WO2020015687A1 - 抗her3人源化单克隆抗体及其制剂 - Google Patents

抗her3人源化单克隆抗体及其制剂 Download PDF

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WO2020015687A1
WO2020015687A1 PCT/CN2019/096418 CN2019096418W WO2020015687A1 WO 2020015687 A1 WO2020015687 A1 WO 2020015687A1 CN 2019096418 W CN2019096418 W CN 2019096418W WO 2020015687 A1 WO2020015687 A1 WO 2020015687A1
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antibody
variable region
chain variable
light chain
heavy chain
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PCT/CN2019/096418
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English (en)
French (fr)
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瞿爱东
梁红远
徐帆洪
李翱翔
吴丽娜
祝婧烨
邱建华
陆瑾
张琳
赵鑫
宋效飞
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上海生物制品研究所有限责任公司
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Priority to US17/260,442 priority Critical patent/US11965037B2/en
Priority to EP19838498.4A priority patent/EP3825334A4/en
Publication of WO2020015687A1 publication Critical patent/WO2020015687A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • 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
    • C07K2317/90—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
    • 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
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention relates to the field of medicine, in particular to an anti-HER3 humanized monoclonal antibody and a preparation thereof.
  • human epidermal growth factor 3 (human epidermal growth factor 3 receptor HER3) plays an important role in the occurrence and progression of tumors.
  • HER3 usually forms heterodimers with EGFR or HER2 molecules and plays a role.
  • EGFR and HER2 overexpression is often accompanied by HER3 overexpression, and HER3 overexpression is considered to be resistant to EGFR and HER2 targeted therapy Play an important role.
  • HRG ligand Heregulin
  • the conformation changes, exposing the binding site to EGFR and HER2, and then forming heterodimers with EGFR and HER2 molecules, activating intracellular signals and promoting tumor cell proliferation.
  • mouse monoclonal antibodies cause human anti-mouse antibody (HAMA) in clinical treatment, they are limited in clinical treatment.
  • Antibody humanization technology can greatly reduce the immunogenicity of mouse monoclonal antibodies.
  • the purpose of the invention is to provide a HER3 humanized antibody with high affinity and high biological activity and its application.
  • a light chain variable region of an antibody the light chain variable region being selected from the group consisting of:
  • substitution, deletion, modification, and / or addition of the amino acid residue does not occur in the CDR region, but in the framework region.
  • the light chain variable region corresponds to a mutation at glycine (G) at position 69 in the sequence shown in SEQ ID NO :: 1.
  • variable region of the light chain corresponds to the mutation of glycine (G) at position 69 in the sequence shown in SEQ ID NO :: 1 to serine (S) (ie, G69S).
  • the light chain variable region corresponds to a mutation in the isoleucine (I) at position 63 in the sequence shown in SEQ ID NO :: 1.
  • variable region of the light chain corresponds to mutation of Ile at position 63 in the sequence shown in SEQ ID NO :: 1 to Val (ie, Ile ⁇ Val).
  • the light chain variable region sequence is shown as SEQ ID NO .: 1 or 3 or 7.
  • a light chain of an antibody having the light chain variable region according to the first aspect of the present invention.
  • the light chain of the antibody further includes a heavy chain constant region.
  • the light chain constant region is of human, mouse or rabbit origin, preferably human origin.
  • a heavy chain variable region of an antibody the heavy chain variable region being selected from the group consisting of:
  • substitution, deletion, modification, and / or addition of the amino acid residue does not occur in the CDR region, but in the framework region.
  • variable region of the heavy chain corresponds to the sequence shown in SEQ ID NO .: 2 and has an amino acid mutation selected from the group consisting of arginine (R) at position 38 and methionine at position 48. Acid (M), arginine (R) at position 67, valine (V) at position 68, or a combination thereof.
  • the mutation of the heavy chain variable region corresponding to the sequence shown in SEQ ID NO .: 2 is selected from the group consisting of R38K, M48I, V68A, R67K, or a combination thereof; preferably, said Mutation is R38K, or R67K + V68A.
  • the heavy chain variable region is mutated at position 67 corresponding to the sequence shown in SEQ ID NO.:2.
  • the mutation of the heavy chain variable region corresponding to the sequence shown in SEQ ID NO :: 2 is selected from the group consisting of: R67K.
