WO2018161872A1 - Anticorps anti-b7-h3, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée - Google Patents

Anticorps anti-b7-h3, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée Download PDF

Info

Publication number
WO2018161872A1
WO2018161872A1 PCT/CN2018/078011 CN2018078011W WO2018161872A1 WO 2018161872 A1 WO2018161872 A1 WO 2018161872A1 CN 2018078011 W CN2018078011 W CN 2018078011W WO 2018161872 A1 WO2018161872 A1 WO 2018161872A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
sequence
cancer
antigen
Prior art date
Application number
PCT/CN2018/078011
Other languages
English (en)
Chinese (zh)
Inventor
闫树德
葛虎
张连山
曹国庆
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN201880003854.1A priority Critical patent/CN109843927B/zh
Publication of WO2018161872A1 publication Critical patent/WO2018161872A1/fr

Links

Images

Classifications

    • 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
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the present invention relates to an anti-B7-H3 antibody immunoreactive to a human B7-H3 receptor, and an antigen-binding fragment thereof, a chimeric antibody, a humanized antibody comprising the CDR region of the anti-B7-H3 antibody, and A pharmaceutical composition comprising a human anti-B7-H3 antibody and an antigen-binding fragment thereof, and use thereof as an anticancer drug.
  • Tumor immunotherapy is a long-term hotspot in the field of cancer treatment, and T cell tumor immunotherapy is at the core. Tumor escape is a huge obstacle to tumor immunotherapy. Most tumors express antigens that can be recognized by the host immune system to varying degrees, but in many cases, due to inefficient activation of effector T cells, an inadequate immune response is triggered. Tumor cells use their own inhibition of the immune system to promote the crazy growth of tumors. Tumor immunotherapy is to fully utilize and mobilize the killing T cells in tumor patients to kill the tumor.
  • B7 superfamily related molecules known as the B7 superfamily.
  • B7 family include B7.1 (CD80), B7.2 (CD86), ligands for inducible stimuli (ICOS-L/B7-H2), and programmed death-1 ligands (PD-L1/ B7-H1), programmed death-2 ligand (PD-L2/B7-DC), B7-H3 and B7-H4, etc., have immunoglobulin V-like domain (IgV) and immunoglobulin C-like structure Domain (IgC) immunoglobulin superfamily members, each encoded by a single exon, are predicted to form back-to-back, non-covalent homodimers on the cell surface.
  • IgV immunoglobulin V-like domain
  • IgC immunoglobulin C-like structure Domain
  • B7-H3 may inhibit T cell activation by NFAT (nuclear factor for activated T cells), NF- ⁇ B (nuclear factor ⁇ B) and AP-1 (activator 1) factors (Yi.KH) Etc., Immunol. Rev. 229: 145-151), and is believed to inhibit Th1, Th2 or Th17 in vivo (Fukushima, A. et al., Immunol. Lett. 113: 52-57; Yi. KH et al., Immunol. Rev. 229:145-151).
  • NFAT nuclear factor for activated T cells
  • NF- ⁇ B nuclear factor ⁇ B
  • AP-1 activator 1
  • B7-H3 is expressed on various cancer cells such as gastric cancer, ovarian cancer, non-small cell lung cancer, and neuroblastoma, and the expression of B7-H3 protein has been detected by immunohistochemistry in tumor cell lines.
  • the present invention provides anti-B7-H3 antibodies with high affinity, high selectivity and high biological activity, monoclonal antibody immunotherapy for tumors and related applications. Drugs, compositions, and methods for the treatment of B7-H3 positive tumors.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, comprising:
  • An antibody light chain variable region comprising at least one LCDR selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8; SEQ ID NO : 14, SEQ ID NO: 15 or SEQ ID NO: 16;
  • An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO :11, SEQ ID NO: 12 or SEQ ID NO: 13.
  • the LCDR preferably includes one or more of LCDR1, LCDR2 and LCDR3, and the amino acid sequence of the LCDR1 is as shown in SEQ ID NO: 6 or SEQ ID NO: 14 in the Sequence Listing; The sequence is as SEQ ID NO: 7 or SEQ ID NO: 15 in the Sequence Listing; the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 8 or SEQ ID NO: 16 in the Sequence Listing.
  • the HCDR comprises one or more of HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 3 or SEQ ID NO: 11 in the Sequence Listing; The SEQ ID NO: 4 or SEQ ID NO: 12 in the Sequence Listing; the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 5 or SEQ ID NO: 13 in the Sequence Listing.
  • an anti-B7-H3 antibody or antigen-binding fragment thereof wherein the antibody light chain variable region comprises the sequence SEQ ID NO: 6, SEQ ID NO :7 and LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 8.
  • an anti-B7-H3 antibody or antigen-binding fragment thereof wherein the antibody light chain variable region comprises the sequence SEQ ID NO: 14, SEQ ID NO : 15 and LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 16.
  • an anti-B7-H3 antibody or antigen-binding fragment thereof wherein the antibody heavy chain variable region comprises the sequence SEQ ID NO: 3, SEQ ID NO, respectively. : 4 and HCDR1, HCDR2 and HCDR3 shown by SEQ ID NO: 5.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the antibody heavy chain variable region comprises the sequence of SEQ ID NO: 11, SEQ ID NO, respectively. : 12 and HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 13.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the antibody light chain variable region comprises a sequence such as:
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8;
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.
  • an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein said antibody heavy chain variable region comprises a sequence such as:
  • HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof may be selected from any of the following:
  • the antibody light chain variable region comprises the sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; the antibody heavy chain variable region comprises sequences such as SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, HCDR1, HCDR2 and HCDR3.
