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WO2021052307A1 - 一种抗b7-h3抗体及其应用 - Google Patents

一种抗b7-h3抗体及其应用 Download PDF

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Publication number
WO2021052307A1
WO2021052307A1 PCT/CN2020/115262 CN2020115262W WO2021052307A1 WO 2021052307 A1 WO2021052307 A1 WO 2021052307A1 CN 2020115262 W CN2020115262 W CN 2020115262W WO 2021052307 A1 WO2021052307 A1 WO 2021052307A1
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seq
amino acid
acid sequence
antibody
chain variable
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PCT/CN2020/115262
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English (en)
French (fr)
Inventor
王勇
赵立文
肖扬
徐瑶
罗成
杨洋
魏爱琪
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南京圣和药业股份有限公司
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Priority to EP20865483.0A priority Critical patent/EP4039702A1/en
Priority to US17/760,738 priority patent/US20220332829A1/en
Publication of WO2021052307A1 publication Critical patent/WO2021052307A1/zh

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07K16/2827—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 against B7 molecules, e.g. CD80, CD86
    • 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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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 invention relates to the technical field of antibody medicines, in particular to an anti-B7-H3 antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising an anti-B7-H3 antibody or an antigen-binding fragment thereof, and their applications.
  • B7-H3 (CD276) belongs to the B7 superfamily and is a transmembrane glycoprotein. Its extracellular domain structure is divided into two types, one is the monovalent 2Ig-B7-H3, and the other is composed of two repeating units. The bivalent 4Ig-B7-H3. B7-H3 is a new type of immune checkpoint. According to the literature, B7-H3 can inhibit T cell proliferation and cytokine release by interacting with a receptor of unknown structure (Suh W K, et al.
  • the B7 family member B7 -H3 preferentially down-regulates T helper type 1-mediated immune responses[J].Nature Immunology,2003,4(9):899), inhibits the lytic activity of NK cells on patient-derived tumor cells with high B7-H3 expression ( Castriconi R,et al.Identification of 4Ig-B7-H3 as a neuroblastoma-associated molecule that exerts a protective role from an NK cell-mediated lysis[J].Proceedings of the National Academy(34) of Science, 2004 12640-12645). Although the B7-H3 receptor is unknown, there have been more and more reports about the negative regulation of the role of B7-H3 and the receptor in tumor immunity in recent years.
  • Tumor cells express B7-H3 to avoid CD8+ T cell immune surveillance.
  • the use of anti-mouse B7-H3 antibodies can enhance the immune function of CD8+ T cells (Yonesaka K, et al. B7- H3 negatively modulates CTL-mediated cancer immunity[J].Clinical Cancer Research,2018,clincanres.2852.2017).
  • knockout mice B7-H3 or the use of anti-mouse B7-H3 antibodies can significantly inhibit tumor growth, and this inhibition depends on the function of CD8+T and NK cells (Lee Y, et al.Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function[J].Cell Research,2017,27(8):1034-1045).
  • Studies in lung cancer patients found that patients with high B7-H3 expression have fewer tumor infiltrating lymphocytes and are more likely to develop lymph node metastasis (Altan M, et al. B7-H3 expression in NSCLC and its association with B7-H4, PD-L1 and Tumor Infiltrating Lymphocytes[J].Clinical Cancer Research,2017,23(17):5202).
  • B7-H3 is abnormally expressed in a variety of tumor cells, tumor stroma, tumor blood vessels, tumor infiltrating macrophages and DC cells.
  • tissue samples from patients with prostate cancer, pancreatic cancer, hepatocellular carcinoma, head and neck cancer, kidney cancer, etc. the expression ratio of B7-H3 all exceeded 90%, and expression of B7-H3 was rarely seen in normal tissues (Seaman S, et al. Eradication of Tumors through Simultaneous Ablation of CD276/B7-H3-Positive Tumor Cells and Tumor Vasculature. [J]. Cancer Cell, 2017, 31(4): 501-515).
  • the clinically researched antibody drugs against the B7-H3 target are all based on the tumor targeting of B7-H3 for drug development.
  • Enoblituzumab a humanized anti-B7-H3 monoclonal antibody, kills tumors by targeting the tumor-mediated ADCC effect of the Fc segment of the antibody.
  • Burtomab is a radioactive iodine-carrying anti-B7-H3 murine antibody drug conjugate. It is used in the clinical treatment of metastatic neuroblastoma and significantly improves the survival of children with tumors.
  • B7-H3 Based on the high expression characteristics of B7-H3 in the tumor environment and its inhibitory activity on immune cells, B7-H3 can be used as an effective target for the development of anti-tumor drugs.
  • an anti-B7-H3 antibody or antigen-binding fragment thereof which has higher affinity and stability, can block the immunosuppression of B7-H3 on tumors, and enhance the killing activity of CD8+ T cells and NK cells on tumors , And cooperate with the ADCC effect of the Fc segment of the antibody to play an anti-tumor effect, thereby having a more excellent anti-tumor activity.
  • an anti-B7-H3 antibody or antigen-binding fragment thereof which comprises a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises the complement of the heavy chain variable region Determining region 1 (HCDR1), complementarity determining region 2 (HCDR2) of the heavy chain variable region, and/or complementarity determining region 3 (HCDR3) of the heavy chain variable region, the light chain variable region comprising the light chain variable region
  • HCDR1 complementarity determining region 1
  • LCDR2 complementarity determining region 2
  • HCDR3 complementarity determining region 3
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the following group:
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from the following group:
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 1, 2 and 3, or the same as SEQ ID NO:
  • the amino acid sequences shown in 1, 2 and 3 have a heavy chain variable region of a CDR with at least 85% sequence identity
  • the LCDR1, LCDR2, and LCDR3 are respectively SEQ ID NO: 4, 5 and 6 or the same as SEQ ID NO. :
  • the amino acid sequences shown in 4, 5 and 6 have the light chain variable region of CDRs with at least 85% sequence identity.
  • the present invention provides an anti-B7-H3 antibody or an antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 7, 8 and 9, or the same as SEQ ID NO:
  • the amino acid sequences shown in 7, 8 and 9 have CDR heavy chain variable regions with at least 85% sequence identity
  • the LCDR1, LCDR2, and LCDR3 are respectively SEQ ID NO: 10, 11 and 12 or the same as SEQ ID NO. :
  • the amino acid sequence shown in 10, 11 and 12 has a light chain variable region of a CDR with at least 85% sequence identity;
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 13, 14 and 15, respectively, or the same as SEQ ID NO:
  • the amino acid sequences shown in 13, 14 and 15 have the heavy chain variable regions of CDRs with at least 85% sequence identity
  • the LCDR1, LCDR2, and LCDR3 are SEQ ID NO: 16, 18, and 19, or the same as SEQ ID NO. :
  • the amino acid sequences shown in 16, 18 and 19 have at least 85% sequence identity in the light chain variable region of the CDR.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 13, 14 and 15, respectively, or the same as SEQ ID NO:
  • the amino acid sequences shown in 13, 14 and 15 have the heavy chain variable regions of CDRs with at least 85% sequence identity
  • the LCDR1, LCDR2, and LCDR3 are SEQ ID NO: 17, 18 and 19, or the same as SEQ ID NO. :
  • the amino acid sequences shown in 17, 18 and 19 have at least 85% sequence identity in the light chain variable region of the CDR.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 20, 21 and 22, or the same as SEQ ID NO:
  • the amino acid sequences shown in 20, 21, and 22 have the heavy chain variable regions of CDRs with at least 85% sequence identity
  • the LCDR1, LCDR2, and LCDR3 are SEQ ID NO: 23, 24, and 25, or the same as SEQ ID NO. :
  • the amino acid sequences shown in 23, 24 and 25 have at least 85% sequence identity in the light chain variable region of the CDR.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof according to the present invention is a monoclonal antibody or antigen-binding fragment thereof.
  • the anti-B7-H3 antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, or a humanized antibody or an antigen-binding fragment thereof .
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence shown in (b1) is obtained by substituting, deleting or adding one or more amino acids and having the same or similar function as the amino acid sequence shown in (b1);
  • amino acid sequence of the light chain variable region is selected from:
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 26, and SEQ ID NO: 26 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 26 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 26, and the amino acid of the light chain variable region
  • the sequence is SEQ ID NO: 27, SEQ ID NO: 27 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 27 or having at least 85% sequence with SEQ ID NO: 27 Amino acid sequence of identity.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 28, and SEQ ID NO: 28 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 28 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 28, and the amino acid of the light chain variable region
  • the sequence is SEQ ID NO: 29, SEQ ID NO: 29 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 29 or having at least 85% sequence with SEQ ID NO: 29 Amino acid sequence of identity.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 30 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 30, and the amino acid of the light chain variable region
  • the sequence is SEQ ID NO: 31, SEQ ID NO: 31 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 31 or having at least 85% sequence with SEQ ID NO: 31 Amino acid sequence of identity.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 30 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 30, and the amino acid of the light chain variable region
  • the sequence is SEQ ID NO: 32
  • SEQ ID NO: 32 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 32 or having at least 85% sequence with SEQ ID NO: 32 Amino acid sequence of identity.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 35, and SEQ ID NO: 35 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 35 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 35, and the amino acid of the light chain variable region
  • the sequence is SEQ ID NO: 36
  • SEQ ID NO: 36 is obtained by substituting, deleting or adding one or more amino acids and having the same function as SEQ ID NO: 36 or having at least 85% sequence with SEQ ID NO: 36 Amino acid sequence of identity.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 26, and SEQ ID NO: 26 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 26 or having at least 85% sequence identity with SEQ ID NO: 26 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 1 , 2 and 3
  • the amino acid sequence of the light chain variable region is SEQ ID NO: 27, SEQ ID NO: 27 is obtained by substituting, deleting or adding one or more amino acids and is identical to SEQ ID
  • the functionally identical amino acid sequence of NO:27 or SEQ ID NO:27 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 4, 5 and 6.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 28, and SEQ ID NO: 28 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 28 or having at least 85% sequence identity with SEQ ID NO: 28 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 7
  • the amino acid sequence shown in, 8 and 9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 29, SEQ ID NO: 29 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID
  • the functionally identical amino acid sequence of NO:29 or SEQ ID NO:29 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:10, 11 and 12.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 30 or having at least 85% sequence identity with SEQ ID NO: 30 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 13 , 14 and 15, and the amino acid sequence of the variable region of the light chain is SEQ ID NO: 31, SEQ ID NO: 31 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID
  • the functionally identical amino acid sequence of NO:31 or SEQ ID NO:31 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 16, 18 and 19.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 30, and SEQ ID NO: 30 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 30 or having at least 85% sequence identity with SEQ ID NO: 30 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 13 , 14 and 15, and the amino acid sequence of the light chain variable region is SEQ ID NO: 32, SEQ ID NO: 32 is obtained by substituting, deleting or adding one or more amino acids and is identical to SEQ ID
  • the functionally identical amino acid sequence of NO:32 or SEQ ID NO:32 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 17, 18 and 19.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 35, and SEQ ID NO: 35 is substituted,
  • An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 35 or having at least 85% sequence identity with SEQ ID NO: 35 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 20
  • the amino acid sequence shown in, 21 and 22, and the amino acid sequence of the light chain variable region is SEQ ID NO: 36, SEQ ID NO: 36 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID
  • the amino acid sequence of NO: 36 is functionally identical or has at least 85% sequence identity with SEQ ID NO: 36 and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 23, 24 and 25.
  • the anti-B7-H3 antibody according to the present invention is a murine antibody, which further contains the heavy chain constant region of murine IgG1, IgG2, IgG3, or IgG4 or a variant thereof, and murine The light chain constant region of a kappa chain or a variant thereof.