  • the heavy chain variable region sequence is as shown in SEQ ID NO .: 2 or 4 or 8.
  • variable region of the heavy chain is SEQ ID No .: 2 and has a mutation selected from the group consisting of:
  • VH38 is Arg ⁇ Lys (R38K)
  • VH48 is Met ⁇ Ile (M48I)
  • VH68 is Val ⁇ Ala (V68A)
  • VH67 is Arg ⁇ Lys (R67K)
  • VH68 is Val ⁇ Ala (V68A).
  • an antibody heavy chain having the heavy chain variable region according to the third aspect of the present invention there is provided an antibody heavy chain having the heavy chain variable region according to the third aspect of the present invention.
  • the heavy chain of the antibody further includes a heavy chain constant region.
  • the heavy chain constant region is of human, mouse or rabbit origin, and is preferably of human origin.
  • an antibody having:
  • the antibody has: a light chain according to the second aspect of the invention; and / or a heavy chain according to the fourth aspect of the invention.
  • the antibody has a light chain variable region as shown in SEQ ID NO .: 1 or 3; and / or a heavy chain variable region as shown in SEQ ID NO .: 2 or 4.
  • the antibody has a mutation selected from the group consisting of:
  • the light chain variable region of the antibody corresponds to a mutation at glycine (G) at position 69 in the sequence shown in SEQ ID NO :: 1; and / or
  • the heavy chain variable region of the antibody corresponds to the sequence shown in SEQ ID NO .: 2 and has an amino acid mutation selected from the group consisting of arginine (R) at position 38 and methionine at position 48 ( M), valine (V) at position 68, or a combination thereof.
  • the light chain variable region of the antibody corresponds to a mutation at glycine (G) at position 69 in the sequence shown in SEQ ID NO.:1.
  • the antibody further includes an amino acid mutation corresponding to the sequence of the heavy chain variable region shown in SEQ ID NO :: 2, which is selected from the group consisting of arginine (R) at position 38, Methionine (M) at position 48, valine (V) at position 68, or a combination thereof.
  • the light chain variable region of the antibody corresponds to the mutation of glycine (G) at position 69 in the sequence shown in SEQ ID NO :: 1 to serine (S), and the heavy chain of the antibody may be The variable region corresponds to mutation of arginine (R) at position 38 in the sequence shown in SEQ ID NO :: 2 to lysine (K).
  • the light chain variable region sequence of the antibody is shown as SEQ ID NO .: 1 or 3; and / or the heavy chain variable region sequence of the antibody is shown as SEQ ID NO .: 2 or 4 shown.
  • the light chain variable region sequence of the antibody is shown in SEQ ID NO .: 3; and the heavy chain variable region sequence of the antibody is shown in SEQ ID NO .: 2 or 4.
  • the antibody has a light chain variable region having the sequence shown in SEQ ID NO.:3; and the antibody has a heavy chain variable region having the sequence shown in SEQ ID NO.:4.
  • the antibody is a humanized antibody.
  • the antibody specifically binds HER3.
  • the KD value (M) of the affinity of the antibody for human HER3 is 1.0E-8 to 2.0E-10.
  • the antibody is a double-chain antibody or a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a bispecific antibody.
  • the antibody is in the form of a drug conjugate.
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes a 6His tag.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • an antibody preparation is provided, and the antibody preparation includes:
  • a carrier which comprises: a buffer, sterile water, and optionally a surfactant.
  • the concentration of the antibody is 5-100 mg / mL; preferably 10-70 mg / mL, and more preferably 20-60 mg / mL.
  • the buffering agent is selected from the group consisting of a citric acid buffer system, a histidine buffer system, or a combination thereof.
  • the buffering agent is a histidine buffering system.
  • the concentration of the histidine buffer system is 0.5-20 mM, preferably 1-10 mM.
  • the citric acid buffer system contains histidine and histidine hydrochloride, and preferably contains 1-10 mM histidine and 1-10 mM histidine hydrochloride. By weight.
  • the buffering agent is a citric acid buffering system.
  • the concentration of the citric acid buffer system is 5-100 mM, preferably 10-50 mM.
  • the citrate buffer system contains sodium citrate and sodium chloride, preferably 10-50 mM sodium citrate and 50-200 mM sodium chloride, based on the total weight of the antibody preparation .
  • the surfactant is selected from the group consisting of Tween 80, Tween 20, or a combination thereof.