  • the antibody light chain variable region comprises the sequences of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; the antibody heavy chain variable region comprises sequences such as : LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof is preferably a murine antibody, a chimeric antibody, a humanized antibody or a human antibody.
  • the antibody light chain variable region of the murine antibody further comprises a light chain FR region of a murine ⁇ , ⁇ chain or variant thereof.
  • the murine antibody further comprises a light chain constant region of a murine kappa, lambda chain or variant thereof.
  • the B7-H3 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the B7-H3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof.
  • the amino acid sequence of the heavy chain variable region of the murine antibody or the chimeric antibody is as shown in SEQ ID NO. 1 of the Sequence Listing, and the light chain variable region is The amino acid sequence is as shown in SEQ ID NO. 2 of the Sequence Listing; or wherein the amino acid sequence of the murine antibody or the heavy chain variable region of the chimeric antibody is as shown in SEQ ID NO. 9 of the Sequence Listing, light chain The amino acid sequence of the variable region is shown in SEQ ID NO. 10 in the Sequence Listing.
  • the antibody light chain variable region of the humanized antibody further comprises a light chain FR region of a human kappa, lambda chain or variant thereof.
  • the light chain FR region sequence on the light chain variable region of the humanized antibody preferably derived from the human germline light chain IGKV1-33 sequence set forth in SEQ ID NO: 24; or derived from SEQ ID NO : Human germline light chain IGKV1-9 sequence shown in 26.
  • the amino acid sequence of the light chain variable region of the humanized antibody is as set forth in SEQ ID NO: 30 or SEQ ID NO: 33 in the Sequence Listing. More preferably, the humanized antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. Further preferably, the light chain sequence of the humanized antibody is the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 20 or a variant thereof; said variant preferably having a light chain variable region
  • the amino acid change of 0-10 is preferably a mutation of amino acid positions 4 and 9, and the amino acid after the 4th mutation is methionine (M).
  • the heavy chain variable region of the humanized antibody further comprises a heavy chain FR region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG1, IgG2 or IgG4 heavy chain FR
  • IgG1 which enhances the toxicity of antibody-dependent cell-mediated cytotoxicity (antibody-dependent cell-mediated cytotoxicity).
  • the heavy chain FR region sequence on the heavy chain variable region of the humanized antibody preferably derived from the human germline heavy chain IGHV3-23 sequence set forth in SEQ ID NO: 23; or derived from SEQ ID NO The human germline heavy chain IGHV1-2 sequence shown in :25.
  • the amino acid sequence of the heavy chain variable region of the humanized antibody is as set forth in SEQ ID NO: 27 or SEQ ID NO: 31 of the Sequence Listing. More preferably, the humanized antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof. Further preferably, the heavy chain sequence of the humanized antibody is the sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 19 or a variant thereof; the variant preferably has a variable region in the heavy chain
  • the amino acid change of 0-10 is preferably a mutation of amino acid positions 9, 13, and 49, the amino acid after the 9th mutation is proline (P), and the amino acid after the 13th mutation is glutamine (Q). The amino acid after the 49th mutation is alanine (A).
  • the humanized antibody is a humanized antibody huA9 or a humanized antibody huA3;
  • the heavy chain variable region sequence of the humanized antibody huA9 is SEQ ID NO: 31 Shown that the light chain variable region sequence is set forth in SEQ ID NO: 33; the heavy chain variable region sequence of the humanized antibody huA3 is set forth in SEQ ID NO:27, and the light chain variable region sequence is SEQ. ID NO: 30 is shown.
  • the humanized antibody huA9 comprises the heavy chain antibody sequence SEQ ID NO: 19, and the light chain antibody sequence SEQ ID NO: 20; wherein the humanized antibody huA3 comprises the heavy chain antibody sequence SEQ ID NO :17, and the light chain antibody sequence SEQ ID NO:18.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is Fab, Fv, sFv, F(ab') 2 , linear antibody , single-chain antibodies, Nanobodies, domain antibodies, and multispecific antibodies.
  • the invention further provides a DNA sequence encoding an anti-B7-H3 antibody or antigen-binding fragment thereof as described above.
  • the invention further provides an expression vector comprising the DNA sequence as described above.
  • the invention further provides a host cell transformed with an expression vector as described above.
  • a host cell as described above said host cell being a bacterium, preferably Escherichia coli.
  • a host cell as described above is a yeast, preferably Pichia pastoris.
  • a host cell as described above is a mammalian cell, preferably a Chinese hamster ovary (CHO) cell or a human embryonic kidney (HEK) 293 cell.
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • the invention also provides a multispecific antibody comprising a light chain variable region and a heavy chain variable region as previously described.
  • the invention also provides a single chain antibody comprising a light chain variable region and a heavy chain variable region as previously described.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-B7-H3 antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, dilution or carrier.
  • the invention further provides the use of an anti-B7-H3 antibody or antigen-binding fragment thereof as described above for the preparation of a medicament for the treatment of a B7-H3 mediated disease or condition; wherein the disease is preferably cancer;
  • the cancer is B7-H3; the cancer is most preferably breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder Cancer, glioblastoma and melanoma.
  • the invention further provides a method of treating and preventing a B7-H3 mediated disease or condition, comprising administering to a patient in need thereof a therapeutically effective amount of an anti-B7-H3 antibody or antigen-binding fragment thereof as described above, or comprising the same
