individual and can be distinguished from a “metastatic tumor” that is present in a distant portion of the individual from the primary tumor.
  • malignant tumor refers to or describes the physiological condition of a mammal, which is typically characterized by unregulated cell growth.
  • exemplary malignancies include: carcinoma, melanoma sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma.
  • malignant tumors include: squamous cell carcinoma (for example, squamous cell carcinoma), including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung cancer of the lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, Gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colon Rectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma , brain cancer and head and neck cancer and related metastases.
  • squamous cell carcinoma for example, squamous cell carcinoma
  • gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblast
  • cancer refers to a composition consisting of a variant epithelial cell or a variant cell having an unknown histological occurrence but having specific molecular or histological features associated with epithelial cells, such as the production of cytokeratin or an intercellular bridge.
  • Invasive malignant tumor Exemplary cancers of the present application include ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, rectal cancer, colon cancer, gastric cancer, pancreatic cancer, islet tumor, adenocarcinoma, adenosquamous carcinoma, neuroendocrine tumor.
  • breast cancer, lung cancer, esophageal cancer oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, glioma, pituitary cancer and bone cancer.
  • lymphomas include: non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ( Macroglobulinemia), spleen marginal lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, intracranial marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse Large B-cell lymphoma, mediastinal (thymus) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, Burkitt's lymphoma, B-cell chronic lymphocytic lymphoma, classic Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia (
  • sarcoma is a malignant tumor derived from a variant cell in one of a variety of tissues developed by the embryonic mesoderm.
  • sarcomas include tumors of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissue.
  • osteosarcoma from bone chondrosarcoma from cartilage, liposarcoma from fat, and leiomyosarcoma from smooth muscle.
  • Exemplary sarcomas include: Askin tumor, grape sarcoma, chondrosarcoma, Ewing's sarcoma-PNET, malignant hemangioendothelioma, malignant schwannomas, osteosarcoma, soft tissue sarcoma.
  • Subclasses of soft tissue sarcoma include: soft tissue acinar sarcoma, angiosarcoma, phyllodes cystosarcoma, cutaneous fibrosarcoma, fibroma, profibrotic small round cell tumor, epithelioid sarcoma, extramedullary chondrosarcoma, extraosseous osteosarcoma, fibrosarcoma , vascular epithelioma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma and synovial sarcoma.
  • leukemia is a malignant tumor of blood or bone marrow characterized by an abnormal increase in white blood cells.
  • Leukemia is a broad term that covers a range of diseases. Therefore, leukemia is part of a broader range of diseases known as hematological malignancies.
  • Leukemia is subdivided into several broad categories; the first is the acute and chronic form of leukemia. Acute leukemia is characterized by a rapid increase in the number of immature blood cells. Due to the accumulation of these cells, the bone marrow cannot produce healthy blood cells.
  • Chronic leukemia is characterized by overproduction of relatively mature, but still abnormal, white blood cells.
  • leukemias include: acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell lymphocytic leukemia, large granular lymphocytic leukemia, juvenile granulocyte-monocytic leukemia, B-cell lymphoblastic leukemia, Burkitt leukemia, and adult T-cell leukemia.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • HCL hairy cell leukemia
  • T-cell lymphocytic leukemia large granular lymphocytic leukemia
  • juvenile granulocyte-monocytic leukemia B-cell lymphoblastic leukemia
  • Burkitt leukemia Burkitt leukemia
  • R represents A or G
  • Y represents C or T
  • M represents A or C
  • K represents G or T
  • S represents C or G
  • W represents A or T
  • H represents A or C or T
  • B represents C or G or T
  • V represents A or C or G
  • D represents A or G or T
  • N represents A or C or G or T.