  • the anti-B7-H3 murine antibody according to the present invention further contains the heavy chain constant region of murine IgG1 or IgG2 or a variant thereof, and the light chain constant of a murine kappa chain or a variant thereof. Area.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the anti-B7-H3 antibody is a humanized antibody, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c4);
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the anti-B7-H3 antibody is a humanized antibody, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • (c3) It has at least 85% sequence identity with the amino acid sequence shown in (c1), and the HCDR1, HCDR2 and HCDR3 are the amino acid sequences shown in SEQ ID NO: 1, 2 and 3; and
  • amino acid sequence of the light chain variable region is selected from:
  • amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c4);
  • (c6) has at least 85% sequence identity with the amino acid sequence shown in (c4), and the LCDR1, LCDR2, and LCDR3 have the amino acid sequences shown in SEQ ID NO: 4, 5, and 6.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the anti-B7-H3 antibody is a humanized antibody, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c4);
  • (c6) has at least 85% sequence identity with the amino acid sequence shown in (c4), and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 10, 11 and 12.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the anti-B7-H3 antibody is a humanized antibody, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c4);
  • (c6) has at least 85% sequence identity with the amino acid sequence shown in (c4), and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NOs: 17, 18 and 19.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the anti-B7-H3 antibody is a humanized antibody, wherein:
  • amino acid sequence of the heavy chain variable region is selected from:
  • amino acid sequence of the light chain variable region is selected from:
  • amino acid sequence shown in (c4) is obtained by substituting, deleting or adding one or more amino acids, and having the same or similar function as the amino acid sequence shown in (c4);
  • (c6) has at least 85% sequence identity with the amino acid sequence shown in (c4), and the LCDR1, LCDR2 and LCDR3 have the amino acid sequences shown in SEQ ID NO: 23, 24 and 25.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 45, and SEQ ID NO: 45 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 45 or having at least 85% sequence identity with SEQ ID NO: 45 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 1
  • the amino acid sequence shown in, 2 and 3 and the amino acid sequence of the light chain variable region is SEQ ID NO: 49, SEQ ID NO: 49 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID
  • the functionally identical amino acid sequence of NO:49 or SEQ ID NO:49 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are the amino acid sequences shown in SEQ ID NO: 4, 5 and 6.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 52, and SEQ ID NO: 52 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 52 or having at least 85% sequence identity with SEQ ID NO: 52 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 7 , 8 and 9, and the amino acid sequence of the light chain variable region is SEQ ID NO: 59, SEQ ID NO: 59 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID
  • the functionally identical amino acid sequence of NO:59 or SEQ ID NO:59 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:10, 11 and 12.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 63, and SEQ ID NO: 63 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 63 or having at least 85% sequence identity with SEQ ID NO: 63 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 13
  • the amino acid sequence shown in, 14 and 15, and the amino acid sequence of the light chain variable region is SEQ ID NO: 65, SEQ ID NO: 65 is obtained by substitution, deletion or addition of one or more amino acids and is the same as SEQ ID
  • the functionally identical amino acid sequence of NO:65 or SEQ ID NO:65 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 17, 18 and 19.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 64, and SEQ ID NO: 64 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 64 or having at least 85% sequence identity with SEQ ID NO: 64 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 13 , 14 and 15, and the amino acid sequence of the light chain variable region is SEQ ID NO: 67, SEQ ID NO: 67 is obtained by substituting, deleting or adding one or more amino acids and is identical to SEQ ID
  • the functionally identical amino acid sequence of NO: 67 or SEQ ID NO: 67 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 17, 18 and 19.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 72, SEQ ID NO: 72 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 72 or having at least 85% sequence identity with SEQ ID NO: 72 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 20 , 21 and 22, and the amino acid sequence of the light chain variable region is SEQ ID NO: 77, SEQ ID NO: 77 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID
  • the functionally identical amino acid sequence of NO:77 or SEQ ID NO:77 has at least 85% sequence identity and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:23, 24 and 25.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 22, and SEQ ID NO: 22 is substituted,
  • the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 22 or having at least 85% sequence identity with SEQ ID NO: 22 and the HCDR1, HCDR2 and HCDR3 are as SEQ ID NO: 1 , 2 and 3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 25, SEQ ID NO: 25 is obtained by substituting, deleting or adding one or more amino acids, and is identical to SEQ ID
  • the amino acid sequence of NO:25 is functionally identical or has at least 85% sequence identity with SEQ ID NO:25, and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO:10, 11 and 12.
  • the present invention provides an anti-B7-H3 humanized antibody or antigen-binding fragment thereof, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof
  • the light chain includes a human-derived kappa, lambda chain or a light chain constant region of a variant thereof.
  • the murine anti-B7-H3 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine ⁇ , ⁇ chain or a variant thereof, and/or further comprise The heavy chain constant region of murine IgG1, IgG2, IgG3 or IgG4 or variants thereof.
  • the antibody light chain of the anti-B7-H3 chimeric antibody or antigen-binding fragment thereof further comprises a light chain constant region of murine ⁇ , ⁇ chain or a mutant sequence thereof.
  • the antibody heavy chain of the anti-B7-H3 chimeric antibody or antigen-binding fragment thereof further comprises a heavy chain constant region of murine IgG1, IgG2, IgG3, gG4 or its mutant sequence, and preferably comprises a human IgG1 or IgG2 heavy chain constant Region, or IgG4 constant region that significantly reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after using amino acid mutations.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the anti-B7-H3 humanized antibody or antigen-binding fragment thereof of the present invention further comprises the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, and human ⁇ , The light chain constant region of the lambda chain or a variant thereof.
  • the anti-B7-H3 humanized antibody or antigen-binding fragment thereof of the present invention further comprises the heavy chain constant region of human IgG1 or IgG2 or a variant thereof, and a human kappa chain or a variant thereof The constant region of the light chain.
  • the present invention provides an anti-B7-H3 antibody or antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fv, sFv or F(ab)2.
  • Another aspect of the present invention provides an isolated nucleic acid encoding the anti-B7-H3 antibody or antigen-binding fragment thereof according to the present invention.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is such as SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84 or SEQ ID NO: 86; the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85 or SEQ ID NO: 87.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the heavy chain variable region SEQ ID NO: 45 is shown in SEQ ID NO: 80; encoding the light chain variable region SEQ ID
  • the nucleotide sequence of NO:49 is shown in SEQ ID NO:81.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the heavy chain variable region SEQ ID NO: 52 is shown in SEQ ID NO: 82; encoding the light chain variable region SEQ ID
  • the nucleotide sequence of NO:59 is shown in SEQ ID NO:83.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the heavy chain variable region SEQ ID NO: 64 is shown in SEQ ID NO: 84; encoding the light chain variable region SEQ ID
  • the nucleotide sequence of NO: 67 is shown in SEQ ID NO: 85.
  • the isolated nucleic acid according to the present invention wherein the nucleotide sequence encoding the heavy chain variable region SEQ ID NO: 72 is shown in SEQ ID NO: 86; encoding the light chain variable region SEQ ID
  • the nucleotide sequence of NO:77 is shown in SEQ ID NO:87.
  • Another aspect of the present invention provides an expression vector that expresses the anti-B7-H3 antibody or antigen-binding fragment thereof of the present invention.
  • the expression vector according to the present invention contains the isolated nucleic acid molecule of the present invention.
  • Another aspect of the present invention provides a host cell transformed with the expression vector as described above.
  • the host cell according to the present invention is selected from prokaryotic cells and eukaryotic cells.
  • the host cell is bacteria, preferably Escherichia coli.
  • the host cell is a mammalian cell.
  • Another aspect of the present invention provides a method for preparing the anti-B7-H3 antibody or antigen-binding fragment thereof of the present invention, which includes the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
  • Another aspect of the present invention provides a pharmaceutical composition comprising the anti-B7-H3 humanized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition, which comprises the anti-B7-H3 humanized antibody or antigen-binding fragment thereof of the present invention, and also contains other active components, such as other antibodies, targeted drugs, and the like.
  • the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • the pharmaceutically acceptable carrier is in the form of liposomes.
  • the anti-B7-H3 humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration.
  • Administration methods include, but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes.
  • the preparation can be administered by any route, for example, by infusion or bolus injection, by the route of absorption through the epithelium or skin mucosa (for example, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the preparation can be prepared by a method known in the art, and contains a carrier, diluent or excipient conventionally used in the field of pharmaceutical preparations.
  • Another aspect of the present invention provides a method for inhibiting the activity of B7-H3, the method comprising administering the anti-B7-H3 antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.
  • Another aspect of the present invention provides the use of the anti-B7-H3 antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in the preparation of drugs for inhibiting the activity of B7-H3.
  • the drug that inhibits B7-H3 activity is used to treat leukemia, lymphoma, breast cancer, lung cancer, stomach cancer, bowel cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer , Glioma and/or melanoma.
  • the present invention provides the use of the above-mentioned anti-B7-H3 antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of anti-tumor drugs.
  • the tumor is selected from leukemia, lymphoma, Breast cancer, lung cancer, stomach cancer, bowel cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma and melanoma.
  • the anti-B7-H3 antibody or its antigen-binding fragment provided by the present invention has higher affinity and stability, has a significant anti-tumor effect, can significantly inhibit tumor growth, and the immunogenicity of the humanized antibody is greatly reduced, which effectively eliminates human immunity
  • the system's rejection of exogenous monoclonal antibodies can be used in the preparation of drugs for the treatment of various tumor diseases, and has broad market prospects.
  • the term "at least 80% sequence identity” means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the term “at least 85% sequence identity” means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. %. Sequence comparison and determination of the percent identity between two sequences can be performed by the BLASTN/BLASTP algorithm on the National Center For Biotechnology Instutute website.
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged at positions relative to each other in a three-dimensional space to form an antigen-binding surface.
  • the antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy chain and light chain are called "complementarity determining regions" or "CDRs".
  • CDRs complementarity determining regions
  • the "antigen-binding fragment” in the present invention refers to Fab fragments, Fab' fragments, F(ab') 2 fragments, and Fv fragments and scFv fragments that bind to human B7-H3 that have antigen binding activity.
  • the Fv fragment contains the variable region of the heavy chain of the antibody and the variable region of the light chain, but does not have the constant region, and has the smallest antibody fragment with all antigen binding sites.
  • an Fv antibody also contains a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding. Different linkers can also be used to connect the variable regions of two antibodies to form a polypeptide chain, which is called single-chain antibody or single-chain Fv (scFv).
  • the antibody in the present invention refers to an immunoglobulin molecule or an immunologically active part thereof, that is, a molecule that contains an antigen binding site that specifically binds to (immunely reacts with) an antigen.
  • "Specific binding” means that an antibody reacts with one or more epitopes of an antigen but does not react with other polypeptides or binds to other polypeptides with a very low affinity (Kd>10 -6 ).
  • Antibodies include but are not limited to polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv and Fab expression libraries.
  • Monoclonal antibody is an antibody obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, and synthesis technology such as CDR grafting or other existing technologies.
  • the "murine antibody” of the present invention is a monoclonal antibody against human B7-H3 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with B7-H3 antigen, and then hybridomas expressing antibodies with desired sequences or functional properties are isolated.
  • the "chimeric antibody” of the present invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma secreting murine-specific monoclonal antibodies, and then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed.
  • the gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the "humanized antibody” of the present invention is also called a CDR grafted antibody, and is an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework (FR).
  • Such variable region framework sequences can be obtained from public DNA databases or public references, for example, from the ImmunoGeneTics (IMGT) website http://imgt.cines.fr or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • Figure 1 is the result of ELISA for the binding activity of anti-B7-H3 humanized antibody to human 4Ig-B7-H3.
  • the abscissa is the antibody concentration ng/ml, and the ordinate is the OD450 value.
  • Figure 2 is the result of ELISA for the binding activity of anti-B7-H3 humanized antibody to human 2Ig-B7-H3.
  • the abscissa is the antibody concentration ng/ml, and the ordinate is the OD450 value.
  • Fig. 3 is the result of ELISA for the binding activity of anti-B7-H3 humanized antibody to monkey B7-H3.
  • the abscissa is the antibody concentration ng/ml, and the ordinate is the OD450 value.
  • Figure 4 is the detection result of IFN ⁇ release level of anti-human B7-H3 mouse monoclonal antibody in the SEB-stimulated PBMC system.
  • PBMC comes from donor B.
  • the abscissa is different antibodies, and the ordinate is the release of IFN ⁇ (pg/ml).
  • Figure 5 is the detection result of IFN ⁇ release level of anti-human B7-H3 murine monoclonal antibody in SEB-stimulated PBMC system.
  • PBMC comes from donor C.
  • the abscissa is different antibodies, and the ordinate is the release of IFN ⁇ (pg/ml).
  • Figure 6 is the detection result of IFN ⁇ release level of anti-B7-H3 humanized antibody hu65 in SEB-stimulated PBMC system.
  • the abscissa is different antibodies, and the ordinate is the release of IFN ⁇ (pg/ml).
  • Figure 7 is the detection result of IFN ⁇ release level of anti-B7-H3 humanized antibody hu51-G34K in SEB stimulated PBMC system.
  • the abscissa is different antibodies, and the ordinate is the release of IFN ⁇ (pg/ml).
  • the amino acid sequence of the extracellular region of the human 4Ig-B7-H3 protein is fused with the amino acid sequence of hIgG1-Fc or his tag.
  • the amino acid sequence design is shown in SEQ ID NO: 91 and SEQ ID NO: 92.
  • the amino acid sequence of the extracellular region of the human 2Ig-B7-H3 protein is fused with the amino acid sequence of hIgG1-Fc or his tag.
  • the amino acid sequence design is shown in SEQ ID NO: 93 and SEQ ID NO: 94.
  • the labeled B7-H3 protein extracellular region gene fragments 4Ig-B7-H3-hFc, 4Ig-B7-H3-his, 2Ig-B7-H3-hFc, 2Ig-B7 were synthesized -H3-his and cloned them into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-4Ig-B7-H3-hFc, pHR-4Ig-B7-H3-his, pHR-2Ig-B7 of the above-mentioned tag proteins. -H3-hFc, pHR-2Ig-B7-H3-his.
  • hBRCA84D Using the humanized antibody hBRCA84D disclosed in the patent application WO2011/109400 as a positive control antibody, the amino acid sequence of hBRCA84D is as follows:
  • hBRCA84D heavy chain amino acid sequence SEQ ID NO: 95;
  • hBRCA84D light chain amino acid sequence: SEQ ID NO: 96.
  • HBRCA84D was prepared according to the method disclosed in WO2011/109400.
  • hBRCA84D is a human IgG1 monoclonal antibody. Compared with natural IgG1, its Fc segment contains specific mutation sites (K214R, L235V, F243L, R292P, Y300L, D356E, L358M, P396L).
  • the amino acid sequence of the hBRCA84D humanized antibody was artificially optimized to obtain the full-length DNA sequence of the heavy chain and light chain of the positive control antibody hBRCA84D.
  • the hBRCA84D heavy chain full-length DNA fragment was cloned into the pHR vector to obtain the eukaryotic expression plasmid pHR-hBRCA84D-8mIgG1.
  • the hBRCA84D light chain full-length DNA fragment was cloned into the pHR vector to obtain the eukaryotic expression plasmid pHR-hBRCA84D-hK.