  • the pharmaceutical composition wherein the disease is preferably cancer; more preferably, a cancer expressing B7-H3; and the cancer is most preferably breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer , gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma and melanoma.
  • FIG 1 is an antibody in vitro ELISA binding experiments, murine antibodies displayed A3 and A9 were purified h-B7H3-Fc-antigen binding activity, EC 50 about 0.03ug / mL.
  • Figure 2 is a graph showing the in vitro binding activity of chimeric antibodies to CHO cells highly expressing B7-H3.
  • Murine antibodies A3 and A9, chimeric antibodies A3C and A9C all showed significant binding activity to target cells at nanomolar (nM) concentration levels.
  • Figure 3 is a graph showing the in vitro binding activity of humanized antibodies to MDA-MB-231 cells highly expressing B7-H3. Both huA3 and huA9 have significant binding activity to target cells at nanomolar levels.
  • antibody 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, ⁇ chain, respectively. , ⁇ chain and ⁇ chain.
  • IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either a kappa chain or a lambda chain by the constant region.
  • Each of the five types of Ig may have a kappa chain or a lambda chain.
  • the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, 2, 3, 4 or a variant 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 (VL) and the heavy chain variable region (VH) 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 VL and VH regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) in number and position, or conform to the Kabat and Chothia numbering rules ( HCDR1).
  • APC antigen presenting cell
  • T cells recognize this complex using the T cell receptor (TCR).
  • APCs include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DC).
  • DC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes B lymphoblasts
  • DC monocyte-derived dendritic cells
  • the term “antigen presentation” refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
  • B7-H3 refers to a member of the human B7 protein family, also known as CD276, which is a type I transmembrane protein with four Ig-like extracellular domains.
  • B7-H3 is one of the immunological checkpoint proteins expressed on the surface of antigen-presenting cells or cancer cells, and has an inhibitory effect on the functional activation of T cells.
  • B7-H3 includes any variant or isoform of B7-H3 naturally expressed by a cell.
  • the antibodies of the invention can be cross-reactive with B7-H3 from non-human species. Alternatively, the antibody may also be specific for human B7-H3 and may not exhibit cross-reactivity with other species.
  • B7-H3, or any variant or isoform thereof can be isolated from cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques common in the art and described herein.
  • the anti-B7-H3 antibody targets human B7-H3 with a normal glycosylation pattern.
  • recombinant human antibody includes human antibodies which are prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as (1) transgenes from human immunoglobulin genes, transgenic chromosomes.
  • Such recombinant human antibodies comprise variable regions and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.
  • murine antibody is in the present invention a monoclonal antibody to human B7-H3 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the B7-H3 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • the murine B7-H3 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a murine kappa, lambda chain or variant thereof, or further comprising a murine IgG1 , heavy chain constant region of IgG2, IgG3 or IgG4 or a variant thereof.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • Human antibodies of the invention can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • human antibody does not include an antibody in which a CDR sequence derived from a germline of another mammalian species, such as a mouse, has been grafted onto a human framework sequence (ie, "humanized antibody”).
  • humanized antibody also referred to as a CDR-grafted antibody, refers to an antibody produced by grafting a CDR sequence of a mouse into a human antibody variable region framework. It is possible to overcome the strong immune response induced by chimeric antibodies by carrying a large amount of mouse protein components. To avoid a decrease in activity while reducing immunogenicity, the human antibody variable region can be subjected to minimal reverse mutation to maintain activity.
  • 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 selected, and then the variable region gene is cloned from the mouse hybridoma cell, and the constant region gene of the human antibody is cloned as needed to change the mouse.
  • the region gene and the human constant region gene are ligated into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC (antibody-dependent) after amino acid mutation Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
  • antigen-binding fragment refers to an antigen-binding fragment of an antibody and an antibody analog, which typically includes at least a portion of an antigen binding or variable region (eg, one or more CDRs) of a parental antibody.
  • the antibody fragment retains at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a molar basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, Nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.
  • a "Fab fragment” consists of a light chain and a heavy chain CH1 and a variable region.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments comprising the CH1 and CH2 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic action of the CH3 domain.
  • a "Fab' fragment” contains a light chain and a portion of a heavy chain comprising a VH domain and a CH1 domain and a region between the CH1 and CH2 domains, thereby being between the two heavy chains of the two Fab' fragments An interchain disulfide bond is formed to form a F(ab')2 molecule.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains.