  • the application provides an antibody that specifically binds to human CD47, comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 sequences and a light chain variable region comprising the LCDR1, LCDR2 and LCDR3 sequences, wherein
  • the HCDR1 sequence is NYWMH
  • the HCDR2 sequence is VIAPSDNYTNYNQKFQG
  • the HCDR3 sequence is GGKYSMDY
  • the LCDR1 sequence is RSSQSIVHSNGNTYLE
  • the LCDR2 sequence is KVSNRFS
  • the LCDR3 sequence is FQGSHVPFT;
  • the HCDR1 sequence is SYWMH
  • the HCDR2 sequence is TIDRSDSYISYNQKFKG
  • the HCDR3 sequence is GGPYGSKMMDN
  • the LCDR1 sequence is HASQNINVWLS
  • the LCDR2 sequence is KASNLHT
  • the LCDR3 sequence is QQGQSYPLT;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPSSGNTKYAQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPSSGNTKYAQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSVLYSSNQKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is GQYYAYPIT;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSLLYSSNKKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is QQFYAYPIS;
  • the HCDR1 sequence is DYYMH
  • the HCDR2 sequence is WIYPGSGNTRYSQKFKD
  • the HCDR3 sequence is REEDYFDY
  • the LCDR1 sequence is KSSQSVLYSSNQKNYLT
  • the LCDR2 sequence is WASTRES
  • the LCDR3 sequence is GQYYAYPIT;
  • HCDR and LCDR sequences are defined according to Kabat.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 23, 28, 30, 32 or 33.
  • amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 24, 29, 31, 34 or 35.
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO:23, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:24;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 28, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 29;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 30, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 31;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 32, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 35;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 34;
  • amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 33, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:35.
  • the application provides an antibody that specifically binds to human CD47, wherein the amino acid sequence of the heavy chain variable region of the antibody has any one of SEQ ID NO: 23, 28, 30, 32 or 33 At least 90% identity, and the amino acid sequence of the light chain variable region of the antibody has at least 90% identity to any of SEQ ID NO: 24, 29, 31, 34 or 35.
  • the application provides an antibody that specifically binds to human CD47, wherein the binding epitope of the antibody to the human CD47 segment of SEQ ID NO: 1 is a discontinuous epitope, including amino acids Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103, E104, G105 and E106, the above residue numbers refer to SEQ ID NO: 1.
  • the antibody is a monoclonal antibody.
  • the antibody binds to and neutralizes human CD47, thereby blocking the CD47-SIRP ⁇ signaling pathway.
  • the antibody promotes phagocytosis of tumor cells by macrophages, inhibits growth of tumor cells in vivo, and does not increase phagocytosis of normal blood cells by macrophages.
  • the antibody has at least one of the following properties:
  • the antibody is a whole antibody, a Fab fragment, a F(ab') 2 fragment or a single chain Fv fragment (scFv).
  • the antibody is a fully human antibody.
  • the antibody further comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and/or comprises a selected from the kappa subtype or the lambda subtype Light chain constant region.
  • the present application provides a nucleic acid molecule encoding the antibody of the first aspect to the third aspect or an antigen binding portion thereof.
  • the nucleic acid molecule is operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody of the first to third aspects and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is for treating a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the pharmaceutical composition may further comprise one or more of the following: a lubricant such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier; a suspending agent; a preservative, Such as benzoic acid, sorbic acid and calcium propionate; sweeteners and / or flavoring agents.
  • a lubricant such as talc, magnesium stearate, and mineral oil
  • a wetting agent such as talc, magnesium stearate, and mineral oil
  • an emulsifier such as benzoic acid, sorbic acid and calcium propionate
  • a preservative such as benzoic acid, sorbic acid and calcium propionate
  • sweeteners and / or flavoring agents such as benzoic acid, sorbic acid and calcium propionate
  • the pharmaceutical compositions of the present application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.
  • the pharmaceutical compositions of the present application can be delivered using any physiologically acceptable administration, including but not limited to: oral administration, parenteral administration, nasal administration, rectal administration. Drug, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.
  • a pharmaceutical composition for therapeutic use may be formulated in a lyophilized formulation or as an aqueous solution by mixing an agent having the desired purity with a pharmaceutically acceptable carrier, excipient, or the like, as appropriate. storage.
  • the application provides the use of the antibodies of the first to third aspects for the preparation of a medicament for the prevention or treatment of a CD47 mediated disease.
  • the disease is a tumor, such as a malignant tumor.
  • the malignancy is Burkitt's lymphoma or multiple myeloma.
  • the present application provides a method of preventing or treating a CD47-mediated disease comprising administering the antibody of the first aspect to the third aspect or the pharmaceutical composition of the fifth aspect to an individual in need thereof.