  • the heavy chain variable region sequence of the positive control antibody hBRCA84D was cloned into the eukaryotic expression plasmid pHR-mG2a containing the murine mG2a heavy chain constant region to obtain the eukaryotic expression plasmid pHR-hBRCA84D-mG2a.
  • the light chain variable region sequence of the positive control antibody hBRCA84D was cloned into the eukaryotic expression plasmid pHR-mK containing the mouse K light chain constant region to obtain the eukaryotic expression plasmid pHR-hBRCA84D-mK.
  • the eukaryotic expression plasmid pTargeT-B7-H3 was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) under a square pulse of 15msec at a voltage of 160V and placed at 37°C, 5% CO 2 Cultivate in a concentration incubator. After 24h, pressurized culture was carried out with a medium containing 1000ug/ml G418 (Gibco, #10131-027).
  • the mixed solution of PEI and FreeStyle 293 expression medium was added to the plasmid, mixed evenly, and then added to the cell culture, placed in a 37°C, 8% CO 2 , humidified CO 2 incubator for cultivation.
  • the cells were fed on the 1st and 3rd day after cell transfection, 2.5ml of glutamine (mother liquor concentration of 200mM) and 5ml of glucose (mother liquor concentration of 180g/L) were added to each bottle.
  • the cell viability dropped to 65%-75%, the cell supernatant was collected.
  • the cell culture was centrifuged at 1500 rpm for 5 min, and the supernatant was collected, and then centrifuged at 8000 rpm for 20 min, and the supernatant was collected.
  • Sample loading sample the cell expression supernatant, retention time 5min;
  • the collection starts when the UV280 reaches about 50mAu, and stops when it drops to about 50mAu. Adjust the pH of the sample to 7.0 with 1M Tris-HCl, pH 9.0;
  • On-line cleaning 0.1M NaOH cleaning for 30min, retention time 5min;
  • 4Ig-B7-H3 antigen and 2Ig-B7-H3 antigen protein with different labels and adjuvants to immunize experimental animals, including SJL strain mice/Balb/c strain mice/SD rats.
  • the immunized animals used 50ug antigen to immunize one animal according to the first immunization, and then used 25ug antigen to immunize one animal in the later period.
  • the immune adjuvant can be Freund's adjuvant (Sigma) or Quick Antibody-Mouse5W (Beijing Boaolong Immunological Technology Co., Ltd.).
  • Use Freund’s adjuvant to emulsify the antigen add 4Ig-B7-H3 or 2Ig-B7-H3 antigen protein samples of different labels to the adjuvant solution drop by drop, vortex while dripping to mix well, the adjuvant dosage reference
  • the instructions are carried out.
  • the mice were immunized after mixing uniformly to form a water-in-oil milk.
  • Quick Antibody-Mouse5W as an adjuvant, mix 4Ig-B7-H3 or 2Ig-B7-H3 antigen protein samples of different labels with Quick Antibody-Mouse5W at a volume ratio of 1:1, and then mix them by intramuscular injection. Way, immunize SD rats.
  • mice were immunized with the stably transfected cell line CHO-K1-B7-H3 expressing B7-H3 to produce antibodies against B7-H3.
  • the CHO-K1-B7-H3 cells obtained in Example 1 were digested with trypsin, and then centrifuged at 1000 rpm for 5 minutes, the cell supernatant was discarded, the cell pellet was resuspended in PBS, the sample was counted with a cell counter, and the remaining cells were centrifuged at 1000 rpm 5min, discard the supernatant, resuspend the cell pellet in PBS, and add an appropriate amount of PBS to obtain a cell suspension of 1 ⁇ 10 8 cells/ml.
  • Each immunization experiments 1x10 7 cells in mice, SD rats per experimental group immunized 2x10 7 cells.
  • the immunization program is shown in Table 2:
  • mice/rats after booster immunization were sacrificed and soaked in 75% alcohol.
  • the spleen was dissected and taken out, ground with a grinding rod, and filtered through a cell sieve to prepare a single cell suspension. Centrifuge the spleen cell suspension at 2000 rpm for 5 min, and discard the supernatant. Add 2mL red blood cell lysate, lyse the red blood cells at room temperature for 2min, add PBS to 20mL, centrifuge at 1500rpm for 7min, discard the supernatant, and count the viable cells after resuspension.
  • the cell suspension was transferred to 15 mL RPMI 1640 complete medium containing 20% FBS, and placed at room temperature for 20 minutes.
  • the fused cells were resuspended in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, and 20% FBS.
  • the cell culture supernatant of the hybridoma parent clone was taken, and the hybridoma parent clones that combined 4Ig-B7-H3-his protein and cynomolgus B7-H3 protein were screened by ELISA. Furthermore, flow cytometry was used to screen out the parent clones that can bind to the stable CHO-K1-B7-H3 cell line. The positive mother clones were expanded to a 24-well plate, and the culture supernatant of the mother clones cultured for 2 days was selected and co-cultured with T cells to detect the release of IFN- ⁇ in the culture supernatant.
  • the specific experimental operation is as follows:
  • CD8+ T cells Resuscitate CD8+ T cells on Day 2 (obtained by induction and expansion with CD3 and CD28, frozen in 90% FBS+10% DMSO), using IMDM+10% FBS+free 100ng/ml SEB+4ng/ml IL-2+100x The cells were resuspended in the medium of the double antibody and cultured in the T25 coated in step 1, at a culture density of 2-3 ⁇ 10 6 cells/ml, and cultured for 48 hours.
  • a 96-well plate was coated in accordance with 1ug/ml anti-CD3e and 10ug/ml 4Ig-B7-H3-his. Set the coating blank group, anti-CD3e group, anti-CD3e+B7-H3-4Ig- The his group was incubated overnight at 4°C.
  • Elisa and FACS were used to screen out parent clones with binding ability, and further evaluate the level of cytokine IFN- ⁇ released by the cell supernatant secreted by the parent clones, and screen out positive parent clones that can promote IFN- ⁇ release.
  • the positive parent clone was subcloned by the limiting dilution method, and after one week of culture, the binding activity of the subclonal supernatant and 4Ig-B7-H3 molecule was detected by ELISA to obtain a monoclonal cell line secreting anti-B7-H3 antibody.
  • the parental monoclonal antibody clones 17, 39, 51, and 65 were identified and expanded.
  • the culture conditions are 1640 medium containing 10% fetal bovine serum, 1x NAEE, 1x sodium pyruvate, and 1% penicillin double antibody.
  • the confluence of cells is greater than> 80%, the cells are subcultured and cultured. Collect the supernatant when it reaches about 50ml, and purify the antibody.
  • the obtained antibody was confirmed to be of good purity by SDS-PAGE gel electrophoresis.
  • the subcloned positive hybridoma cells were expanded and cultured, and the appropriate amount of cells was extracted according to the RNeasy Plus Mini Kit (Qiagen, 74134) kit instructions to extract the total RNA, and the Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) was used for reverse transcription.
  • the kit synthesizes the first strand of cDNA.
  • Design specific primers according to the variable region of the mouse antibody subtype (the 5'end contains the homologous arm sequence for homologous recombination with eukaryotic expression vector), and use cDNA as a template for PCR amplification of antibody variable region genes , So as to obtain the gene fragments of the variable region of the mouse antibody light chain and heavy chain respectively; design primers (references: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods, 1997,201:35–55; 2.
  • the mouse antibody heavy chain variable region gene fragment obtained by sequencing in Table 4 was cloned into the eukaryotic expression plasmid containing the heavy chain constant region of the specific mutation site (K214R, L235V, F243L, R292P, Y300L, D356E, L358M, P396L) (pHR-8mIgG1), clone the mouse antibody light chain variable region gene fragments obtained by sequencing in Table 4 into the eukaryotic expression plasmid (pHR-hK) containing the human light chain constant region.
  • the constructed antibody heavy and light chain plasmids were transformed into E. coli DH5 ⁇ competent cells, and the mixture was evenly spread on the surface of the agar plate containing the corresponding antibiotics.
  • the positive clones with correct sequencing were inoculated into 2 ⁇ YT liquid medium containing the corresponding antibiotics, cultured with shaking at 37°C for more than 12 hours, and then collected the bacterial cells for plasmid extraction to obtain the expression plasmids of the chimeric antibody light chain and heavy chain , Use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the chimeric antibody heavy and light chain plasmids were co-transfected into HEK293E cells, and the antibodies were obtained by expression and purification.
  • the chimeric antibodies were named SHS007-17-CHI, SHS007-39-CHI, SHS007-51-CHI, SHS007-65-CHI, Then carry out purity test, activity analysis and affinity test.
  • the chimeric antibodies SHS007-17-CHI, SHS007-39-CHI, SHS007-51-CHI, and SHS007-65-CHI were genetically mutated to screen for better antibodies.
  • the 34th G mutation of SHS007-51-CHI light chain CDR is K, and the stability of the antibody is improved.
  • the label is SHS007-51-G34K-CHI.
  • the chimeric antibody sequencing results are shown in Table 5.
  • SHS007-17-CHI, SHS007-39-CHI, SHS007-51-G34K-CHI, SHS007-65-CHI were selected for humanized antibody transformation.
  • the humanized transformation of antibodies is first performed by comparing with the mouse antibody sequence in the immune gene database (IMGT) to confirm SHS007-17-CHI, SHS007-39-CHI, SHS007-51-G34K-CHI, SHS007-
  • the murine germline of the variable region of 65-CHI antibody after homology comparison, the heavy chain of SHS007-17-CHI, SHS007-39-CHI, SHS007-51-G34K-CHI, SHS007-65-CHI antibody is variable
  • the FR regions of the region sequence are respectively the most similar to the mouse antibody germline genes (IGHV1-69*02, IGHV1-2*02, IGHV3-21*01, IGHV3-48*01); the FR sequence of the antibody light chain variable region They are most similar to mouse antibodies (IGKV2-28*01, IGKV3-11*01, IGKV2-30*02, IGKV3-11*01).
  • SHS007-17-CHI, SHS007-39-CHI, SHS007-51-G34K-CHI, SHS007-65-CHI antibody framework region sequence FR1-FR3 as templates look for 3D structure similar but immunogenic in the human framework region library
  • the lower full human framework replaces the FR1-FR3 sequence of SHS007-17-CHI, SHS007-39-CHI, SHS007-51-G34K-CHI, SHS007-65-CHI, and the heavy chain/light chain full-length sequence is used for 3D modeling
  • the structure comparison and analysis of the original antibody heavy chain/light chain sequence were carried out, and the antigenicity and 3D structural similarity were considered comprehensively, and finally the 4 humanized heavy chain variable regions of SHS007-17-CHI were selected (see SEQ ID NO: 45, 46, 47, 48) and 3 humanized light chain variable regions (see SEQ ID NO: 49, 50, 51), and 6 humanized heavy chain variable regions of SHS007-39-CHI (see SEQ ID
  • the humanized antibody light chain and heavy chain variable region amino acid sequences designed above are reverse transcribed into corresponding nucleotide sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments are generated by OverlapPCR
  • the oligonucleotide fragments are annealed and joined together, and then specific primers (5' end containing homology arm sequences for homologous recombination with eukaryotic expression vectors) are used to amplify the complete light chain and heavy chain Nucleotide fragment of the region; the purified nucleotide fragment of the light chain variable region and the linearized eukaryotic expression plasmid (pHR-hK) containing the constant region of the human K light chain were co-transformed into E.
  • the purified heavy chain variable region nucleotide fragment and the eukaryotic expression plasmid (pHR-IgG1) of the human IgG1 heavy chain constant region were co-transformed into E. coli DH5 ⁇ competent cells, and the competent cells of the transformed plasmid were uniformly coated on The surface of the agar plate containing the corresponding antibiotics was cultured in a constant temperature incubator at 37°C overnight, and then several single colonies were picked for DNA sequencing.
  • Transfect the plasmid into HEK293E cells express and purify to obtain a large number of antibodies, and conduct purity testing, activity analysis and affinity testing.
  • ELISA was used to analyze the binding activity of antibodies. Coat human 4Ig-B7-H3-His protein or 2Ig-B7-H3-His protein (1ug/well, prepared in Examples 1 and 2) onto a 96-well microtiter plate, and incubate overnight at 4°C. After washing 3 times with 1xPBST, it was blocked with 5% skimmed milk at 37°C for 2h. After washing 3 times with 1xPBST, the anti-B7-H3 antibody provided by the present invention starts from 2 ⁇ g/mL as the primary antibody, and adds 5-fold dilutions to the ELISA plate.
  • a total of 8 concentrations are 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, 0.0256ng/mL, incubate at 37°C for 2h, the positive control antibody is hBRCA84D; after washing 5 times with 1xPBST, use Anti -Human IgG HRP (Jackson, 109-035-003, 1:5000), incubate at 37°C for 1h. After washing 5 times with 1xPBST, add the color developing solution TMB, and use the microplate reader (thermo, Multiskan FC) to read the OD450 value after termination. Use GraphPad to generate EC50, and the results are shown in Figures 1 and 2.
  • Protein-based Elisa was used to analyze the binding activity of antibodies. Cynomolgus monkey B7-H3-His (0.5 ⁇ g/well, Sino Biological, Cat. No. 90806-C08H) was coated with a 96-well microtiter plate.
  • the anti-B7-H3 antibody provided by the present invention starts from 2 ⁇ g/mL as a primary antibody, and adds 5-fold gradient dilution to the ELISA plate. A total of 8 concentrations are 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, respectively.