  • the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both heavy and light chains, but lacks a constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies having multi-epitope specificity.
  • These multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multi-epitope specificity; having two or more Antibodies of the VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region Different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked Along with antibody fragments and the like.
  • single-chain antibody is a single-stranded recombinant protein joined by a heavy chain variable region (VH) and a light chain variable region (VL) of a antibody via a ligation peptide, which is the smallest with complete antigen binding sites.
  • VH heavy chain variable region
  • VL light chain variable region
  • domain antibody fragment is an immunologically functional immunoglobulin fragment containing only a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently joined to a peptide linker to form a bivalent domain antibody fragment.
  • the two VH regions of a bivalent domain antibody fragment can target the same or different antigens.
  • binding to B7-H3 in the present invention means that it can interact with human B7-H3.
  • antigen binding site refers to a three-dimensional spatial site that is discrete on an antigen and is recognized by an antibody or antigen-binding fragment of the present invention.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • An epitope can be formed by an adjacent amino acid, or a non-adjacent amino acid juxtaposed by a tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
  • the terms “specifically bind” and “selectively bind” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody has an equilibrium solution of less than about 10 -7 M or even less when measured by surface plasmon resonance (SPR) techniques in an instrument.
  • the isolating constant (K D ) binds to a predetermined antigen, and its affinity for binding to a predetermined antigen is at least twice its affinity for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen (such as BSA, etc.).
  • the term “antibody recognizing an antigen” can be used interchangeably herein with the term “specifically bound antibody”.
  • cross-reactive refers to the ability of an antibody of the invention to bind to B7-H3 from a different species.
  • an antibody of the invention that binds to human B7-H3 can also bind to B7-H3 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (eg, SPR and ELISA), or binding or functional interactions with cells that physiologically express B7-H3.
  • binding assays eg, SPR and ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • inhibiting or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • the inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blockade.
  • Inhibition and blockade are also intended to include a decrease in any measurable ligand binding affinity when contacted with an anti-B7-H3 antibody, compared to a ligand not contacted with an anti-B7-H3 antibody.
  • inhibiting growth eg, involving cells
  • inhibiting growth is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a particular antigen.
  • inducing for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • the "ADCC” described in the present invention is an antibody-dependent cell-mediated cytotoxicity, which means that a cell expressing an Fc receptor directly kills an antibody by Fc segment of the recognition antibody.
  • Target cells The ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc fragment on IgG. Said modification refers to mutations in the heavy chain constant region of the antibody.
  • a mouse can be immunized with human B7-H3 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 engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody 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 are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified and collected by a conventional technique.
  • 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 -70 ° C, or lyophilized.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention 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 Contact of the test subject, cell, tissue, organ or biological fluid.
  • 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 fluid, wherein the fluids 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 treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, such as a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases, whether by inducing such symptoms to degenerate or inhibiting the progression of such symptoms to any degree of clinical right measurement.
  • 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 in 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 invention may be ineffective in alleviating the symptoms of a target disease in each patient, but according to any statistical test methods known in the art such as Student's t test, chi-square test, according to Mann It was determined by Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test that it should alleviate the target disease symptoms in a statistically significant number of patients.
  • a binding compound consisting essentially of the amino acid sequence recited may also include one or more amino acids that do not significantly affect the properties of the binding compound.
  • naturally occurring refers to the fact that the object can be found in nature.
  • a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a natural source and that has not been intentionally modified in the laboratory by humans is naturally occurring.
  • 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.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the route and dosage of the method of administration, 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 organism, cell, or human body depending on the background.
  • Endogenous refers to a substance produced in a cell, organism or human body depending on the background.