  • the disease is a tumor, such as a malignant tumor.
  • the application further provides a vector comprising an isolated nucleic acid molecule encoding an antibody of the invention or an antigen binding portion thereof, and a host cell comprising the nucleic acid molecule or vector.
  • the present application also provides methods of producing the antibodies of the present application.
  • a method of producing an antibody comprises culturing a host cell to facilitate expression of the nucleic acid.
  • the method of producing an antibody further comprises recovering the antibody from the host cell culture medium.
  • a variety of different recombinant proteins are required for the preparation of anti-CD47 mAb, including human CD47 extracellular domain D1 (hCD47, SEQ ID NO: 1), mouse CD47 extracellular domain D1 (mCD47, SEQ ID NO: 2). , a high affinity mutant of the macaque CD47 extracellular domain D1 (mmCD47, SEQ ID NO: 3) and the human CD172a extracellular domain immunoglobulin-like domain (hCD172a-D1M1, SEQ ID NO: 4). These proteins have post-translational modifications (such as glycosylation or disulfide bonds, etc.), and thus the use of mammalian cell expression systems is more conducive to maintaining the structure and function of the recombinant protein.
  • post-translational modifications such as glycosylation or disulfide bonds, etc.
  • the His tag His, SEQ ID NO: 5
  • the Fc segment of human antibody IgG1 Fc, SEQ ID NO
  • the antibody heavy chain constant region can be a human IgG1 subtype (SEQ ID NO: 8), a human IgG2 subtype (SEQ ID NO: 9), a human IgG4 subtype (SEQ ID NO: 10) or a murine IgG1 subtype (SEQ ID NO: 11), murine IgG2a subtype (SEQ ID NO: 12); light chain constant region may be human kappa subtype (SEQ ID NO: 13), human lambda subtype (SEQ ID NO: 14) or murine kappa Type (SEQ ID NO: 15), murine lambda subtype (SEQ ID NO: 16).
  • the genes of the above various recombinant proteins were designed and synthesized based on the amino acid sequence of the recombinant protein for various purposes of the Uniprot database.
  • the various recombinant protein genes synthesized are cloned into a suitable eukaryotic expression vector (such as invitrogen pcDNA3.1, etc.) using conventional molecular biology techniques, and then using liposomes (such as 293fectin of Invitrogen, etc.) or other cations.
  • the recombinant protein expression plasmid prepared by transfection reagent (such as PEI) is transfected into HEK293 cells (such as HEK293F of Invitrogen) and cultured in serum-free suspension culture for 3-4 days. The culture supernatant is then harvested by centrifugation or the like.
  • the recombinant protein expressed by the His-tag fusion is subjected to one-step purification of the recombinant protein in the culture supernatant by a metal chelate affinity chromatography column (such as GE's HisTrap FF).
  • the recombinant protein expressed by fusion of Fc and mFc was subjected to one-step purification using a Protein A/G affinity chromatography column (e.g., Mabselect SURE, GE).
  • the recombinant protein storage buffer is then replaced with PBS (pH 7.0) or other suitable buffer using a desalting column (eg, Hitrap desaulting, GE, etc.). If necessary, the antibody samples can be sterilized by filtration and then stored separately at -20 °C.
  • mice of 6-8 weeks old were taken, and the mice were subjected to tail vein blood sampling to leave the background serum before immunization.
  • the hCD47-mFc fusion protein was immunized for the first time and emulsified with complete Freund's adjuvant.
  • Each mouse was intraperitoneally injected with 50 ⁇ g of fusion protein.
  • the immunization was boosted at intervals of two weeks, and the hCD47-mFc fusion protein was emulsified with incomplete adjuvant.
  • Each mouse was intraperitoneally injected with 50 ⁇ g of fusion protein, and blood was taken from the tail before injection for two booster immunizations.
  • the fourth immunization was changed to shock immunization, and the anti-adjuvant hCD47-mFc recombinant antigen was used as an immunogen.
  • Each mouse was intraperitoneally injected with 50 ⁇ g of fusion protein, and the mice were sacrificed 3 days after the immunization to collect spleen cells.
  • mice spleen lymphocytes were separated using mouse lymphocyte separation solution (Dakko, #DKW33-R0100), and the isolated lymphocytes were subjected to total RNA using a total RNA extraction kit (Tiangen, #DP430). extract.