  • the positive control antibody is hBRCA84D.
  • Use Anti-Human IgG HRP Jackson, 109-035-003, 1:10000
  • TMB color developing solution
  • TMB thermo, Multiskan FC
  • the experimental results show that the humanized anti-B7-H3 antibodies hu51-G34K-12 and hu65-13 provided by the present invention have the ability to bind to cynomolgus monkey B7-H3, and the binding ability is equivalent to the positive control antibody hBRCA84D.
  • FACS was used to analyze the binding activity of the antibody to B7-H3 on the surface of tumor cells.
  • the mean fluorescence intensity (MFI) and EC50 detected by FACS indicate the binding activity of the antibody to the cell.
  • MFI mean fluorescence intensity
  • the above-mentioned cells were plated on a cell plate in a manner of 1 ⁇ 10 5 cells per well.
  • the anti-B7-H3 antibody provided by the present invention was used as the primary antibody, starting from 20 ⁇ g/ml, and added to the cell plate in gradient dilutions, with a total of 8 concentrations, each at a concentration of 20000ng/ mL, 10000ng/mL, 2000ng/mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, incubate at 4°C for 1h, the positive control antibody is hBRCA84D; the secondary antibody uses PE- Anti-Human IgG (Biolegend, Cat. No.
  • the affinity of the humanized anti-B7-H3 antibody prepared in Example 4 was determined using Biacore.
  • the antigen 4Ig-B7-H3-His was coupled to the surface of the CM5 chip (GE, Cat. No. BR100012), and the active groups of the uncoupled protein were blocked with ethanolamine.
  • hu39-01, hu65-13, and hBRCA84D antibodies are respectively diluted in equal proportions.
  • the diluted antibody proteins of each concentration are placed on the sampling tray of the instrument in order, and the program is set to inject samples separately.
  • PBMC peripheral blood mononuclear cells
  • the SEB (Toxin techonology, BT202) solution with a final concentration of 100ng/ml was prepared with the culture medium and added to the corresponding wells of the 96-well plate. After the above 96-well plate was co-cultured in a constant temperature incubator for 72 hours, the IFN- ⁇ concentration in the above-mentioned culture supernatant was detected according to the DuoSet detection kit (R&D, DY285B) instructions.
  • the results of Figures 4 and 5 show that the anti-human B7-H3 mouse single Anti-SHS007-39, 51, 59, 65, 72, etc., in the PBMC-SEB system derived from donor B and donor C, can increase the release of cytokine IFN- ⁇ in the system.
  • Figures 6 and 7 show that the anti-human B7-H3 humanized antibodies hu51 and hu65 enhance the release of IFN- ⁇ in the PBMC-SEB experimental system.
  • Human kidney cancer cell 786-0 highly expresses human B7-H3, and 786-0 cells are spread on a 96-well plate with 20,000 cells per well.
  • Heat-inactivated fetal bovine serum (FBS, Gibco, Cat. No. 10091-148) was used to prepare RPMI-1640 medium containing 2% FBS.
  • SHS007-17CHI, SHS007-39CHI, SHS007-51CHI, SHS007-65CHI, hBRCA84D antibodies were dissolved in the above medium in 10-fold dilutions, and incubated in a constant temperature incubator at 37°C for 1 hour. According to the instructions of the Ficoll (GE Healthcare, Cat. No.
  • the experimental results show that compared with the positive control hBRCA84D antibody, the antibody provided by the present invention has a better killing EC50 value and greatly improves the ADCC killing activity.
  • Example 11 Anti-tumor test of a mouse transplanted tumor model with human tumor cells
  • Human renal carcinoma cell 786-0 cells were purchased from the American Type Culture Collection (ATCC);
  • Human triple-negative breast cancer MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC);
  • SCID-Beige mice female, 6-8 weeks old, weighing 18-20 grams, purchased from Shanghai Lingchang Experimental Animal Co., Ltd.;
  • the negative control substance Isotype IgG1 (Cat. No. C0001-4) was purchased from Sino-American Crown Biotechnology Co., Ltd. In the mouse xenograft model, hBRCA84D-IgG1 is much better than hBRCA84D, so hBRCA84D-IgG1 is used as a positive control.
  • hBRCA84D-IgG1 is a human IgG1 monoclonal antibody constructed using the variable region sequence of the target antibody hBRCA84D, and the constant region is the same as the human IgG1 chimeric antibody of the present invention.
  • the anti-B7-H3 chimeric antibodies of the present invention (SHS007-39-IgG1, SHS007-65-IgG1) were prepared with PBS at 1 mg/mL, and Isotype IgG1 and hBRCA84D-IgG1 were prepared at 1 mg/mL.
  • the human tumor cell U87 cells were inoculated on the right back of the mouse, and the number of inoculated cells was 5 ⁇ 10 6 /mouse.
  • the tail vein is administered twice a week.
  • the tumor diameter is measured with a vernier caliper twice a week to calculate the tumor volume.
  • the tumor volume measured for 24 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. The results are shown in Table 10.
  • the human tumor cell 786-0 cells were inoculated on the right back of the mouse, and the number of inoculated cells was 1 ⁇ 10 7 /mouse.
  • the tumor masses grown from the 786-O cell inoculation were removed with a sterile surgical instrument, and the tumor tissues in the vigorous growth period were cut into about 30mm 3 in normal saline.
  • the right back of the mouse When the tumor grows to an average volume of 160mm 3 , start grouping.
  • the tail vein is administered twice a week, and the tumor diameter is measured with a vernier caliper twice a week to calculate the tumor volume.
  • the tumor volume measured for 19 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. The results are shown in Table 11.
  • the human tumor cell MDA-MB-231 cells were inoculated on the right back of the mouse, and the number of inoculated cells was 8 ⁇ 10 6 /mouse. After 2 weeks of inoculation, use sterile surgical instruments to remove the tumor masses grown from the inoculation of MDA-MB-231 cells, cut the tumor tissues in the vigorous growth period into about 30mm 3 in normal saline, and inoculate them under aseptic conditions. On the right back of the mouse. When the tumor grows to an average volume of 150mm 3 , start grouping. The tail vein is administered twice a week, and the tumor diameter is measured with a vernier caliper twice a week to calculate the tumor volume.
  • the tumor volume measured for 21 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. The results are shown in Table 12.
  • the experimental results show that the antibody provided by the present invention has a SCID-Beige mouse xenograft model inoculated with human malignant glioma U87 cells, human renal cancer cells 786-0 cells, and human triple-negative breast cancer cells MDA-MB-231. Inhibit tumor growth.
  • the anti-tumor effect of antibodies SHS007-39-CHI and SHS007-65-IgG1 is better than the control antibody hBRCA84D-IgG1.
  • the anti-B7-H3 antibody provided by the present invention has a more significant effect of inhibiting tumor growth.
  • Example 12 Anti-tumor test of a mouse transplanted tumor model with human tumor cells
  • Human renal carcinoma cell 786-0 cells were purchased from the American Type Culture Collection (ATCC);
  • Human triple-negative breast cancer MDA-MB-231 cells were purchased from the American Type Culture Collection (ATCC);
  • SCID-Beige mice female, 6-8 weeks old, weighing 18-20 grams, purchased from Shanghai Lingchang Experimental Animal Co., Ltd.;
  • the reference substance Isotype IgG1 (Cat. No. C0001-4) was purchased from Sino-American Crown Biotechnology Co., Ltd. and used as a negative control substance;
  • the reference substance hBRCA84D-IgG1 is a human IgG1 monoclonal antibody constructed using the variable region sequence of the target antibody hBRCA84D , The constant region is the same as the humanized antibody of the present invention, which is used as a positive control;
  • the humanized anti-B7-H3 antibodies hu65-13 and hu51-G34K-12 of the present invention were prepared with PBS at 1 mg/mL or 2 mg/ml, and Isotype IgG1 and hBRCA84D-IgG1 were prepared at 1 mg/mL or 2 mg. /ml.
  • the human tumor cell 786-0 cells were inoculated on the right back of the mouse, and the number of inoculated cells was 1 ⁇ 10 7 /mouse.
  • the tumor masses grown by the 786-O cell inoculation were removed with sterile surgical instruments, and the vigorously growing tumor tissue was cut into about 15 mm 3 in normal saline.
  • the tumor was inoculated into small The right back of the mouse.
  • the tail vein administration is administered twice a week, and the administration is to the 21st day. The frequency of administration is reduced to once a week.
  • the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated.
  • the tumor volume measured for 36 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. The results are shown in Table 13.
  • the human tumor cell MDA-MB-231 cells were inoculated on the right back of the mouse, and the number of inoculated cells was 8 ⁇ 10 6 /mouse. After 2 weeks of inoculation, use sterile surgical instruments to remove the tumor masses grown from the inoculation of MDA-MB-231 cells, cut the tumor tissues in the vigorous growth period into about 30mm 3 in normal saline, and inoculate them under aseptic conditions. On the right back of the mouse. When the tumor grows to an average volume of 100mm 3 , start grouping. The tail vein is administered twice a week, and the tumor diameter is measured with a vernier caliper twice a week to calculate the tumor volume.
  • the tumor volume measured for 25 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. The results are shown in Table 14.
  • mice were transplanted into SCID-Beige mice inoculated with human kidney cancer cell 786-0 cell and human triple-negative breast cancer cell MDA-MB-231
  • the tumor model has the effect of inhibiting tumor growth.
  • the anti-tumor effect of hu51-G34K-12 and hu65-13 is better than the control antibody hBRCA84D-IgG1.
  • the anti-B7-H3 antibody provided by the present invention has a more significant effect of inhibiting tumor growth.
  • the anti-B7-H3 antibody provided by the present invention has a significant anti-tumor effect and can significantly inhibit tumor growth, suggesting that the antibody can be used in the preparation of anti-tumor drugs and has a predictable market prospect.

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Abstract

提供了一种抗B7-H3抗体或其抗原结合片段、包含所述抗B7-H3抗体或其抗原结合片段的药物组合物及其应用。本发明的抗B7-H3抗体或其抗原结合片段具有高亲和力和稳定性，可用于制备抗肿瘤药物。

Description

[根据细则26改正30.09.2020]　一种抗B7-H3抗体及其应用 技术领域
本发明涉及抗体药物技术领域，尤其涉及一种抗B7-H3抗体或其抗原结合片段，包含抗B7-H3抗体或其抗原结合片段的药物组合物以及它们的应用。
背景技术
B7-H3(CD276)属于B7超家族，是一种跨膜糖蛋白，其胞外区结构分为2种，一种是单价的2Ig-B7-H3，一种是由2个重复单元结构组成的双价的4Ig-B7-H3。B7-H3作为一种新型免疫检查点，文献报导中B7-H3通过与一种结构未知的受体作用，能够抑制T细胞增殖及细胞因子释放(Suh W K,et al.The B7 family member B7-H3 preferentially down-regulates T helper type 1-mediated immune responses[J].Nature Immunology,2003,4(9):899)，抑制NK细胞对B7-H3高表达的患者来源的肿瘤细胞的裂解活性(Castriconi R,et al.Identification of 4Ig-B7-H3 as a neuroblastoma-associated molecule that exerts a protective role from an NK cell-mediated lysis[J].Proceedings of the National Academy of Sciences,2004,101(34):12640-12645)。虽然B7-H3受体未知，但是近年来关于B7-H3与受体作用在肿瘤免疫中的负调控的报导越来越多。肿瘤细胞表达B7-H3，使其规避CD8+T细胞的免疫监视，小鼠模型中，抗鼠B7-H3抗体的使用，能增强CD8+T细胞的免疫功能(Yonesaka K,et al.B7-H3 negatively modulates CTL-mediated cancer immunity[J].Clinical Cancer Research,2018,clincanres.2852.2017)。在Lee Y等人研究中，基因敲除小鼠B7-H3或者使用抗鼠B7-H3抗体均能显著抑制肿瘤的生长，这种抑制作用依赖于CD8+T和NK细胞的功能(Lee Y,et al.Inhibition of the B7-H3 immune checkpoint limits tumor growth by enhancing cytotoxic lymphocyte function[J].Cell Research,2017,27(8):1034-1045)。在肺癌患者中的研究发现，B7-H3高表达患者肿瘤浸润性淋巴细胞少且更容易发生淋巴结转移(Altan M,et al.B7-H3 expression in NSCLC and its association with B7-H4,PD-L1  and Tumor Infiltrating Lymphocytes[J].Clinical Cancer Research,2017,23(17):5202).