  • “Homology” refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When positions in both comparison sequences are occupied by the same base or amino acid monomer subunit, for example if each position of two DNA molecules is occupied by adenine, then the molecule is homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homology positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologs in 10 positions in the two sequences, then the two sequences are 60% homologous. In general, comparisons are made when the maximum sequence of homology is obtained by aligning the two sequences.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, 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.
  • the human B7-H3 (h-B7H3-his) sequence encoding the His tag, and the human B7-H3 (h-B7H3-Fc) sequence encoding the huFc tag were synthesized by Integrated DNA Technology (IDT) (recombinant B7-H3 above)
  • IDTT Integrated DNA Technology
  • the proteins were all designed into the pTT5 vector (Biovector) by the design of the template sequence of the present invention.
  • the recombinant B7-H3 protein was purified by Example 2 after expression in 293T cells. The purified protein can be used in the experiments of the following examples.
  • the supernatant sample of the HEK293 cell sheet (purchased from the American type culture collection, ATCC) was 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 applied to the 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 further purified by ion exchange (SP column).
  • Configure solution A 0.01 M PB, pH 8.0.
  • Configure liquid B liquid A + 1 M NaCl.
  • the imidazole in PBS solution was eluted to the A solution, and the SP column was equilibrated with the A solution.
  • the concentration of the B solution was 0-100%, and 10 column volumes were eluted to collect the elution peaks.
  • the obtained protein was electrophoresed, peptide map, and LC-MS was identified and used.
  • the supernatant sample expressed by HEK293 cells was centrifuged at high speed to remove impurities, and the buffer was exchanged for PBS.
  • the Protein A affinity column was equilibrated with 10 mM phosphate buffer and rinsed 2-5 column volumes. The displaced supernatant sample was applied to the column. Rinse the column with 25 column volumes of buffer until the A280 reading drops to baseline.
  • the target protein was eluted with 0.8% acetate buffer at pH 3.5, and the elution peak was collected. Immediately after the addition, the mixture was neutralized with 1 M Tris-Cl pH 8.0 buffer, and then the solution was replaced with Millipore's Amico-15 filter column. .
  • the obtained protein was electrophoresed, peptide map, and LC-MS was identified and used.
  • the full-length sequence encoding the human or cynomolgus B7-H3 protein was synthesized by Integrated DNA Technology (IDT) (the above B7-H3 recombinant proteins were designed by the template sequence of the present invention) and cloned into pcDNA3.1/, respectively. Puro (Invitrogen #V79020).
  • CHO-S (ATCC) cells were cultured to 0.5 x 10 6 /ml in CD-CHO medium (Life Technologies, #10743029).
  • 10 ⁇ g of the vector encoding the huB7H3 or cyB7H3 gene was mixed with 50 ul of LF-LTX (Life Technologies, #A12621) in 1 ml Opti-MEM medium (Life Technologies, #31985088), incubated for 20 minutes at room temperature, and then added to CHO cell culture medium. And put it into a carbon dioxide incubator for cultivation. After 24 hours, the new medium was replaced and 10 ⁇ g/ml of puromycin was added. After that, the new culture solution was changed every 2-3 days, and after 10-12 days of screening, a stable CHO-S cell pool was obtained.
  • LF-LTX Life Technologies, #A12621
  • Opti-MEM medium Life Technologies, #31985088
  • Anti-human B7H3 monoclonal antibodies are produced by immunizing mice.
  • the experiment used Swiss Webster white mice, female, 6 weeks old (Charles River). 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%.
  • the immunizing antigen is the Fc-tagged human B7H3 recombinant protein (huB7H3-Fc).
  • Titermax Sigma Lot Num: T2684
  • Titermax was used as an adjuvant.
  • the ratio of antigen to adjuvant (titermax) was 1:1, and the antigen was emulsified and inoculated for 0, 21, 35, 49, and 63 days.
  • IP intraperitoneal
  • splenocytes were fused to mice with high antibody titers in serum and titers to the platform, and spleen lymphocytes and myeloma cells Sp2/0 cells (ATCC) were optimized using an electrofusion procedure.
  • CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • the conjugated hybridoma cells were cultured for 7-14 days, the supernatant of the culture medium was taken, and the hybridoma supernatant was subjected to antibody screening using the B7-H3 recombinant protein huB7H3-Fc.
  • the positive antibody strain obtained was further stably expressed B7.
  • -H3 CHO-S cells comparing blank CHO-S cells to exclude non-specific binding antibody hybridoma strains, and screening by flow sorting method, thereby selecting two hybridomas that bind to recombinant protein and also bind to cell-expressing antigen .
  • the cDNA obtained by reverse transcription was subjected to PCR amplification using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and then sequenced, and finally the sequences of two murine mouse antibodies A3 and A9 were obtained.
  • the heavy and light chain variable region sequences of murine mAb A3 are as follows:
  • HCDR1 RYGMS SEQ ID NO: 3
  • HCDR2 ISSGGGSIYYPDTVKG SEQ ID NO: 4
  • HCDR3 TRHYLLFEMDY SEQ ID NO: 5
  • LCDR1 KASQNVNTAVA SEQ ID NO: 6
  • LCDR2 SASNRYT SEQ ID NO:7
  • LCDR3 QQYSSSLT SEQ ID NO:8
  • the heavy and light chain variable region sequences of A9 are as follows:
  • the heavy and light chain variable regions of each mouse antibody were cloned into pTT vector plasmid (Biovector) containing human IgG1 heavy chain constant region and kappa light chain constant region, respectively, and then transiently transfected into HEK293 cells to obtain anti-resistant
  • the chimeric antibodies A3C and A9C of B7-H3 were purified, identified, and assayed for activity as described in Example 2 (purification with Fc-tag protein).
  • the neutralizing avidin for binding to biotin was diluted to 1 ⁇ g/ml with PBS buffer, added to a 96-well plate in a volume of 100 ⁇ l/well, and left at 4 ° C for 16 h to 20 h.
  • PBST pH 7.4 PBS containing 0.05% Tween-20
  • 120 ⁇ l/well of PBST/1% milk was added, and the mixture was incubated for 1 hour at room temperature for blocking.
  • 1 ⁇ g/ml of biotin-labeled h-B7H3-Fc diluted with PBST/1% milk was added and incubated for 1 h at room temperature.