  • a total RNA extraction kit Tiangen, #DP430. extract.
  • the first strand cDNA synthesis kit (Thermo scientific, #K1621) was used to synthesize the heavy chain variable region and the light chain variable region, respectively, and the reverse transcription primers were gene-specific primers, and the primer pairing region was used.
  • the prepared mouse single-chain antibody gene was cloned into the vector pADSCFV-S (experimental technical procedure can be found in Chinese Patent Application No. 201510097117.0) to construct a scFv library.
  • the library capacity of this antibody library reached 1.2 ⁇ 10E7, and the correct rate was about 85%.
  • Phage-ELISA showed that hCD172a-D1M1 could compete with four of the murine monoclonal antibodies (S1F9/S2C3/S2H2/S4D12) for binding to CD47, while the binding of murine monoclonal antibody S2D10 to CD47 was not blocked by hCD172a-D1M1.
  • the murine single-chain antibody S4D12 (SEQ ID NO: 19, VH and VK sequences are SEQ ID NOS: 20 and 21, respectively), S2H2 (SEQ ID NO: 22, VH and VK sequences are SEQ ID NO: 23 and 24, respectively) were selected.
  • S2C3 SEQ ID NO: 25, VH and VK sequences are SEQ ID NOS: 26 and 27, respectively
  • a recombinant human IgG4-kappa form of murine-human chimeric antibody is prepared by molecular biological methods, with reference to US Patent Application No. US9017675B2
  • humanized anti-CD47 mAb Hu5F9-G4 was prepared (VH sequence and VK sequence are SEQ ID NO: 17 and SEQ ID NO: 18, respectively).
  • Recombinant anti-CD47 mAb blocks hCD47 binding to hCD172a
  • the antigen hCD47-mFc was coated, and the S2C3/S2H2/S4D12 chimeric antibody was serially diluted with a fixed concentration of hCD172a-D1M1-His, and CD172a-D1M1- was detected with HRP-mouse-anti-his IgG (Kangwei Century, CW0285M). Binding of His and hCD47-mFc.
  • the results of the ELISA analysis (Fig. 1) showed that the ability of the monoclonal antibodies S2C3, S2H2 and S4D12 to block the interaction of CD47 and CD172a-D1M1 was not much different, and the IC 50 was in the range of 2-5 nM (Table 1).
  • Table 1 Three different anti-CD47 mAb IC 50 inhibition of CD47 and CD172a-D1M1 binding
  • the affinity of the IgG4 chimeric antibody was determined using Biacore X100.
  • Amino coupling kit, human antibody capture kit, CM5 chip and related reagents and consumables such as 10 ⁇ HBS-EP of pH 7.4 were purchased from GE healthcare.
  • the antibody against the human Fc segment was conjugated to the surface of the CM5 chip according to the instructions in the kit, and the antibody protein was diluted to a suitable concentration to ensure that about 300 RU of the antibody was captured by the antibody against human Fc.
  • huCD47-D1-his was placed in a series of concentration gradients (500 nM, 167 nM, 56 nM, 18.5 nM, 6.2 nM) through the surface of the stationary phase, and the affinity of each monoclonal antibody was determined at 25 °C. The results are shown in Table 2.
  • the surface of the red blood cells highly expresses CD47, so the anti-CD47 mAb can specifically bind to CD47 on the surface of red blood cells. Since each antibody has two antigen binding sites, anti-CD47 mAb may cause red blood cell agglutination. Monoclonal antibodies that cause red blood cell agglutination cause side effects such as anemia and decreased red blood cell count in the body. Whether the anti-CD47 mAb causes erythrocyte agglutination depends mainly on the recognition epitope of the mAb on the CD47 molecule. This example utilizes classical hemagglutination experiments to analyze the ability of CD47 monoclonal antibody to cause red blood cell agglutination.
  • the anti-CD47 antibody (40 ⁇ g/ml) selected in the previous examples was diluted with PBS for a series of dilutions, and then mixed with the prepared 1% red blood cell suspension in a microhemagglutination plate and shaken on a micro-oscillator. Mix for 1 min.