B7-H3异常表达于多种肿瘤细胞、肿瘤基质、肿瘤血管及肿瘤浸润的巨噬细胞和DC细胞。在来自病人的前列腺癌、胰腺癌、肝细胞癌、头颈癌、肾癌等组织样本中，B7-H3的表达比率均超过90％，而在正常组织中鲜见表达(Seaman S,et al.Eradication of Tumors through Simultaneous Ablation of CD276/B7-H3-Positive Tumor Cells and Tumor Vasculature.[J].Cancer Cell,2017,31(4):501-515)。基于B7-H3的肿瘤特异性表达趋向，临床在研抗B7-H3靶点的抗体类药物，均是借助B7-H3的肿瘤靶向性来进行药物开发。Enoblituzumab，人源化抗B7-H3单克隆抗体，通过抗体靶向肿瘤介导抗体Fc段的ADCC效应来杀伤肿瘤。Burtomab是携带放射性碘的抗B7-H3鼠源抗体药物偶联物，在临床中治疗转移性神经母细胞瘤，显著提高儿童肿瘤患者生存期。
基于B7-H3在肿瘤环境中的高表达特性及其对免疫细胞的抑制活性特点，B7-H3可作为抗肿瘤药物开发的有效的靶点。需要提供一种抗B7-H3抗体或其抗原结合片段，其具有更高的亲和力和稳定性，可阻断B7-H3对肿瘤的免疫抑制，增强CD8+T细胞和NK细胞对肿瘤的杀伤活性，并协同抗体Fc段的ADCC效应发挥抗肿瘤作用，从而具有更优异的抗肿瘤活性。
发明内容
本发明一方面提供一种抗B7-H3抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(HCDR1)、重链可变区的互补决定区2(HCDR2)和/或重链可变区的互补决定区3(HCDR3)，所述轻链可变区包含轻链可变区的互补决定区1(LCDR1)、轻链可变区的互补决定区2(LCDR2)和/或轻链可变区的互补决定区3(LCDR3)。
在一些实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
(1)所述重链可变区包含选自如下组的HCDR1、HCDR2和HCDR3：
(a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
(a2)如SEQ ID NO:7、8和9所示的氨基酸序列；
(a3)如SEQ ID NO:13、14和15所示的氨基酸序列；和
(a4)如SEQ ID NO:20、21和22所示的氨基酸序列；
(a5)与(a1)、(a2)、(a3)或(a4)所示的氨基酸序列具有至少85％序列同一性的CDR；和
(2)所述轻链可变区包含选自如下组的LCDR1、LCDR2和LCDR3：
(a6)如SEQ ID NO:4、5和6所示的氨基酸序列；
(a7)如SEQ ID NO:10、11和12所示的氨基酸序列；
(a8)如SEQ ID NO:16、18和19所示的氨基酸序列；
(a9)如SEQ ID NO:17、18和19所示的氨基酸序列；
(a10)如SEQ ID NO:23、24和25所示的氨基酸序列；和
(a11)与(a6)、(a7)、(a8)、(a9)或(a10)所示的氨基酸序列具有至少85％序列同一性的CDR。
在一个具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:10、11和12或与SEQ ID NO:10、11和12所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区；
在一个具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:13、14和15或与SEQ ID NO:13、14和15所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:16、18和19或与SEQ ID NO:16、18和19所示的氨基酸序列具有至少85％序列同一性 的CDR的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:13、14和15或与SEQ ID NO:13、14和15所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:17、18和19或与SEQ ID NO:17、18和19所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区。
在一个具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其具有所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:20、21和22或与SEQ ID NO:20、21和22所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:23、24和25或与SEQ ID NO:23、24和25所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区。
在一些具体的实施方案中，根据本发明的抗B7-H3抗体或其抗原结合片段是单克隆抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的抗B7-H3抗体或其抗原结合片段是鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段或人源化抗体或其抗原结合片段。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中
(1)所述重链可变区的氨基酸序列选自：
(b1)如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:35所示的氨基酸序列；
(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(b4)如SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:32、 SEQ ID NO:36所示的氨基酸序列；
(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:31，SEQ ID NO:31经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:31功能相同的氨基酸序列或与SEQ ID NO:31具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可 变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:35，SEQ ID NO:35经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:35功能相同的氨基酸序列或与SEQ ID NO:35具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:36，SEQ ID NO:36经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:36功能相同的氨基酸序列或与SEQ ID NO:36具有至少85％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:4、5和6所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:7、8和9所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:10、11和12所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片 段，其中所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:13、14和15所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:31，SEQ ID NO:31经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:31功能相同的氨基酸序列或与SEQ ID NO:31具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:16、18和19所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:13、14和15所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:17、18和19所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:35，SEQ ID NO:35经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:35功能相同的氨基酸序列或与SEQ ID NO:35具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:20、21和22所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:36，SEQ ID NO:36经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:36功能相同的氨基酸序列或与SEQ ID NO:36具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:23、24和25所示的氨基酸序列。
在一些具体的实施方案中，根据本发明的抗B7-H3抗体为鼠源抗体，其还含有鼠源的IgG1、IgG2、IgG3、或IgG4或其变体的重链恒定区，和鼠源的κ链或其变体的轻链恒定区。
在一些优选的实施方案中，根据本发明的抗B7-H3鼠源抗体还含有鼠源的IgG1或IgG2或其变体的重链恒定区，和鼠源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述抗B7-H3抗体为人源化抗体，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:72或SEQ ID NO:73所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述抗B7-H3抗体为人源化抗体，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47或SEQ ID NO:48所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:4、5和6所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述抗B7-H3抗体为人源化抗体，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:7、8和9所示的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61或SEQ ID NO:62所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:10、11和12所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述抗B7-H3抗体为人源化抗体，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:63或SEQ ID NO:64所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:13、14和15所示的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69或SEQ ID NO:70所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:17、18和19所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述抗B7-H3抗体为人源化抗体，其中：
(1)所述重链可变区的氨基酸序列选自：
(c1)如SEQ ID NO:72或SEQ ID NO:73所示的氨基酸序列；
(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c3)与(c1)所示的氨基酸序列具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:20、21和22所示的氨基酸序列；和
(2)所述轻链可变区的氨基酸序列选自：
(c4)如SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79所示的氨基酸序列；
(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
(c6)与(c4)所示的氨基酸序列具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:23、24和25所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:45，SEQ ID NO:45经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:45功能相同的氨基酸 序列或与SEQ ID NO:45具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:49，SEQ ID NO:49经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:49功能相同的氨基酸序列或与SEQ ID NO:49具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:4、5和6所示的的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:52，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:7、8和9所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:59，SEQ ID NO:59经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:59功能相同的氨基酸序列或与SEQ ID NO:59具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:10、11和12所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:63，SEQ ID NO:63经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:63功能相同的氨基酸序列或与SEQ ID NO:63具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:13、14和15所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:65，SEQ ID NO:65经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:65功能相同的氨基酸序列或与SEQ ID NO:65具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:17、18和19所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:64，SEQ ID NO:64经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:64功能相同的氨基酸序列或与SEQ ID NO:64具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:13、14和15所示的氨基酸序列，且所述轻链可变区的氨 基酸序列为SEQ ID NO:67，SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:67功能相同的氨基酸序列或与SEQ ID NO:67具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:17、18和19所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:72，SEQ ID NO:72经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:72功能相同的氨基酸序列或与SEQ ID NO:72具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:20、21和22所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:77，SEQ ID NO:77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:77功能相同的氨基酸序列或与SEQ ID NO:77具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:23、24和25所示的氨基酸序列。
在一些具体的实施方案中，本发明提供一种抗B7-H3抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:22，SEQ ID NO:22经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:22功能相同的氨基酸序列或与SEQ ID NO:22具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:25，SEQ ID NO:25经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:25功能相同的氨基酸序列或与SEQ ID NO:25具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:10、11和12所示的氨基酸序列。
在一些实施方案中，本发明提供一种抗B7-H3人源化抗体或其抗原结合片段，其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，所述轻链包含人源的κ、λ链或其变体的轻链恒定区。
在本发明一个优选的实施方案中，所述的鼠源抗B7-H3抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，和/或进一步包含鼠源IgG1，IgG2，IgG3或IgG4或其变体的重链恒定区。