  • the B7-H3 antibody to be tested diluted with PBST/1% milk was added to an appropriate concentration, and incubated at room temperature for 1.5 h.
  • the reaction system was removed, and the plate was washed three times with PBST, and then a horseradish peroxidase (HRP)-labeled anti-mouse antibody secondary antibody (The Jackson Laboratory) diluted with PBST/1% milk was added at 100 ⁇ l/well. Incubate for 1 h at room temperature. After washing the plate 3 times with PBST, 100 ⁇ l/well of TMB was added and incubated for 5-10 min at room temperature.
  • HRP horseradish peroxidase
  • the reaction was stopped by the addition of 100 ⁇ l/well of 1 MH 2 SO 4 , and the absorbance was read at 450 nm, and the ELISA binding EC 50 value was calculated.
  • the EC 50 of the antibodies A3 and A9 were both about 0.03 ⁇ g/mL, and the murine antibodies A3 and A9 all had binding activity to the purified h-B7H3-Fc antigen.
  • CHO-S cells highly expressing huB7-H3 were centrifuged at 1000 rpm for 5 minutes, and the precipitate was collected and suspended in 10-15 ml of pre-cooled flow buffer, and the cells were counted.
  • the cells were collected by centrifugation at 1000 rpm for 5 minutes in a 50 ml centrifuge tube, the supernatant was discarded, and the pellet was resuspended in a pre-cooled blocking buffer at a density of 0.5-1.0 x 10 7 cells/ml. After incubating for 30 minutes at 4 ° C, it was resuspended to 100 ⁇ l per well and added to a 96-well plate.
  • A3, A9 and their corresponding chimeric antibodies all showed significant binding to huB7H3 high expressing cells at nanomolar (nM) concentration level, and their binding was stronger than that of Macrogenics' reference antibody BRCA84D.
  • CHO-S cells with high expression of cyB7-H3 were used to detect the binding of each antibody to cynomolgus B7-H3 at a single concentration of 10 nM.
  • the results are shown in Table 3 below.
  • A3C has obvious binding to monkey antigen.
  • Signal, while A9C and the reference antibody BRCA84D have no binding.
  • This experiment was measured by surface plasmon resonance (SPR) method.
  • the anti-mouse IgG polyclonal antibody was covalently linked to a CM5 (GE) chip using a standard amino coupling method using a kit supplied by Biacore, and then the purified mouse antibody to be tested of the present invention was captured to the stationary phase using the antibody. .
  • Humanization of murine anti-human B7-H3 monoclonal antibodies was performed as disclosed in many literatures in the art. Briefly, the human constant domain is used in place of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the homology of the murine antibody and the human antibody.
  • the present invention humanizes the candidate molecules A3 and A9.
  • the heavy and light chain variable region sequences were compared with the human antibody germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from a human kappa light chain gene
  • the human germline heavy chain framework region is derived from a human heavy chain
  • the antibody of the present invention is preferably a human germline antibody template shown below.
  • A3 is preferably a human germline heavy chain template IGHV3-23 (SEQ ID NO: 23):
  • A3 preferably human germline light chain template IGkV1-33 (SEQ ID NO: 24):
  • A9 is preferably a human germline heavy chain template IGHV1-2 (SEQ ID NO: 25):
  • A9 is preferably a human germline light chain template IGkV1-9 (SEQ ID NO: 26):
  • the CDR regions of murine antibodies A3 and A9 are grafted onto the selected corresponding humanized template, the humanized variable region is replaced, and recombined with the IgG constant region (preferably the heavy chain is IgG1 and the light chain is ⁇ ). Then, based on the three-dimensional structure of the murine antibody, the residues which have an important interaction with the CDRs and the residues of the CDRs, and the residues which have an important influence on the conformation of VL and VH are subjected to back mutation and the CDR regions are The chemically labile amino acid residues were optimized, and antibodies assembled from the following humanized light and heavy chain variable region sequences were designed and tested.
  • the final humanized huA3 (using the H1 heavy chain and the L2 light chain) and the huA9 antibody molecule (using the H1 heavy chain and the L1 light chain) were selected by expression test and back mutation number comparison, the respective sequences of which are SEQ ID NO: 17-20.
  • the huA9 antibody light chain sequence encodes a gene sequence (wherein the first 60 nucleotides of the following sequence are not involved in the coding of the final huA9 antibody light chain):
  • a cDNA fragment was synthesized based on the gene sequences of the above humanized antibody light and heavy chains, and inserted into a pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2 and placed in a CO 2 incubator for incubation 4- 5 days. After the expressed antibody was recovered by centrifugation, antibody purification was carried out by the method of Example 2 (purification with Fc-tag protein) to obtain a humanized antibody protein of the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Botany (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un anticorps anti-B7-H3, un fragment de liaison à l'antigène de celui-ci, et une composition pharmaceutique comprenant l'anticorps ou le fragment de liaison à l'antigène. L'invention concerne également une utilisation de l'anticorps ou du fragment de liaison à l'antigène dans la préparation d'un médicament pour le traitement d'une maladie ou d'un trouble induit par B7-H3. L'anticorps peut être un anticorps murin, un anticorps chimérique, un anticorps humanisé ou un anticorps humain. La composition pharmaceutique de l'invention peut être utilisée pour traiter des cancers.
PCT/CN2018/078011 2017-03-06 2018-03-05 Anticorps anti-b7-h3, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée WO2018161872A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201880003854.1A CN109843927B (zh) 2017-03-06 2018-03-05 抗b7-h3抗体、其抗原结合片段及其医药用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710129023 2017-03-06
CN201710129023.6 2017-03-06