  • the content of the surfactant is 0.005 to 0.2% by weight, based on the total weight of the antibody preparation.
  • the surfactant is Tween 80, and the concentration is 0.02-0.1 wt%.
  • the pH range of the formulation is 5.0-7.5, preferably 5.5-7.
  • the preparation is an injection preparation.
  • the preparation includes: the antibody according to the fifth aspect of the present invention, a histidine buffer system, a surfactant, and a balance of sterile water for injection.
  • the preparation includes:
  • the antibody according to the fifth aspect of the present invention 20-60 mg / mL;
  • Histidine buffer system 1–10 mM: Histidine buffer system: 1-10 mM;
  • Tween 80 0.02-0.1wt%; Tween 80: 0.02-0.1wt%;
  • the pH range of the preparation is preferably 5.5-6.5.
  • kits containing the antibody preparation according to the seventh aspect of the present invention there is provided a kit containing the antibody preparation according to the seventh aspect of the present invention, and a container containing the antibody preparation.
  • a CAR construct is provided.
  • the scFv segment of the antigen-binding region of the CAR construct is a binding region that specifically binds to HER3, and the scFv has The light chain variable region described above and the heavy chain variable region according to the third aspect of the invention.
  • a recombinant immune cell is provided, and the immune cell expresses an exogenous CAR construct according to the ninth aspect of the present invention.
  • the immune cells are selected from the group consisting of NK cells and T cells.
  • the immune cells are from a human or non-human mammal (such as a mouse).
  • an antibody drug conjugate is provided, and the antibody drug conjugate contains:
  • a coupling moiety coupled to the antibody moiety being selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.
  • the antibody portion and the coupling portion are coupled by a chemical bond or a linker.
  • an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the present invention, and a heavy chain variable region according to the second aspect of the present invention.
  • a recombinant protein, an immune cell according to the tenth aspect of the present invention, an antibody drug conjugate according to the eleventh aspect of the present invention, or a combination thereof, the active ingredient is used for
  • the tumor is selected from the group consisting of a hematological tumor, a solid tumor, or a combination thereof.
  • the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and diffuse large B-cell lymphoma (DLBCL), Hodgkin's lymphoma, or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • Hodgkin's lymphoma or a combination thereof.
  • the solid tumor is selected from the group consisting of gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • gastric cancer gastric cancer peritoneal metastasis
  • liver cancer leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colorectal cancer, Cervical cancer, ovarian cancer, lymphoma, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, or a combination thereof.
  • NSCLC non-small
  • the tumor is a tumor that highly expresses HER3.
  • the medicine or preparation is used for preparing a medicine or preparation for preventing and / or treating a disease related to HER3 (positive expression).
  • the antibody is in the form of a drug conjugate (ADC).
  • ADC drug conjugate
  • the detection reagent or kit is used for diagnosing HER3-related diseases.
  • the detection reagent or kit is used for detecting HER3 protein in a sample.
  • the detection reagent is a detection sheet.
  • a pharmaceutical composition is provided, and the pharmaceutical composition contains:
  • an active ingredient selected from the group consisting of a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, and a third aspect of the invention
  • Light chain variable region, the light chain according to the fourth aspect of the present invention, or the antibody according to the fifth aspect of the present invention, the recombinant protein according to the sixth aspect of the present invention, or the tenth aspect of the present invention Immune cells, the antibody drug conjugate according to the eleventh aspect of the invention, or a combination thereof;
  • the pharmaceutical composition is a liquid formulation.
  • the pharmaceutical composition is an injection.
  • the pharmaceutical composition is used for treating tumors.
  • the tumor is a tumor that highly expresses HER3.
  • a fourteenth aspect of the present invention provides a polynucleotide, which encodes a polypeptide selected from the group consisting of:
  • a vector containing the polynucleotide according to the fourteenth aspect of the present invention is provided.
  • the vector includes: a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus, or other vectors.
  • a sixteenth aspect of the present invention there is provided a genetically engineered host cell containing the vector according to the fifteenth aspect of the present invention or a genome integrated with the vector according to the fourteenth aspect of the present invention.
  • a seventeenth aspect of the present invention provides a method for in vitro detection (including diagnostic or non-diagnostic) of a HER3 protein in a sample, the method comprising the steps:
  • a detection plate includes: a substrate (support plate) and a test strip, wherein the test strip contains the antibody according to the fifth aspect of the present invention, or The antibody-drug conjugate according to the eleventh aspect of the present invention.