在本发明一个优选的实施方案中，所述的抗B7-H3嵌合抗体或其抗原结合 片段的抗体轻链进一步包含鼠源κ、λ链或其突变序列的轻链恒定区。所述的抗B7-H3嵌合抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2、IgG3、gG4或其突变序列的重链恒定区，优选包含人源IgG1或IgG2重链恒定区，或者使用氨基酸突变后显著降低ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的IgG4恒定区。
在一些具体的实施方案中，本发明的抗B7-H3人源化抗体或其抗原结合片段还包含人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，和人源κ、λ链或其变体的轻链恒定区。在一些优选的实施方案中，本发明的抗B7-H3人源化抗体或其抗原结合片段还包含人源IgG1或IgG2或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。
在一些实施方案中，本发明提供抗B7-H3抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、Fv、sFv或F(ab)2。
本发明的另一方面提供一种分离的核酸，其编码根据本发明的抗B7-H3抗体或其抗原结合片段。
在一些具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84或SEQ ID NO:86所示；编码轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85或SEQ ID NO:87所示。
在一个具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区SEQ ID NO:45的核苷酸序列如SEQ ID NO:80所示；编码轻链可变区SEQ ID NO:49的核苷酸序列如SEQ ID NO:81所示。
在一个具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区SEQ ID NO:52的核苷酸序列如SEQ ID NO:82所示；编码轻链可变区SEQ ID NO:59的核苷酸序列如SEQ ID NO:83所示。
在一个具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区SEQ ID NO:64的核苷酸序列如SEQ ID NO:84所示；编码轻链可变区SEQ ID NO:67的核苷酸序列如SEQ ID NO:85所示。
在一个具体的实施方案中，根据本发明的分离的核酸，其中编码重链可变区SEQ ID NO:72的核苷酸序列如SEQ ID NO:86所示；编码轻链可变区SEQ ID  NO:77的核苷酸序列如SEQ ID NO:87所示。
本发明的另一方面提供一种表达载体，其表达本发明的抗B7-H3抗体或其抗原结合片段。根据本发明的表达载体其包含本发明的分离的核酸分子。
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。
本发明的另一方面提供制备本发明的抗B7-H3抗体或其抗原结合片段的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。
本发明的另一方面提供一种药物组合物，其包含本发明的抗B7-H3人源化抗体或其抗原结合片段和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗B7-H3人源化抗体或其抗原结合片段，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。
可以将本发明的抗B7-H3人源化抗体或其抗原结合片段与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注，通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。
本发明的另一方面提供抑制B7-H3活性的方法，所述方法包括向有此需要的个体施用本发明的抗B7-H3抗体或其抗原结合片段或本发明的药物组合物。
本发明的另一方面提供本发明的抗B7-H3抗体或其抗原结合片段或本发明的药物组合物在制备抑制B7-H3活性的药物中的应用。在一些实施方案中，所述抑制B7-H3活性的药物用于治疗白血病、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和/或黑素 瘤。在一些实施方案中，本发明提供上述抗B7-H3抗体或其抗原结合片段或本发明的药物组合物在制备抗肿瘤的药物中的应用，优选地，所述肿瘤选自白血病、淋巴瘤、乳腺癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、肾癌、膀胱癌、胰腺癌、神经胶质瘤和黑素瘤。
本发明提供的抗B7-H3抗体或其抗原结合片段具有更高的亲和力和稳定性，抗肿瘤作用显著，可明显抑制肿瘤增长，人源化后的抗体免疫原性大大降低，有效消除人体免疫系统对外源性单抗的排异反应，可在制备用于治疗各类肿瘤疾病的药物中应用，具有广阔的市场前景。
定义
除非另有定义，本文中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。本文中所述的细胞和组织培养、分子生物学以及蛋白质和寡或多核苷酸化学及杂交中使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和组织培养与转化(如电穿孔、脂质转染)，使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等，Molecular Cloning:A Laboratory Manual)(第2版，Cold Spring Harbor Laboratory Press，纽约冷泉港(1989))。本文所述的分析化学、合成有机化学以及医学和药学化学中使用的命名以及实验室方法和技术是本领域公知且普遍使用的。
在本发明中，术语“至少80％序列同一性”是指至少80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在本发明中，术语“至少85％序列同一性”是指至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在一些优选的实施方案中，本发明所述的序列同一性可以至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Instutute网站上的BLASTN/BLASTP算法来进行。
在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中以相对彼此的位置排列以形成抗原结合表面。抗原结合表面与所结合抗原的三维表面互补，且每条重链和轻链的三个高变区均被称作“互补决定区”或“CDR”。氨基酸向每个结构域的分配是根据Kabat《免疫学感兴趣的蛋白质的序列》(国立卫生研究院，马里兰州贝塞斯达(1987和1991))或Chothia和Lesk，J.Mol.Biol.196:901-917(1987)，Chothia等，Nature 342:878-883(1989)定义。
本发明所述的“抗原结合片段”是指具有抗原结合活性的Fab片段、Fab’片段、F(ab’) 2片段及与人B7-H3结合的Fv片段、scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体或单链Fv(scFv)。
本发明所述的抗体指免疫球蛋白分子或其免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10 -6)结合其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。
本发明所述的“鼠源抗体”为根据本领域知识和技能制备的对人B7-H3的单克隆抗体。制备时用B7-H3抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
本发明所述的“嵌合抗体”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。
本发明所述的“人源化抗体”也称为CDR移植抗体，是将小鼠的CDR序列移植到人的抗体可变区框架(FR)中产生的抗体。此类可变区框架序列可以从公共的DNA数据库或公开的参考文献获得，例如从ImMunoGeneTics(IMGT)网站http://imgt.cines.fr得到或从免疫球蛋白杂志，2001ISBN012441351上获得。
附图说明
图1是抗B7-H3人源化抗体与人4Ig-B7-H3结合活性测定(ELISA)结果，横坐标为抗体浓度ng/ml，纵坐标为OD450值。
图2是抗B7-H3人源化抗体与人2Ig-B7-H3结合活性测定(ELISA)结果，横坐标为抗体浓度ng/ml，纵坐标为OD450值。
图3是抗B7-H3人源化抗体与猴B7-H3结合活性测定(ELISA)结果，横坐标为抗体浓度ng/ml，纵坐标为OD450值。
图4是抗人B7-H3鼠单抗在SEB刺激的PBMC体系中IFNγ释放水平检测结果，PBMC来自donor B。横坐标为不同抗体，纵坐标为IFNγ释放量(pg/ml)。
图5是抗人B7-H3鼠单抗在SEB刺激的PBMC体系中IFNγ释放水平检测结果，PBMC来自donor C。横坐标为不同抗体，纵坐标为IFNγ释放量(pg/ml)。图6是抗B7-H3人源化抗体hu65在SEB刺激的PBMC体系中IFNγ释放水平检测结果。横坐标为不同抗体，纵坐标为IFNγ释放量(pg/ml)。
图7是抗B7-H3人源化抗体hu51-G34K在SEB刺激的PBMC体系中IFNγ释放水平检测结果。横坐标为不同抗体，纵坐标为IFNγ释放量(pg/ml)。
具体实施方式
下面代表性的实施例是为了更好地说明本发明，而非用于限制本发明的保护范围。以下实施例中未注明条件的实验方法通常按照常规条件，如冷泉港的抗体技术实验手册、分子克隆手册等，或按照原料或商品制造厂商所建议的条件进行。实施例中使用的材料、试剂如无特殊说明均为商购获得。
实施例1 抗原蛋白及阳性对照抗体的制备
1、抗原蛋白及阳性对照抗体的表达载体构建
(1)抗原蛋白的表达载体构建
合成编码4Ig-B7-H3蛋白胞外区全长的基因片段，氨基酸序列设计如SEQ ID  NO:88所示。合成编码2Ig-B7-H3蛋白的基因片段，氨基酸序列设计如SEQ ID NO:89所示。将4Ig-B7-H3的全长DNA序列(SEQ ID NO:90)克隆到真核表达质粒pTargeT上，获得其表达质粒pTargeT-4Ig-B7-H3。
将人4Ig-B7-H3蛋白胞外区氨基酸序列与hIgG1-Fc或his标签的氨基酸序列进行融合，氨基酸序列设计如SEQ ID NO:91和SEQ ID NO:92所示。将人2Ig-B7-H3蛋白胞外区氨基酸序列与hIgG1-Fc或his标签氨基酸序列进行融合，氨基酸序列设计如SEQ ID NO:93和SEQ ID NO:94所示。对上述氨基酸序列进行密码子优化后合成带有标签的B7-H3蛋白胞外区基因片段4Ig-B7-H3-hFc、4Ig-B7-H3-his、2Ig-B7-H3-hFc、2Ig-B7-H3-his并将其分别克隆至真核表达质粒pHR中，分别获得上述标签蛋白的表达质粒pHR-4Ig-B7-H3-hFc、pHR-4Ig-B7-H3-his、pHR-2Ig-B7-H3-hFc、pHR-2Ig-B7-H3-his。
(2)阳性对照抗体hBRCA84D的表达载体构建
使用专利申请WO2011/109400中公开的人源化抗体hBRCA84D作为阳性对照抗体，hBRCA84D的氨基酸序列如下所示：
hBRCA84D重链氨基酸序列：SEQ ID NO:95；
hBRCA84D轻链氨基酸序列：SEQ ID NO:96。
根据WO2011/109400公开的方法制备hBRCA84D。hBRCA84D为人IgG1型单克隆抗体，与天然IgG1相比，其Fc段含有特定突变位点(K214R,L235V,F243L,R292P,Y300L,D356E,L358M,P396L)。对hBRCA84D人源化抗体的氨基酸序列进行密码子人工优化，获得阳性对照抗体hBRCA84D的重链及轻链全长DNA序列。将hBRCA84D重链全长DNA片段克隆pHR载体上，获得真核表达质粒pHR-hBRCA84D-8mIgG1。将hBRCA84D轻链全长DNA片段克隆到pHR载体上，获得真核表达质粒pHR-hBRCA84D-hK。
(3)鼠源阳性对照抗体BRCA84D的表达载体构建
将阳性对照抗体hBRCA84D的重链可变区序列克隆至含鼠mG2a重链恒定区的真核表达质粒pHR-mG2a上，获得pHR-hBRCA84D-mG2a的真核表达质粒。将阳性对照抗体hBRCA84D的轻链可变区序列克隆至含鼠K轻链恒定区的真核表达质粒pHR-mK上，获得pHR-hBRCA84D-mK的真核表达质粒。
2、抗原蛋白及阳性对照抗体的表达与纯化
(1)表达抗原蛋白的稳转细胞株构建
将真核表达质粒pTargeT-B7-H3在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置于37℃，5％CO 2浓度的培养箱中培养。24h后采用含1000ug/ml G418(Gibco，#10131-027)的培养基进行加压培养。转染16天后采用流式细胞术检测转染pool的阳性率，将阳性率较高的pool的细胞进行铺板(按照1x10 6个/ml的细胞密度，100ul/孔，铺96孔板)，采用hBRCA84D抗体和Goat pAb to Hu IgG(PE)(Abcam,ab98596)抗体与细胞孵育，以流式细胞仪(ACEABIO,Novocyte 2060R)检测585nm波长下mean值，使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆，挑选出克隆化的CHO-K1细胞株，该细胞株高水平表达B7-H3分子，命名为CHO-K1-B7-H3。
(2)标签抗原蛋白及阳性对照抗体的表达
在1L细胞培养瓶中接种密度为0.5x 10 6个细胞/ml的293E细胞，加入新鲜的预热的FreeStyle 293表达培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的CO 2培养箱中培养过夜。取8.5mL FreeStyle 293表达培养基，加入1mg/ml的PEI溶液500ul，混合均匀，取250ug待转染质粒加入8.5ml FreeStyle 293表达培养基中，混合均匀，其中标签抗原蛋白质粒pHR-4Ig-B7-H3-hFc、pHR-4Ig-B7-H3-his、pHR-2Ig-B7-H3-hFc、pHR-2Ig-B7-H3-his分别转染；阳性对照抗体hBRCA84D重链质粒pHR-hBRCA84D-8mIgG1和轻链质粒pHR-hBRCA84D-hK共同转染；鼠源阳性对照抗体BRCA84D重链质粒pHR-hBRCA84D-mG2a和轻链质粒pHR-hBRCA84D-mK共同转染。将PEI与FreeStyle 293表达培养基的混合溶液加入到质粒中，混合均匀，然后加入细胞培养物中，置37℃，8％CO 2，加湿的CO 2培养箱中培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入2.5ml的谷氨酰胺(母液浓度为200mM)和5ml的葡萄糖(母液浓度为180g/L)。当细胞活力降至65％～75％时，收集细胞上清。将细胞培养物1500rpm离心5min，收集上清，再8000rpm离心20min，收集上清。
(3)亲和层析柱纯化
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用不同的亲和层析柱进 行纯化(不同蛋白适配的亲和层析柱见表1)，具体纯化步骤如下：
表1不同蛋白适配的亲和层析柱
Figure PCTCN2020115262-appb-000001
清洗：超纯水清洗设备及管路2min，流速10mL/min，后用0.1M NaOH清洗层析系统；
接柱：将层析柱接入层析设备，并用超纯水冲洗5min；后0.1M NaOH冲洗30min，保留时间5min；
平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV(柱体积)；
上样：将细胞表达上清上样，保留时间5min；
后平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV；
洗脱：50mM醋酸，pH＝3.4洗脱，保留时间5min。UV280至50mAu左右时开始收集，降至50mAu左右时停止收集。用1M Tris-HCl，pH 9.0将样品pH调节至7.0；
再平衡：20mM PB+0.15M NaCl，pH 7.2平衡3个CV，保留时间5min；
在线清洗：0.1M NaOH清洗30min，保留时间5min；
清洗保存：纯化水清洗10min，后20％乙醇2个CV。
实施例2 单克隆抗体的制备
1、杂交瘤单克隆的制备
(1)动物免疫
采用不同标签的4Ig-B7-H3抗原和2Ig-B7-H3抗原蛋白与佐剂共同免疫实验动物，实验动物包括SJL品系小鼠/Balb/c品系小鼠/SD大鼠。免疫动物按照首次免疫使用50ug抗原免疫一只动物，后期均使用25ug抗原免疫一只动物。免疫佐剂可以是弗氏佐剂(Sigma)或Quick Antibody-Mouse5W(北京博奥龙免疫技术有限公司)。采用弗氏佐剂乳化抗原，将不同标签的4Ig-B7-H3或者2Ig-B7-H3 抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫小鼠。采用Quick Antibody-Mouse5W作为佐剂，将不同标签的4Ig-B7-H3或者2Ig-B7-H3抗原蛋白样品与Quick Antibody-Mouse5W按照1:1的体积比进行混合，混匀后即采用肌肉注射的方式，免疫SD大鼠。
采用表达B7-H3的稳定转染细胞系CHO-K1-B7-H3免疫SD大鼠和Balb/c小鼠，使之产生抗B7-H3的抗体。用胰蛋白酶消化处理实施例1中获得的CHO-K1-B7-H3细胞，然后按照1000rpm离心5min，丢弃细胞上清，用PBS重悬细胞沉淀，取样用细胞计数仪计数，剩余细胞按照1000rpm离心5min，弃上清，用PBS重悬细胞沉淀，加入适量的PBS以获得1x10 8个细胞/ml的细胞悬液。实验组小鼠每只免疫1x10 7个细胞，实验组SD大鼠每只免疫2x10 7个细胞。
免疫方案如表2所示：
表2 动物免疫方案
组别 抗原 动物 佐剂 免疫途径
1 PBS SJL/Balb/c/SD大鼠 无 s.c./i.m.
2 4Ig-B7-H3-his/4Ig-B7-H3-hFc SJL/Balb/c 弗氏佐剂 s.c./i.m.
3 2Ig-B7-H3-his/2Ig-B7-H3-hFc Balb/c 弗氏佐剂 s.c./i.m.
4 4Ig-B7-H3-his/4Ig-B7-H3-hFc SD大鼠 Quick Antibody-Mouse5W i.m.