  • the micro-hemagglutination reaction plate was allowed to stand at 25 ° C for 1 h, and the degree of erythrocyte agglutination was recorded by photography, and the micro-hemagglutination reaction plate was tilted at an angle of 45° for several minutes, and the degree of red blood cell agglutination was further determined by the rate of red blood cell flow.
  • the degree of erythrocyte agglutination can be divided into 4 grades: 4 condensed into a uniform thin layer, the slant does not flow; 3 condenses into a uniform thin layer, the slant has a slight flow; 2 condenses into a small thin layer, the slanting flow is faster; 1 has no thin layer Formed, the slope is the same as the flow velocity of the control well.
  • the results showed (Fig. 2) that human-mouse chimeric mAbs S2C3 and S2H2 did not cause erythrocyte agglutination, whereas human-mouse chimeric mAb S4D12 and humanized antibody Hu5F9 had strong erythrocyte agglutination.
  • Recombinant anti-CD47 mAb can promote macrophage phagocytosis of tumor cells
  • PBMCs peripheral blood mononuclear cells
  • CD14 + monocytes were sorted from PBMCs by CD14 magnetic beads (anti-human CD14 magnetic particles - DM, BD).
  • CD14 + monocytes were seeded in a 12-well plate at a density of 8 ⁇ 10 5 /well, and induced with 10 ng/ml MCSF for about 7 days to differentiate into mature macrophages.
  • hS2C3 was subjected to in vitro affinity maturation.
  • the main strategy for in vitro affinity maturation is to introduce mutations in the heavy chain CDRs and light chain CDRs in turn, construct a light chain or heavy chain mutation library, and then based on the dual vector phage display system (for specific operations, refer to the Chinese patent application filed by the applicant before).
  • Example 5 of No. 201510097117.0 screens for high affinity mutants of heavy and light chains, respectively.
  • the mutation library of hS2C3 heavy chain variable region was constructed by conventional molecular biology method, and the storage capacity was 6.5 ⁇ 10E5. The correct rate of the mutation library was about 95%.
  • the primers required for introducing mutations into HCDR1 and HCDR2 are shown in Table 5.
  • the hS2C3-HCDR12 mutant library was screened by solid-phase screening strategy for 3 rounds, and finally two mutants H10C7 (SEQ ID NO: 32) and H11E5 (SEQ ID NO:) with further improved affinity were obtained. 33).
  • erythrocyte agglutination experiment showed (Fig. 7) that four humanized anti-CD47 monoclonal antibodies with increased affinity did not cause erythrocyte agglutination in the concentration range of 0-133 nM, and all four humanized mutants could promote macrophage to CD47. + Phagocytosis of tumor cells (Daudi) ( Figure 8).
  • mice subcutaneously inoculated into male NSG mice at a dose of 1 ⁇ 10 4 cells/mouse, and randomly divided into 5 groups with a tumor volume of 100 cm 3 , 8 mice per group (1) vehicle control group; (2) 20 mg/kg H10C7+L26A6 group; (3) 10 mg/kg H10C7+L26A6 group; (4) 3 mg/kg H10C7+L26A6 group; (5) 10 mg/kg hu5F9-IgG4 group . It was administered intraperitoneally twice a week for a total of 3 weeks. Tumor size and animal body weight were measured three times a week during the administration to evaluate the antitumor effect of the anti-CD47 antibody.
  • Fig. 9 The experimental results are shown in Fig. 9.
  • the tumor-bearing mice in which the abscissa is the raji tumor received the drug treatment time, and the ordinate was the tumor volume.
  • the results showed that the humanized anti-CD47 monoclonal antibody H10C7+L26A6 inhibited tumor growth compared with the vehicle control group, and its inhibitory ability was comparable to that of the control antibody hu5F9-IgG4.
  • mice were divided into 5 groups of 8 mice each: (1) vehicle control group (ipBIW*4, poQD*4); (2) 10 mg/kg daratumumab (ipBIW*4) group; (3) 10 mg/kg H10C7+L26A6 (ipBIW*4) group; (4) 50 mg/kg lenolidomide (poQD*4) group; 510 mg/kg dalimumab (ipBIW*4)+ 50 mg/kg lenalidomide (poQD*4) group.