  • a kit in a nineteenth aspect of the present invention, includes:
  • the kit contains a detection plate according to the eighteenth aspect of the present invention.
  • a method for preparing a recombinant polypeptide includes:
  • a method for a HER3-related disease comprising: administering to a subject in need the antibody according to the fifth aspect of the present invention, the antibody-drug conjugate of the antibody Or CAR-T cells expressing the antibody, or a combination thereof.
  • Figure 1 shows the HER3 mouse monoclonal antibody and human Germline template variable region sequences.
  • a is a light chain variable region
  • b is a heavy chain variable region.
  • the upper part is the sequence of the variable region of the mouse monoclonal antibody (SEQ ID No .: 5 or 6), and the lower part is the sequence of the variable region after CDR transplantation (SEQ ID No .: 9 or 10).
  • the underlined part is the CDR region and the gray part is marked. Amino acids are different from those of the mouse backbone and human Germline template backbone regions.
  • Figure 2 shows the results of the solubility test using the PEG precipitation method.
  • the arrows indicate the wells where precipitation started.
  • Figure 3 shows the endogenous fluorescence detection pattern of the humanized antibody of the present invention.
  • Fig. 4 shows the tumor suppressor effect of the humanized antibody of the present invention in animals (A549).
  • Fig. 5 shows the inhibitory effect of the humanized antibody hu1044-7 of the present invention on tumors (Fadu) in animals.
  • Fig. 6 shows the inhibitory effect of the humanized antibody hu1044-7 of the present invention on tumors (BT-474) in animals.
  • the inventors have unexpectedly obtained an anti-HER3 humanized antibody with excellent affinity and good structural stability.
  • a humanized framework region taking into account the similarity and the frequency of human use, the framework regions of two sequences of IGKV3-20 * 01 and IGHV1-69 * 01 were selected for humanization, and the obtained human Mutation screening of sourced antibodies.
  • Humanized antibodies, especially mutated humanized antibodies have similar affinity to chimeric antibodies.
  • Preliminary studies on the solubility and endogenous fluorescence of humanized antibodies have confirmed that the humanized has a preliminary It is drug-forming, and humanized monoclonal antibody drugs that are further developed into targeted therapy will be developed in the future.
  • a preliminary drugability study was performed on the humanized antibody, and the formulation of the anti-HER3 humanized antibody was screened and determined. The present invention has been completed on this basis.
  • amino acid three-letter codes and one-letter codes used in the present invention are described in J. biol. Chem, 243, p3558 (1968).
  • administering refers to the application of an exogenous drug, therapeutic agent, diagnostic agent, or composition to an animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering may refer to treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes the contact of reagents with cells, the contact of reagents with fluids, and the contact of fluids with cells.
  • administering also mean in vitro and ex vivo treatment by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, animal, or research subject refers to the treatment, prevention, or prophylactic measures, research, and diagnosis; including anti-HER3 antibodies and human or animal, subject, cell, tissue, and physiological regions Chamber or contact with physiological fluids.
  • treatment refers to the administration of an internal or external therapeutic agent to a patient, comprising any one of the anti-HER3 antibodies of the present invention and a composition thereof, said patient having one or more symptoms of the disease, and said treatment is known
  • the agent has a therapeutic effect on these symptoms.
  • a patient is administered in an amount (a therapeutically effective amount) of a therapeutic agent effective to alleviate the symptoms of one or more diseases.
  • the term “optionally” or “optionally” means that an event or situation described subsequently can occur but does not have to occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be, but not necessarily, one, two, or three.
  • sequence identity indicates the degree of identity between two nucleic acids or two amino acid sequences when there is optimal alignment and comparison in the case of mutations such as appropriate substitutions, insertions, or deletions.
  • sequence identity between the sequence described in the present invention and the sequence having the same identity may be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 100%.
  • Human epidermal growth factor 3 (human epidermal growth factor, receptor 3, HER3) plays an important role in the occurrence and progression of tumors.
  • HER3 usually forms heterodimers with EGFR or HER2 molecules and plays a role.
  • EGFR and HER2 overexpression is often accompanied by HER3 overexpression, and HER3 overexpression is considered to be resistant to EGFR and HER2 targeted therapy Play an important role.