5 CHO-K1-B7-H3 Balb/c/SD大鼠 无 i.p.
*i.m.肌内注射；s.c.皮下注射；i.p.腹腔注射。
(2)杂交瘤融合
脾细胞的获取和制备：将加强免疫后的小鼠/大鼠处死后浸泡75％的酒精中。解剖取出脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min，弃上清。加入2mL红细胞裂解液，室温裂解红细胞2min，加入PBS至20mL，1500rpm离心7min，弃上清，重悬后进行活细胞计数。收集培养瓶中的Sp2/0细胞，1000rpm离心5min后弃上清，重悬后进行活细胞计数。按脾细胞：Sp2/0细胞＝1:1的比例混合细胞，1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞，1500rpm离心7min。弃上清，重复一次。分别用适量电转缓冲液重悬细胞，保证细胞浓度2×10 7个细胞/mL左右。把细胞 悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20％FBS的15mL RPMI 1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μl/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。
(3)杂交瘤及亚克隆筛选
融合一周后，取杂交瘤母克隆的细胞培养上清，通过ELISA筛选结合4Ig-B7-H3-his蛋白和食蟹猴B7-H3蛋白的杂交瘤母克隆。进一步通过流式细胞术筛选出能结合CHO-K1-B7-H3稳转细胞株的母克隆。将筛选阳性的母克隆扩至24孔板，选择培养2天的母克隆培养上清，与T细胞进行共培养，检测培养上清中IFN-γ的释放量。具体实验操作如下所述：
1.Day 1以1ug/ml anti-CD3e(R&D)包被T25细胞培养瓶，4℃条件下孵育过夜，于实验前弃去液体，用PBS缓冲液洗3次，待用。
2.Day 2复苏CD8+T细胞(用CD3和CD28诱导扩增获得，90％FBS+10％DMSO冻存)，采用IMDM+10％FBS+游离100ng/ml SEB+4ng/ml IL-2+100x双抗的培养基重悬细胞，于步骤1包被的T25中培养，培养密度2-3×10 6cells/ml，培养48h。
3.Day 3按照1ug/ml anti-CD3e和10ug/ml 4Ig-B7-H3-his共同包被96孔板，设置包被空白组，anti-CD3e组，anti-CD3e+B7-H3-4Ig-his组，4℃条件下过夜孵育。
4.Day 4将杂交瘤培养上清按照100ul/孔加至上一步的96孔板中，对照孔加入100ul杂交瘤培养基，于37℃孵育30min。
5.取出已经刺激2天的CD8+T，将细胞密度调整至1×10 6cell/ml，按照100ul/孔加入到96孔板，培养48h。
6.Day 6培养基上清中IFN-γ释放的检测，按照R&D的duset(DY285B)检测试剂盒，检测培养基上清中IFN-γ。
通过Elisa和FACS筛选出具有结合能力的母克隆，进一步评估母克隆分泌的细胞上清诱导细胞因子IFN-γ释放的水平，筛选出能促进IFN-γ释放的阳性 母克隆。利用有限稀释法将阳性母克隆进行亚克隆，培养一周后利用ELISA检测亚克隆上清与4Ig-B7-H3分子的结合活性，进而获得分泌抗B7-H3抗体的单克隆细胞株。
2、亚型的鉴定
参照鼠抗体亚型鉴定试剂盒SBA Clonotyping Systerm-C57BL/6-HRP(SouthernBiotech，货号：5300-05B)说明书对抗体进行亚型鉴定，结果如表3所示：
表3 抗体亚型鉴定结果
命名 抗体亚型
SHS007-17 IgG/k
SHS007-39 IgG/k
SHS007-51 IgG/k
SHS007-65 IgG/k
3、单克隆抗体的制备
根据亚克隆上清活性分析结果确定单克隆抗体母克隆株17、39、51、65，将其扩大培养。培养条件是含有10％胎牛血清、1x NAEE、1x丙酮酸钠、1％青链霉素双抗的1640培养基，待细胞汇合度大于>80％时，进将细胞传代扩培，待培养至约50ml时收集上清，纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。
4、单克隆抗体测序
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。
根据小鼠抗体亚型可变区设计特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得小鼠抗体轻链与重链可变区的基因片段；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester et al.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods, 1997,201:35–55；2.Simon KorenMiha
Figure PCTCN2020115262-appb-000002
AnjaColjaVenturinietal.Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列，测序结果见表4。
表4 抗B7-H3鼠源单克隆抗体序列表
抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHS007-17 SEQ ID NO:26 SEQ ID NO:27
SHS007-39 SEQ ID NO:28 SEQ ID NO:29
SHS007-51 SEQ ID NO:30 SEQ ID NO:31
SHS007-59 SEQ ID NO:33 SEQ ID NO:34
SHS007-65 SEQ ID NO:35 SEQ ID NO:36
SHS007-72 SEQ ID NO:37 SEQ ID NO:38
SHS007-90 SEQ ID NO:39 SEQ ID NO:40
SHS007-95 SEQ ID NO:41 SEQ ID NO:42
SHS007-108 SEQ ID NO:43 SEQ ID NO:44
实施例3 嵌合抗体的构建
将表4测序获得的小鼠抗体重链可变区基因片段克隆至含有特定突变位点(K214R,L235V,F243L,R292P,Y300L,D356E,L358M,P396L)的重链恒定区的真核表达质粒(pHR-8mIgG1)上，将表4测序获得的小鼠抗体轻链可变区基因片段克隆至含人轻链恒定区的真核表达质粒(pHR-hK)上。分别将构建的抗体重轻链质粒转化大肠杆菌DH5α感受态细胞，将混合液均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得嵌合抗体轻链与重链的表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将嵌合抗体重轻链质粒共转染HEK293E细胞，表达纯化获得抗体，将嵌合抗体分别命名为SHS007-17-CHI、SHS007-39-CHI、SHS007-51-CHI、SHS007-65-CHI，然后进行纯度检测、活性分析及亲和力的检测。
利用定点突变的方法，将嵌合抗体SHS007-17-CHI、SHS007-39-CHI、 SHS007-51-CHI、SHS007-65-CHI进行基因突变，以筛选更优的抗体。SHS007-51-CHI轻链CDR第34位G突变为K，抗体稳定性提高，标记为SHS007-51-G34K-CHI。嵌合抗体测序结果见表5。
表5 抗B7-H3嵌合抗体序列表
嵌合抗体 重链可变区氨基酸序列 轻链可变区氨基酸序列
SHS007-17-CHI SEQ ID NO:26 SEQ ID NO:27
SHS007-39-CHI SEQ ID NO:28 SEQ ID NO:29
SHS007-51-CHI SEQ ID NO:30 SEQ ID NO:31
SHS007-51-G34K-CHI SEQ ID NO:30 SEQ ID NO:32
SHS007-65-CHI SEQ ID NO:35 SEQ ID NO:36
实施例4 人源化抗体的构建及生产
根据嵌合抗体的免疫活性和杀伤活性分析，选择SHS007-17-CHI、SHS007-39-CHI、SHS007-51-G34K-CHI、SHS007-65-CHI进行人源化抗体改造。
抗体的人源化改造，首先是通过与免疫基因数据库(IMGT)中的小鼠抗体序列进行比对，确认SHS007-17-CHI、SHS007-39-CHI、SHS007-51-G34K-CHI、SHS007-65-CHI抗体可变区的鼠源种系，经过同源比对，SHS007-17-CHI、SHS007-39-CHI、SHS007-51-G34K-CHI、SHS007-65-CHI抗体的重链可变区序列的FR区分别与小鼠抗体种系基因(IGHV1-69*02、IGHV1-2*02、IGHV3-21*01、IGHV3-48*01)最为相似；抗体轻链可变区的FR序列则分别与小鼠抗体(IGKV2-28*01、IGKV3-11*01、IGKV2-30*02、IGKV3-11*01)最为相似。以SHS007-17-CHI、SHS007-39-CHI、SHS007-51-G34K-CHI、SHS007-65-CHI抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代SHS007-17-CHI、SHS007-39-CHI、SHS007-51-G34K-CHI、SHS007-65-CHI的FR1-FR3序列，重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，最终选择SHS007-17-CHI的4条人源化重链可变区(参见SEQ ID NO:45、46、47、48)和3条人源化轻链可变区(参见SEQ ID NO:49、50、51)、SHS007-39-CHI的6条人源化重链可变区(参见SEQ ID NO:52、53、54、55、56、57)和5条人 源化轻链可变区(参见SEQ ID NO:58、59、60、61、62)、SHS007-51-G34K-CHI的2条人源化重链可变区(参见SEQ ID NO:63、64)和6条人源化轻链可变区(参见SEQ ID NO:65、66、67、68、69、70)及SHS007-65-CHI的3条人源化重链可变区(参见SEQ ID NO:71、72、73)和6条人源化轻链可变区(参见SEQ ID NO:74、75、76、77、78、79)进行下一步优化。SHS007-17-CHI、SHS007-39-CHI、SHS007-51-G34K-CHI、SHS007-65-CHI的人源化抗体非CDR区序列均达到95％以上人源化。
将以上设计好的人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过OverlapPCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有人K轻链恒定区的真核表达质粒(pHR-hK)共转化大肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与人IgG1重链恒定区的真核表达质粒(pHR-IgG1)共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有相应抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。
将测序正确的阳性克隆接种于含有相应抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。
挑选纯度、活性和亲和力均较好的人源化抗体，标记为hu17-00、hu39-01、hu65-13、hu51-G34K-12，序列见表6。
表6 抗B7-H3人源化抗体序列表
Figure PCTCN2020115262-appb-000003
Figure PCTCN2020115262-appb-000004
实施例5 与人B7-H3结合活性测定(ELISA)
采用ELISA分析抗体的结合活性。将人4Ig-B7-H3-His蛋白或2Ig-B7-H3-His蛋白(1ug/孔，实施例1、2中制得)包被到96孔酶标板，于4℃条件下孵育过夜。用1xPBST清洗3次后用5％的脱脂牛奶37℃封闭2h。用1xPBST清洗3次后，本发明提供的抗B7-H3抗体作为一抗从2μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0.0256ng/mL，37℃孵育2h，阳性对照抗体为hBRCA84D；用1xPBST清洗5次后，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:5000)，37℃孵育1h。用1xPBST清洗5次后，加入显色液TMB，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如图1、2所示。
实验结果显示，本发明提供的人源化抗B7-H3抗体hu51-G34K-12、hu65-13均具有与人4Ig-B7-H3、2Ig-B7-H3结合的能力，且结合能力与阳性对照抗体hBRCA84D相当。
实施例6 与食蟹猴B7-H3结合活性测定(ELISA)
采用protein based Elisa分析抗体的结合活性。食蟹猴B7-H3-His(0.5μg/孔，Sino Biological，Cat.No.90806-C08H)包被96孔酶标板。本发明提供的抗B7-H3抗体作为一抗从2μg/mL开始，5倍梯度稀释加入酶标板，共8个浓度，浓度分别为2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL、0.0256ng/mL，37℃孵育2h，阳性对照抗体为hBRCA84D。二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:10000)，加入显色液TMB(3,3',5,5'-四甲基联苯胺)，终止后利用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如图3所示。
实验结果显示，本发明提供的人源化抗B7-H3抗体hu51-G34K-12、hu65-13均具有与食蟹猴B7-H3结合的能力，且结合能力与阳性对照抗体hBRCA84D相当。
实施例7 与肿瘤细胞表面B7-H3结合的测定(FACS)
采用FACS分析抗体与肿瘤细胞表面B7-H3的结合活性。FACS检测的平均荧光强度(MFI)和EC50表示抗体与细胞的结合活性。786-0、MDA-MB-231细胞消化后，用2％FBS-PBS的溶液重悬，计数。raji-B7-H3细胞收集细胞后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1x10 5个细胞的方式铺细胞板，本发明提供的抗B7-H3抗体作为一抗从20μg/ml开始，梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL，4℃条件下孵育1h，阳性对照抗体为hBRCA84D；二抗使用PE-Anti-Human IgG(Biolegend，Cat.No.409303，1.25μl/孔)，洗涤后使用流式细胞仪检测抗体与细胞表面结合产生的荧光强度。使用GraphPad计算EC50，结果如表7a和7b所示。
表7a 与肿瘤细胞表面B7-H3结合的测定
Figure PCTCN2020115262-appb-000005
表7b 与肿瘤细胞表面B7-H3结合的测定
Figure PCTCN2020115262-appb-000006
实验结果显示，本发明提供的人源化抗B7-H3抗体hu51-G34K-12、hu65-13均可与肿瘤细胞表面B7-H3结合，与肿瘤细胞的结合密度显著高于对照抗体 hBRCA84D，结合EC50和Emax均明显优于对照抗体hBRCA84D。
实施例8 固定抗体法Biacore亲和力测定
利用Biacore对实施例4中制得的人源化抗B7-H3抗体进行亲和力测定。先将抗原4Ig-B7-H3-His偶联至CM5芯片(GE，Cat.No.BR100012)表面，并用乙醇胺封闭未偶联蛋白的活性基团。待基线平稳后，分别将hu39-01、hu65-13、hBRCA84D抗体进行等比梯度稀释，稀释后的各浓度抗体蛋白按顺序放置于仪器的进样盘上，设置程序分别进样。按Biacore X100的操作规程进行hu51-G34K-12、hu65-13、hBRCA84D抗体与4Ig-B7-H3-His抗原亲和力测定，具体参数及实验结果如表8所示。
表8 与人B7-H3蛋白的亲和力测定
Figure PCTCN2020115262-appb-000007
实验结果显示，hu51-G34K-12和hu65-13与人4Ig-B7-H3蛋白结合具有良好的亲和力，且亲和力数值与阳性对照抗体hBRCA84D相当。
实施例9 IFN-γ释放检测B7-H3鼠单抗、人源化抗体的阻断活性
采用Ficoll(GE Healthcare,Cat.No.10268731)分离得到人外周血单个核细胞(PBMC)，将分离获得的PBMC用含10％FBS的IMDM(Life technologies，12440053)完全培养基重悬，调整密度为1*10 6cell/mL。按照100μL/孔将PBMC细胞加入到96孔板中，静置30min。将待检测抗体、Isotype抗体用相同培养基配制为终浓度20μg/ml，加入到96孔板，于37℃恒温培养箱中培养1h。用培养基配制终浓度为100ng/ml的SEB(Toxin techonology，BT202)溶液，加入96孔板对应孔中。上述96孔板于恒温培养箱中共培养72h后，按照DuoSet检测试剂盒(R&D，DY285B)说明书检测上述培养上清中的IFN-γ浓度，如图4、5结果显示抗人B7-H3鼠单抗SHS007-39、51、59、65、72等，在donor B和donor C来源的PBMC-SEB体系中，均能增加该体系中细胞因子IFN-γ的释放。图6、7结果显示抗人B7-H3人源化抗体hu51、hu65增强PBMC-SEB实验体系中IFN-γ的释放。
实施例10 抗体依赖细胞介导的细胞毒ADCC实验(LDH法)
人肾癌细胞786-0高表达人B7-H3，将786-0细胞按照20000个细胞每孔铺96孔板。用热灭活的胎牛血清(FBS，Gibco，Cat.No.10091-148)配置含2％FBS的RPMI-1640培养基。将SHS007-17CHI、SHS007-39CHI、SHS007-51CHI、SHS007-65CHI、hBRCA84D抗体10倍梯度稀释溶于上述培养基，于37℃恒温培养箱孵育1h。按照Ficoll(GE Healthcare,Cat.No.10268731)试剂盒说明，提取人外周血PBMC作为效应细胞，将PBMC细胞和上述抗体溶液加入到铺有786-0细胞的96孔细胞板。将细胞板在37℃培养箱内培养4h，按照Cytotoxicity LDH Assay
Figure PCTCN2020115262-appb-000008
(同仁化学，CK12)说明书，检测细胞毒性。计算本发明抗体杀伤EC50降低倍数(即阳性对照hBRCA84D的EC50值与本发明提供抗体EC50值的比值)，实验结果如表9所示。
表9 嵌合抗体ADCC杀伤活性测定
Figure PCTCN2020115262-appb-000009
实验结果显示，与阳性对照hBRCA84D抗体相比，本发明提供的抗体具有更优异的杀伤EC50值，大大提高了ADCC杀伤活性。
实施例11 人源肿瘤细胞小鼠移植瘤模型的抗肿瘤试验
1、实验材料
(1)实验细胞及动物
人恶性胶质瘤U87细胞购自美国典型培养物保藏中心(ATCC)；
人肾癌细胞786-0细胞购自美国典型培养物保藏中心(ATCC)；
人三阴性乳腺癌MDA-MB-231细胞购自美国典型培养物保藏中心(ATCC)；
SCID-Beige小鼠，雌性，6-8周龄，体重18-20克，购自上海灵畅实验动物有限公司；
(2)供试品及对照品
阴性对照品Isotype IgG1(货号C0001-4)购自中美冠科生物技术有限公司。在小鼠移植瘤模型中hBRCA84D-IgG1效果远优于hBRCA84D，故使用hBRCA84D-IgG1作为阳性对照品。hBRCA84D-IgG1为利用对标抗体hBRCA84D可变区序列构建的人IgG1形式单克隆抗体，恒定区与本发明的人IgG1嵌合抗体相同。
试验前，将本发明的抗B7-H3嵌合抗体(SHS007-39-IgG1、SHS007-65-IgG1)用PBS配制为1mg/mL，Isotype IgG1和hBRCA84D-IgG1配制为1mg/mL。
2、实验方法
(1)U87移植瘤模型