Publications (1)

Publication Number Publication Date
WO2018161872A1 true WO2018161872A1 (fr) 2018-09-13

Family

ID=63447283

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/078011 WO2018161872A1 (fr) 2017-03-06 2018-03-05 Anticorps anti-b7-h3, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée

Country Status (3)

Country Link
CN (1) CN109843927B (fr)
TW (1) TWI673287B (fr)
WO (1) WO2018161872A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020018964A1 (fr) 2018-07-20 2020-01-23 Fred Hutchinson Cancer Research Center Compositions et procédés pour réguler l'expression de récepteurs spécifiques à l'antigène
WO2020151384A1 (fr) * 2019-01-22 2020-07-30 苏州旭光科星抗体生物科技有限公司 Procédé de préparation et procédé de test immunohistochimique pour anticorps monoclonal anti-b7-h3 humain, utilisation et kit d'anticorps monoclonal anti-b7-h3 humain
WO2021052307A1 (fr) * 2019-09-16 2021-03-25 南京圣和药业股份有限公司 Anticorps anti-b7-h3 et son application
WO2021244590A1 (fr) * 2020-06-02 2021-12-09 明慧医药（上海）有限公司 Anticorps anti-b7-h3 et sa préparation et son utilisation
WO2022117040A1 (fr) * 2020-12-02 2022-06-09 迈威(上海)生物科技股份有限公司 Anticorps anti-b7-h3 humain et utilisation associée
EP4043491A4 (fr) * 2019-10-09 2022-12-07 Dartsbio Pharmaceuticals Ltd. Nanocorps b7-h3 et son procédé de préparation et son utilisation
WO2023131193A1 (fr) * 2022-01-07 2023-07-13 苏州旭光科星抗体生物科技有限公司 Anticorps monoclonal humanisé ciblant la molécule b7-h3 humaine et son utilisation
WO2023236949A1 (fr) * 2022-06-07 2023-12-14 映恩生物制药（苏州）有限公司 Conjugué anticorps-médicament anti-b7h4 et son utilisation
EP4061847A4 (fr) * 2019-11-18 2024-01-10 Univ Texas Anticorps monoclonal anti-b7-h3 et ses procédés d'utilisation

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111454357B (zh) * 2019-08-14 2022-03-15 康诺亚生物医药科技（成都）有限公司 一种含有抗体的肿瘤治疗剂的开发和应用
CN117024590A (zh) * 2020-04-22 2023-11-10 复星凯特生物科技有限公司 抗人b7-h3的单克隆抗体及其应用
CN113527493B (zh) * 2021-07-20 2023-10-27 广州爱思迈生物医药科技有限公司 一种b7-h3抗体及其应用
CN116178553B (zh) * 2022-02-25 2023-11-17 南京蓬勃生物科技有限公司 针对人b7-h3的抗体及其变体