  • HRG Heregulin
  • the conformation changes, exposing the binding site to EGFR and HER2, and then forming heterodimers with EGFR and HER2 molecules, activating intracellular signals and promoting tumor cell proliferation.
  • Anti-HER3 murine monoclonal antibody 1044 (application number 2014104015453) can block the binding of HER3 molecule to its ligand HRG, thereby inhibiting it from forming heterodimers with other molecules of the HER family. Inhibiting the proliferation of human epidermal squamous cell carcinoma A431 cells has the potential to treat a variety of HER3 overexpressing tumors.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by an interchain disulfide bond.
  • the amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , Alpha, and epsilon chains.
  • Igs of the same type can be divided into different subclasses according to the difference in amino acid composition and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain according to the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.
  • the light chain of the antibody of the present invention may further comprise a light chain constant region, and the light chain constant region comprises a human or murine ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the present invention may further include a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of 3 CDR regions and 4 FR regions.
  • the sequence from amino terminal to end is: FR1, CDR1, FR2 , CDR2, FR3, CDR3 and FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • mouse antibody in the present invention is a monoclonal antibody against HER3 prepared according to the knowledge and skill in the art. Test subjects are injected with the HER3 antigen during preparation, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the mouse-derived HER3 antibody or antigen-binding fragment thereof may further include a light chain constant region of a mouse-derived ⁇ , ⁇ chain or a variant thereof, or further include a mouse-derived IgG1, IgG2 , IgG3 or a variant of the heavy chain constant region thereof.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can alleviate the immune response response induced by the murine antibody.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of murine CDR sequences into the human variable region framework, that is, different types of human germline antibodies Antibodies produced in framework sequences. Humanized antibodies can overcome the heterogeneous response induced by chimeric antibodies because they carry a large amount of murine protein components. Such framework sequences can be obtained from a public DNA database including germline antibody gene sequences or published references. In order to avoid the decrease in activity caused by the decrease in immunogenicity, the human antibody variable region framework sequence may be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • antigen-binding fragment of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, HER3). It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of the antibody. Examples of binding fragments included in the term "antigen-binding fragment of an antibody” include
  • Fab fragment a monovalent fragment consisting of VL, VH, CL, and CH1 domains
  • the Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but has no constant region, and has the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also include a polypeptide linker between the VH and VL domains, and can form the structure required for antigen binding.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A et al. (1991) Sequences of protein of immunological interest. NIH Publication 91-3242).
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on the HER3 molecule).
  • An epitope usually includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation.
  • the terms “specifically bind”, “selectively bind”, “selectively bind” and “specifically bind” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is less than about 10 -7 M, such as about less than 1O -8 M, 1O -9 M or lO -10 M or smaller affinity (KD) binding.
  • competitive binding refers to an antibody that recognizes the same epitope (also called an epitope) or a portion of the same epitope on the extracellular region of HER3 as the monoclonal antibody of the invention and binds to the antigen.
  • An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of HER3 recognized by the monoclonal antibody of the present invention.
  • KD dissociation equilibrium constant of a particular antibody-antigen interaction.
  • the antibodies of the present invention is less than about 10 -7 M, such as less than about 1O -8 M, 1O -9 M or lO -10 M or less, a dissociation equilibrium constant (KD) bind HER3.
  • antigenic determinant refers to a three-dimensional spatial site on an antigen that is discontinuous and is recognized by an antibody or antigen-binding fragment of the invention.
  • the present invention includes not only intact antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed between antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives, and analogs of the antibodies.
  • antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art.
  • Recombinant antibodies such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombinant techniques well known in the art.
  • the term "monoclonal antibody” refers to an antibody secreted from a clone derived from a single cell. Monoclonal antibodies are highly specific, being directed against a single epitope.
  • the cells may be eukaryotic, prokaryotic or phage cloned cell lines.
  • the antibody may be monospecific, bispecific, trispecific, or more multispecific.
  • the antibodies of the present invention also include conservative variants thereof, which means that there are at most 10, preferably at most 8 and more preferably at most 5 compared with the amino acid sequence of the antibody of the present invention Up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • conservatively mutated polypeptides are preferably produced by amino acid substitutions according to Table A.