将人肿瘤细胞U87细胞接种于小鼠的右后边，接种细胞数目为5×10 6/只。待肿瘤生长至平均体积达140mm 3时开始分组，尾静脉给药每周2次，每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。记录连续给药24天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线，结果如表10所示。
表10 人恶性胶质瘤U87移植瘤模型抗肿瘤试验结果
Figure PCTCN2020115262-appb-000010
(2)786-0移植瘤模型
将人肿瘤细胞786-0细胞接种于小鼠的右后边，接种细胞数目为1×10 7/只。接种2周后，用无菌手术器械将786-O细胞接种长成的瘤块取下，在生理盐水中将生长旺盛期的瘤组织切成30mm 3左右，在无菌条件下，接种于小鼠右后背。待肿瘤生长至平均体积达160mm 3时开始分组，尾静脉给药每周2次，每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2， a和b分别表示肿瘤的长径和短径。记录连续给药19天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线，结果如表11所示。
表11 人肾癌细胞786-0移植瘤模型抗肿瘤试验结果
Figure PCTCN2020115262-appb-000011
(3)MDA-MB-231移植瘤模型
将人肿瘤细胞MDA-MB-231细胞接种于小鼠的右后边，接种细胞数目为8×10 6/只。接种2周后，用无菌手术器械将MDA-MB-231细胞接种长成的瘤块取下，在生理盐水中将生长旺盛期的瘤组织切成30mm 3左右，在无菌条件下，接种于小鼠右后背。待肿瘤生长至平均体积达150mm 3时开始分组，尾静脉给药每周2次，每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。记录连续给药21天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线，结果如表12所示。
表12 人三阴性乳腺癌细胞MDA-MB-231移植瘤模型抗肿瘤试验结果
Figure PCTCN2020115262-appb-000012
实验结果显示，本发明提供的抗体在人恶性胶质瘤U87细胞、人肾癌细胞786-0细胞、人三阴性乳腺癌细胞MDA-MB-231接种的SCID-Beige小鼠移植瘤模型中具有抑制肿瘤生长的作用。抗体SHS007-39-CHI和SHS007-65-IgG1的抑瘤作用优于对照抗体hBRCA84D-IgG1。本发明提供的抗B7-H3抗体具有更加显著的抑制肿瘤生长的作用。
实施例12 人源肿瘤细胞小鼠移植瘤模型的抗肿瘤试验
1、实验材料
(1)实验细胞及动物
人肾癌细胞786-0细胞购自美国典型培养物保藏中心(ATCC)；
人三阴性乳腺癌MDA-MB-231细胞购自美国典型培养物保藏中心(ATCC)；
SCID-Beige小鼠，雌性，6-8周龄，体重18-20克，购自上海灵畅实验动物有限公司；
(2)供试品及对照品
对照品Isotype IgG1(货号C0001-4)购自中美冠科生物技术有限公司，用作阴性对照品；对照品hBRCA84D-IgG1为利用对标抗体hBRCA84D可变区序列构建的人IgG1形式单克隆抗体，恒定区与本发明的人源化抗体相同，用作阳性对照；
试验前，将本发明的人源化抗B7-H3抗体hu65-13、hu51-G34K-12分别用PBS配制为1mg/mL或者2mg/ml，Isotype IgG1和hBRCA84D-IgG1配制为1mg/mL或者2mg/ml。
2、实验方法
(1)786-0移植瘤模型
将人肿瘤细胞786-0细胞接种于小鼠的右后边，接种细胞数目为1×10 7/只。接种2周后，用无菌手术器械将786-O细胞接种长成的瘤块取下，在生理盐水中将生长旺盛期的瘤组织切成15mm 3左右，在无菌条件下，接种于小鼠右后背。待肿瘤生长至平均体积达100mm 3时开始分组，尾静脉给药每周2次，给药至第21天，给药频率降低为一周给药一次。每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。记录连续给药36天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线，结果如表13所示。
表13 人肾癌细胞786-0移植瘤模型抗肿瘤试验结果
Figure PCTCN2020115262-appb-000013
Figure PCTCN2020115262-appb-000014
(2)MDA-MB-231移植瘤模型
将人肿瘤细胞MDA-MB-231细胞接种于小鼠的右后边，接种细胞数目为8×10 6/只。接种2周后，用无菌手术器械将MDA-MB-231细胞接种长成的瘤块取下，在生理盐水中将生长旺盛期的瘤组织切成30mm 3左右，在无菌条件下，接种于小鼠右后背。待肿瘤生长至平均体积达100mm 3时开始分组，尾静脉给药每周2次，每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。记录连续给药25天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线，结果如表14所示。
表14 人三阴性乳腺癌细胞MDA-MB-231移植瘤模型抗肿瘤试验结果
Figure PCTCN2020115262-appb-000015
实验结果显示，本发明提供的人源化抗体hu51-G34K-12和hu65-13在人肾癌细胞786-0细胞、人三阴性乳腺癌细胞MDA-MB-231接种的SCID-Beige小鼠移植瘤模型中具有抑制肿瘤生长的作用。hu51-G34K-12和hu65-13的抑瘤作用优于对照抗体hBRCA84D-IgG1。本发明提供的抗B7-H3抗体具有更加显的著抑制肿瘤生长的作用。
以上实施例证明本发明提供的抗B7-H3抗体具有显著的抗肿瘤作用，可明显抑制肿瘤增长，提示该抗体可在制备抗肿瘤药物中的应用，具有可预期的市场前景。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。

Claims (16)

  1. 一种抗B7-H3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：
    (1)所述重链可变区包含选自如下组的HCDR1、HCDR2和HCDR3：
    (a1)如SEQ ID NO:1、2和3所示的氨基酸序列；
    (a2)如SEQ ID NO:7、8和9所示的氨基酸序列；
    (a3)如SEQ ID NO:13、14和15所示的氨基酸序列；
    (a4)如SEQ ID NO:20、21和22所示的氨基酸序列；和
    (a5)与(a1)、(a2)、(a3)或(a4)所示的氨基酸序列具有至少85％序列同一性的CDR；和
    (2)所述轻链可变区包含选自如下组的LCDR1、LCDR2和LCDR3：
    (a6)如SEQ ID NO:4、5和6所示的氨基酸序列；
    (a7)如SEQ ID NO:10、11和12所示的氨基酸序列；
    (a8)如SEQ ID NO:16、18和19所示的氨基酸序列；
    (a9)如SEQ ID NO:17、18和19所示的氨基酸序列；
    (a10)如SEQ ID NO:23、24和25所示的氨基酸序列；和
    (a11)与(a6)、(a7)、(a8)、(a9)或(a10)所示的氨基酸序列具有至少85％序列同一性的CDR。
  2. 如权利要求1所述的抗B7-H3抗体或其抗原结合片段，其具有：
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:4、5和6或与SEQ ID NO:4、5和6所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区；
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:7、8和9或与SEQ ID NO:7、8和9所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:10、11和12或与SEQ ID NO:10、11和12所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可 变区；
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:13、14和15或与SEQ ID NO:13、14和15所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:16、18和19或与SEQ ID NO:16、18和19所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区；
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:13、14和15或与SEQ ID NO:13、14和15所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:17、18和19或与SEQ ID NO:17、18和19所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区；或
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:20、21和22或与SEQ ID NO:20、21和22所示的氨基酸序列具有至少85％序列同一性的CDR的重链可变区，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:23、24和25或与SEQ ID NO:23、24和25所示的氨基酸序列具有至少85％序列同一性的CDR的轻链可变区。
  3. 如权利要求1或2所述的抗B7-H3抗体或其抗原结合片段，其中所述抗体是鼠源单克隆抗体、人鼠嵌合抗体或人源化抗体。
  4. 如权利要求1-3之任一项所述的抗B7-H3抗体或其抗原结合片段，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (b1)如SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:35所示的氨基酸序列；
    (b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b3)与(b1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (b4)如SEQ ID NO:27、SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:36所示的氨基酸序列；
    (b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (b6)与(b4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  5. 如权利要求4所述的B7-H3抗体或其抗原结合片段，其中所述抗体还含有鼠源的IgG1、IgG2或其变体的重链恒定区，鼠源的k链或其变体的轻链恒定区。
  6. 如权利要求1-3之任一项所述的抗B7-H3抗体或其抗原结合片段，其中
    所述重链可变区的氨基酸序列为SEQ ID NO:26，SEQ ID NO:26经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:26功能相同的氨基酸序列或与SEQ ID NO:26具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:27，SEQ ID NO:27经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:27功能相同的氨基酸序列或与SEQ ID NO:27具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:28，SEQ ID NO:28经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:28功能相同的氨基酸序列或与SEQ ID NO:28具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:29，SEQ ID NO:29经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:29功能相同的氨基酸序列或与SEQ ID NO:29具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:31，SEQ ID NO:31经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:31功能相同的氨基酸序列或与SEQ ID NO:31具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:30，SEQ ID NO:30经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:30功能相同的氨基酸序列或与SEQ ID NO:30具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:32，SEQ ID NO:32经取代、缺失或添加一个或多 个氨基酸获得的且与SEQ ID NO:32功能相同的氨基酸序列或与SEQ ID NO:32具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:35，SEQ ID NO:35经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:35功能相同的氨基酸序列或与SEQ ID NO:35具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:36，SEQ ID NO:36经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:36功能相同的氨基酸序列或与SEQ ID NO:36具有至少85％序列同一性的氨基酸序列。
  7. 如权利要求1-3之任一项所述的抗B7-H3抗体或其抗原结合片段，其中所述抗B7-H3抗体为人源化抗体，其中：
    (1)所述重链可变区的氨基酸序列选自：
    (c1)如SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:72或SEQ ID NO:73所示的氨基酸序列；
    (c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (c3)与(c1)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列；和
    (2)所述轻链可变区的氨基酸序列选自：
    (c4)如SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:68、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:74、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79所示的氨基酸序列；
    (c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列；和
    (c6)与(c4)所示的氨基酸序列具有至少80％序列同一性的氨基酸序列。
  8. 如权利要求7所述的抗B7-H3抗体或其抗原结合片段，其中
    所述重链可变区的氨基酸序列为SEQ ID NO:45，SEQ ID NO:45经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:45功能相同的氨基酸序列或与SEQ ID NO:45具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:49，SEQ ID NO:49经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:49功能相同的氨基酸序列或与SEQ ID NO:49具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:52，SEQ ID NO:52经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:52功能相同的氨基酸序列或与SEQ ID NO:52具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:59，SEQ ID NO:59经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:59功能相同的氨基酸序列或与SEQ ID NO:59具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:63，SEQ ID NO:63经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:63功能相同的氨基酸序列或与SEQ ID NO:63具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:65，SEQ ID NO:65经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:65功能相同的氨基酸序列或与SEQ ID NO:65具有至少85％序列同一性的氨基酸序列；
    所述重链可变区的氨基酸序列为SEQ ID NO:64，SEQ ID NO:64经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:64功能相同的氨基酸序列或与SEQ ID NO:64具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:67，SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:67功能相同的氨基酸序列或与SEQ ID NO:67具有至少85％序列同一性的氨基酸序列；或
    所述重链可变区的氨基酸序列为SEQ ID NO:72，SEQ ID NO:72经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:72功能相同的氨基酸序列或与SEQ ID NO:72具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:77，SEQ ID NO:77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:77功能相同的氨基酸序列或与SEQ ID NO:77 具有至少85％序列同一性的氨基酸序列。
  9. 一种分离的核酸，其编码权利要求1-8之任一项所述的抗B7-H3抗体或其抗原结合片段。
  10. 如权利要求9所述的核酸，其中：
    (1)编码所述重链可变区氨基酸序列的核苷酸序列如SEQ ID NO:80、SEQ ID NO:82、SEQ ID NO:84或SEQ ID NO:86所示；
    (2)编码所述轻链可变区氨基酸序列的核苷酸序列如SEQ ID NO:81、SEQ ID NO:83、SEQ ID NO:85或SEQ ID NO:87所示。
  11. 一种表达载体，其包含如权利要求9或10所述的核酸。
  12. 一种宿主细胞，其转化如权利要求11所述的表达载体，所述宿主细胞选自原核细胞和真核细胞，优先为哺乳动物细胞。
  13. 制备权利要求1-8任一项所述的抗B7-H3抗体或其抗原结合片段的方法，包括在如权利要求12所述的宿主细胞中表达抗体，以及从宿主细胞中分离所述抗体的步骤。
  14. 一种药物组合物，其包含权利要求1-8之任一项所述的抗B7-H3抗体或其抗原结合片段和药学可接受的载体。
  15. 如权利要求1-8之任一项所述的抗B7-H3抗体或其抗原结合片段或如权利要求14的药物组合物在制备用于抑制B7-H3活性的药物中的应用。
  16. 如权利要求15所述的应用，所述抑制B7-H3活性的药物用于治疗乳腺癌、肾癌、肺癌、胃癌、肠癌、食管癌、卵巢癌、宫颈癌、膀胱癌、胰腺癌、神经胶质瘤、白血病、淋巴瘤和/或黑素瘤。
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