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103687945A (zh) * 2011-04-25 2014-03-26 第一三共株式会社 抗-b7-h3抗体
WO2016033225A2 (fr) * 2014-08-27 2016-03-03 Memorial Sloan Kettering Cancer Center Anticorps, compositions et leurs utilisations
CN106279416A (zh) * 2010-03-04 2017-01-04 宏观基因有限公司 与b7‑h3反应性的抗体、其免疫学活性片段及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RS60033B1 (sr) * 2009-11-24 2020-04-30 Medimmune Ltd Ciljano vezujući agensi usmereni na b7-h1
MX2015017341A (es) * 2013-07-09 2016-07-06 Anexxon Inc Anticuerpos anti-factor del complemento c1q y uso de los mismos.
TW201909926A (zh) * 2017-08-04 2019-03-16 大陸商江蘇恆瑞醫藥股份有限公司 Ｂ７ｈ３抗體－藥物偶聯物及其醫藥用途
AU2019351427A1 (en) * 2018-09-30 2021-04-15 Changzhou Hansoh Pharmaceutical Co., Ltd. Anti-B7H3 antibody-exatecan analog conjugate and medicinal use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106279416A (zh) * 2010-03-04 2017-01-04 宏观基因有限公司 与b7‑h3反应性的抗体、其免疫学活性片段及其用途
CN103687945A (zh) * 2011-04-25 2014-03-26 第一三共株式会社 抗-b7-h3抗体
WO2016033225A2 (fr) * 2014-08-27 2016-03-03 Memorial Sloan Kettering Cancer Center Anticorps, compositions et leurs utilisations

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020018964A1 (fr) 2018-07-20 2020-01-23 Fred Hutchinson Cancer Research Center Compositions et procédés pour réguler l'expression de récepteurs spécifiques à l'antigène
WO2020151384A1 (fr) * 2019-01-22 2020-07-30 苏州旭光科星抗体生物科技有限公司 Procédé de préparation et procédé de test immunohistochimique pour anticorps monoclonal anti-b7-h3 humain, utilisation et kit d'anticorps monoclonal anti-b7-h3 humain
WO2021052307A1 (fr) * 2019-09-16 2021-03-25 南京圣和药业股份有限公司 Anticorps anti-b7-h3 et son application
EP4043491A4 (fr) * 2019-10-09 2022-12-07 Dartsbio Pharmaceuticals Ltd. Nanocorps b7-h3 et son procédé de préparation et son utilisation
JP2022551318A (ja) * 2019-10-09 2022-12-08 ダーツバイオ ファーマシューティカルズ リミテッド Ｂ７－ｈ３ナノ抗体、その製造方法および使用
EP4061847A4 (fr) * 2019-11-18 2024-01-10 Univ Texas Anticorps monoclonal anti-b7-h3 et ses procédés d'utilisation
WO2021244590A1 (fr) * 2020-06-02 2021-12-09 明慧医药（上海）有限公司 Anticorps anti-b7-h3 et sa préparation et son utilisation
WO2022117040A1 (fr) * 2020-12-02 2022-06-09 迈威(上海)生物科技股份有限公司 Anticorps anti-b7-h3 humain et utilisation associée
WO2023131193A1 (fr) * 2022-01-07 2023-07-13 苏州旭光科星抗体生物科技有限公司 Anticorps monoclonal humanisé ciblant la molécule b7-h3 humaine et son utilisation
WO2023236949A1 (fr) * 2022-06-07 2023-12-14 映恩生物制药（苏州）有限公司 Conjugué anticorps-médicament anti-b7h4 et son utilisation

Also Published As

Publication number Publication date
CN109843927A (zh) 2019-06-04
TW201833141A (zh) 2018-09-16
TWI673287B (zh) 2019-10-01
CN109843927B (zh) 2022-06-21

Similar Documents

Publication Publication Date Title
WO2018161872A1 (fr) Anticorps anti-b7-h3, fragment de liaison à l'antigène de celui-ci, et utilisation pharmaceutique associée
US11472882B2 (en) Anti-B7-H4 antibody, antigen-binding fragment thereof and pharmaceutical use thereof
US11525005B2 (en) Anti-CD40 antibody, antigen binding fragment thereof and medical use thereof
WO2019141268A1 (fr) Anticorps anti-4-1bb, fragment de liaison à l'antigène de celui-ci et utilisation médicale associée
CN112243443B (zh) 抗trop-2抗体、其抗原结合片段及其医药用途
TWI714895B (zh) 抗csf-1r抗體、其抗原結合片段及其醫藥用途
WO2019184935A1 (fr) Anticorps anti-cd27, fragment de liaison à l'antigène de celui-ci et utilisation médicale associée
CN110461874B (zh) 抗gitr抗体、其抗原结合片段及其医药用途
CN115298216A (zh) 抗体或其抗原结合片段、其制备方法及医药用途
RU2792748C2 (ru) Антитело к b7-h4, его антигенсвязывающий фрагмент и его фармацевтическое применение
WO2021209066A1 (fr) Molécule spécifique de liaison à l'antigène, procédé de préparation correspondant et utilisation pharmaceutique associée
RU2779128C2 (ru) Антитело к cd40, его антигенсвязывающий фрагмент и его медицинское применение

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18764083

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18764083

Country of ref document: EP

Kind code of ref document: A1