  • the present invention provides an anti-HER3 humanized antibody (hereinafter referred to as HER3 antibody). Specifically, the present invention provides a highly specific and high-affinity humanized antibody against HER3, which includes a heavy chain and a light chain, the heavy chain containing a heavy chain variable region (VH) amino acid sequence, the light The chain contains the light chain variable region (VL) amino acid sequence.
  • HER3 antibody anti-HER3 humanized antibody
  • the present invention provides a highly specific and high-affinity humanized antibody against HER3, which includes a heavy chain and a light chain, the heavy chain containing a heavy chain variable region (VH) amino acid sequence, the light The chain contains the light chain variable region (VL) amino acid sequence.
  • human backbone regions in antibody humanization There are usually two choices of human backbone regions in antibody humanization, one is a known mature antibody, and the other is a human Germline sequence.
  • Known mature antibody backbone regions often contain somatic mutation sites, which may bring potential immunogenicity.
  • the backbone region of human Germline sequence is theoretically less immunogenic, and the structure is more flexible, plastic, and easily accepts different CDR regions.
  • the use frequency of human antibody Germline gene in the human body has a certain bias. Selecting humanized antibodies with a high use frequency of the Germline skeleton region has the advantages of low immunogenicity, high expression, and stable structure.
  • the present invention The Germline sequence with the highest similarity to the mouse-derived antibody was not selected when it was sourced, but the similarity and the frequency of human use were taken into consideration. After a large number of experiments, the IGKV3-20 * 01 and IGHV1-69 * 01 sequences were selected District is humanized. In the present invention, the human antibody Germline backbone region is used for CDR transplantation. The humanized antibody constructed in this way has more stable structure, high expression, low immunogenicity, and higher drugability.
  • VL1, VL4 sites retain mouse monoclonal antibody amino acids
  • VH74 site retains mouse monoclonal antibody amino acid
  • VL1, VL4, VL69 sites retain murine monoclonal antibody amino acids
  • VH38 Humanized antibody heavy chain variable region (VH38, VH74 sites retain mouse monoclonal antibody amino acids) (SEQ ID NO.:4)
  • the constant region of the humanized antibody is the same as the constant region of anti-HER3 murine monoclonal antibody 1044 (application number 2014104015453).
  • the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence preferably has a homology of at least 80%, preferably at least 85%, more preferably at least 90 %, Optimally at least 95% of the amino acid sequence.
  • the antibody of the present invention may be a double-chain or single-chain antibody, and may preferably be a fully humanized antibody.
  • the antibody derivatives of the present invention may be single-chain antibodies and / or antibody fragments, such as: Fab, Fab ', (Fab') 2 or other known antibody derivatives in the field, and IgA, IgD, Any one or more of IgE, IgG, and IgM antibodies or antibodies of other subtypes.
  • the antibodies of the invention may be humanized antibodies, CDR grafted and / or modified antibodies that target HER3.
  • the number of amino acids added, deleted, modified, and / or substituted is preferably not more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably not more than 35%, and more preferably 1-33%. , More preferably 5-30%, more preferably 10-25%, and still more preferably 15-20%.
  • the present invention successfully humanized the HER3 mouse monoclonal antibody, and the humanized antibody reached a affinity close to that of the chimeric antibody.
  • the preliminary study of the solubility and endogenous fluorescence of the humanized antibody confirmed that the human source Chemicals have a preliminary drug-making ability, and in the future, there will be further development of humanized monoclonal antibody drugs for targeted therapy.
  • any method suitable for producing monoclonal antibodies can be used to produce the HER3 antibody of the present invention.
  • animals can be immunized with a linked or naturally occurring HER3 protein or a fragment thereof.
  • Appropriate immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used.
  • HER3 can be used as an immunogen (antigen) for generating non-human antibodies specific to HER3 and screening for the biological activity of said antibodies.
  • the immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. Immunogens can be purified from natural sources or produced in genetically modified cells.
  • the DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in origin. DNA encoding the immunogen can be expressed using suitable genetic vectors, including, but not limited to, adenoviral vectors, baculovirus vectors, plasmids, and non-viral vectors.
  • the humanized antibody may be selected from any kind of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE.
  • immunoglobulin including IgM, IgD, IgG, IgA, and IgE.
  • any type of light chain can be used in the compounds and methods herein.
  • kappa, lambda chains or variants thereof are useful in the compounds and methods of the invention.