  • Tumor size and animal body weight were measured 3 times a week during the administration.
  • Dalemuzumab is a marketed monoclonal antibody against multiple myeloma
  • lenalidomide is a routine drug for clinical multiple myeloma.
  • Fig. 10 The experimental results are shown in Fig. 10.
  • the abscissa is the time when the NOD-SCID tumor-bearing mice of the subcutaneous xenograft RMPI 8226 tumor were treated with the drug, and the ordinate was the tumor volume, which was humanized compared with the vehicle control group.
  • Anti-CD47 monoclonal antibody H10C7+L26A6 can significantly inhibit tumor growth, and its tumor inhibition ability is significantly higher than the combination of dalimumab, lenalidomide and dalimumab + lenalidomide.
  • HEK293 cells were used to prepare recombinant H10C7+L26A6 Fab fragment and recombinant human CD47 extracellular domain D1 (hCD47, SEQ ID NO: 1) to form Fab- hCD47 complex, forming complex crystals under 2.0M ammonium sulfate + 5% isopropanol, X-ray diffraction experiments on complex crystals, resolution
  • the data (Table 9) and the crystal structure of the CD47 antigen-antibody complex were analyzed by molecular replacement.
  • the resulting structural model includes the CD47 extracellular domain 1-116 aa (SEQ ID NO: 1), the antibody heavy chain 1-219 aa, and the light chain 1-219 aa.
  • the last amino acid residue of the H chain and L chain of the antibody is not visible in the electron density, suggesting that it has no fixed structure in the crystal.
  • CD47 exists in a monomeric form and there is visible glycosylation at the N16, N32, N55 and N93 sites.
  • the interaction area between CD47 and Fab is about
  • the interaction area between CD47 and heavy chain is about
  • the area of interaction with the light chain is approximately
  • the interaction mainly involves the N-terminus of the CD47, the BC loop, the ⁇ -chain F, the FG loop and the ⁇ -strand G, while the HCDR1, HCDR2, HCDR3 of the antibody heavy chain variable region, and the LCDR1 and LCDR3 of the light chain variable region are involved.
  • Identification of CD47 ( Figure 11).
  • the interactions include hydrogen bonds, salt bonds, van der Waals forces, and hydrophobic interactions (Table 10).
  • humanized anti-CD47 mAb H10C7+L26A6 recognizes a discontinuous (spatial) epitope of human CD47, mainly including amino acids Q1, E29, A30, Q31, N32, E35, E97, E100, L101, T102, R103, E104, G105 and E106.
  • the antigen-binding epitope of anti-human CD47 monoclonal antibody hu5F9 monoclonal antibody includes amino acids Q1, E29, K39, K41, E97, T99, R103 and E104; and the epitope of anti-human CD47 monoclonal antibody 2A1 includes amino acid Y37. K39, K41, K43, G44, R45, D46, D51, H90, N93, E97, T99, E104 and E106.
  • the epitopes of the anti-human CD47 mAbs H10C7+L26A6, hu5F9 and 2A1 partially overlap, but they are not identical.
  • BIAcore determined the affinity of humanized anti-CD47 mAb H10C7+L26A6 for these hCD47 mutants and compared it to native hCD47 (Table 11). The results showed that the L101A point mutation resulted in complete no binding, the Q1A mutation resulted in a decrease in affinity of about 100-fold, while the E35A mutation resulted in an approximately 4-fold increase in affinity.
  • SIRPa Signal-regulatory protein a
  • CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.
  • CD47 is upregulated on cir-culating hematopoietic stem cells and leukemia cells to avoid phagocytosis.

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Abstract

La présente invention concerne un anticorps ou une fraction de liaison à l'antigène de celui-ci qui se lie de manière spécifique à CD47 humain, un polynucléotide codant pour l'anticorps ou la fraction de liaison à l'antigène de celui-ci, un vecteur comprenant le polynucléotide, une cellule hôte comprenant le polynucléotide ou le vecteur, un procédé de préparation et de purification de l'anticorps, et une application de l'anticorps ou de la fraction de liaison à l'antigène de celui-ci.
PCT/CN2018/100262 2017-09-01 2018-08-13 Anticorps dirigé contre cd47 humain et son utilisation WO2019042119A1 (fr)

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