  • Example 1 An exemplary method for humanizing the HER3 antibody of the invention is described in Example 1.
  • sequence of the DNA molecule of the antibody or the fragment of the present invention can be obtained by conventional techniques, such as using PCR amplification or genomic library screening.
  • the coding sequences of the light and heavy chains can also be fused together to form a single chain antibody.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually cloned into a vector, and then transferred into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.
  • synthetic methods can also be used to synthesize related sequences, especially when the fragment length is short.
  • long fragments can be obtained by synthesizing multiple small fragments first and then ligating them.
  • This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art.
  • nucleic acid molecule refers to both DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operatively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which an additional DNA segment can be ligated.
  • the invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the term "host cell” refers to a cell into which an expression vector has been introduced.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant or animal cell (such as a mammalian cell).
  • the steps of transforming host cells with recombinant DNA described in the present invention can be performed using techniques well known in the art.
  • the obtained transformant can be cultured by a conventional method, and the transformant expresses a polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultured under appropriate conditions using a conventional medium.
  • the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. Then use conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
  • immunoglobulin purification steps such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography.
  • the antibodies of the present invention can be purified by conventional separation and purification means well known to those skilled in the art.
  • the obtained monoclonal antibodies can be identified by conventional means.
  • the binding specificity of a monoclonal antibody can be determined by immunoprecipitation or an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies have different stability in different formulation buffers, manifested by changes in charge heterogeneity, degradation and polymerization of antibody molecules, etc. These changes in qualitative properties are related to the physicochemical properties of the antibody itself. Therefore, in the development of antibody drugs In the process, it is necessary to select a suitable preparation buffer according to the physicochemical properties of different antibodies.
  • commonly used antibody preparation buffer systems include phosphate buffer, citrate buffer, histidine buffer, etc.
  • salt ions or excipients such as sorbitol, trehalose, and sucrose are added according to the nature of the antibody, and An appropriate amount of surfactant such as Tween 20 or Tween 80 to maintain the stability of the antibody.
  • the antibody preparation of the present invention is as described in the seventh aspect of the present invention.
  • the antibody-medicine combination preparation of the present invention can effectively inhibit the side reactions such as aggregation, precipitation, hydrolysis, oxidation, and deamidation of the humanized antibody of the present invention, and can effectively improve the product under pressure (high temperature, strong light irradiation, freeze-thaw, etc.) Stability under accelerated and long-term refrigeration conditions.
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or an active fragment thereof or a fusion protein thereof or an ADC or a corresponding CAR-T cell, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, and preferably about 6-8, although the pH may vary with The nature of the formulation and the condition to be treated will vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.
  • the antibody of the present invention may also be used for cell therapy by expressing a nucleotide sequence in a cell, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) and the like.
  • CAR-T chimeric antigen receptor T cell immunotherapy
  • the pharmaceutical composition of the present invention can be directly used to bind the HER3 protein molecule, and thus can be used to prevent and treat HER3-related diseases.
  • other therapeutic agents can be used simultaneously.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99% by weight, preferably 0.01-90% by weight, more preferably 0.1-80% by weight) of the above-mentioned monoclonal antibody (or a conjugate thereof) of the present invention and a pharmacy Acceptable carrier or excipient.
  • a safe and effective amount such as 0.001-99% by weight, preferably 0.01-90% by weight, more preferably 0.1-80% by weight
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1
  • a safe and effective amount of the pharmaceutical composition is administered to a mammal, wherein the safe and effective amount is usually at least about 10 ⁇ g / kg body weight, and in most cases does not exceed about 50 mg / kg body weight, compared with Preferably the dose is from about 10 micrograms / kg body weight to about 20 mg / kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health, and other factors, which are all within the skill of a skilled physician.
  • the antibodies of the invention can be used in detection applications, such as for detecting samples, thereby providing diagnostic information.
  • the samples (samples) used include cells, tissue samples, and biopsy specimens.
  • biopsy as used in the present invention shall include all kinds of biopsies known to those skilled in the art.
  • the biopsy used in the present invention may therefore include, for example, a tissue sample prepared by an endoscopic method or a puncture or needle biopsy of an organ.
  • Samples used in the present invention include fixed or preserved cell or tissue samples.
  • the present invention also provides a kit containing the antibody (or a fragment thereof) of the present invention.
  • the kit further includes a container, an instruction manual, a buffer, and the like.
  • the antibody of the present invention can be immobilized on a detection plate.
  • the humanized antibody of the present invention has a typical structure with good tryptophan and tyrosine embedding.
  • the screened human VH and VL framework regions have a stable template structure and are well adapted to the CDR region of a mouse monoclonal antibody.
  • the light and heavy chain variable regions can be paired together well, with high affinity and stable structure.
  • the humanized antibody of the present invention has excellent biological activity and specificity, and while retaining the affinity comparable to HER3, it has lower immunogenicity and higher expression level.
  • the humanized antibody of the present invention has high solubility and good drug-forming properties.
  • the humanized antibody of the present invention has obvious characteristics of inhibiting tumor growth in the A549 tumor model, and the tumor growth inhibitory activity is related to the therapeutic dose, which proves that the humanized anti-HER3 monoclonal antibody still has good Biological activity.
  • the present invention takes into account the similarity and the frequency of human use when selecting the humanized backbone region.
  • the humanized antibodies have lower immunogenicity and higher expression (especially in mammalian cells, Such as in CHO-K1 cells), the structure is more stable, and the drugability is better.
  • the humanized antibody of the present invention is used to construct a stable expression cell line in CHO-K1 cells.
  • the selected stable cell line is subjected to Fed Batch culture in a shake flask, and the antibody expression level reaches 4-5 g / L.
  • E.coli DH5 ⁇ competent cells were prepared by our laboratory; FreeStyle 293-F cells and expression medium were purchased from Gibco; pSGHV0 eukaryotic expression plasmid was donated by Dr. DJ Leahy, Department of Biophysics, Johns Hopkins University School of Medicine; Babl / c nude mice were purchased from Shanghai Slark Experimental Animal Co., Ltd.
  • Site-directed mutagenesis kits were purchased from Beijing Cybex Gene Technology Co., Ltd .; MabselectSure affinity media were purchased from GE; rituximab (batch number H0709) and trastuzumab (batch number N3674) were purchased from Shanghai Roche Pharmaceutical Co., Ltd.
  • HER3 extracellular domain recombinant protein was recombinantly expressed by CHO cells; goat anti-human antibodies were purchased from Jackson ImmunoResearch; Polyethylenimine (PEIMW 25000) was purchased from Polysciences; polyethylene glycol 8000 (PEG 8000) was purchased from Sigma-Aldrich SDS-MW Analysis Kit was purchased from Beckman Coulter Company; OPM-CHO CD07, PFF05, and CDF16 medium were purchased from Shanghai Opima Biotechnology Co., Ltd .; 1044 mouse monoclonal antibody was the antibody 1044 in 2014104015453.
  • PEIMW 25000 Polyethylenimine
  • PEG 8000 polyethylene glycol 8000
  • Sigma-Aldrich SDS-MW Analysis Kit was purchased from Beckman Coulter Company
  • OPM-CHO CD07, PFF05, and CDF16 medium were purchased from Shanghai Opima Biotechnology Co., Ltd .
  • 1044 mouse monoclonal antibody was the antibody 1044 in 2014104015453.
  • the humanized variable region gene sequence was synthesized by Shanghai Boshang Biotechnology Co., Ltd., and then the heavy chain and light chain variable region genes were constructed to contain the human IgG1 heavy chain, respectively. Constant region and kappa chain constant region on the pSGHV0 vector. Site-directed mutagenesis kit was used to backmutate the humanized antibody backbone regions VH38, VH48, VH68, VL69 and confirmed by sequencing. 1044 human-mouse chimeric antibodies and each back-mutated humanized antibody were transiently transfected with PEI in 293F cells.
  • the transiently transfected cell culture supernatant was centrifuged at 2000 rpm and the affinity was measured using ProteOn XPR36.
  • the final humanized antibody sequence was selected by Shanghai Opima Biotechnology Co., Ltd. to construct a CHO-K1 stable cell line.
  • the stable cell line was used to express the humanized antibody hu1044.
  • the selected stable cell line was subjected to Fed Batch culture in shake flasks. Stable cell lines were inoculated into culture flasks at a cell density of 5 ⁇ 10 5 / ml.
  • the basic medium was OPM-CHO CD07.
  • the culture volume was supplemented by 3%, 5%, 7%, 5%, 3% of PFF05 feed medium, and culture day 5, 7, 9, and 11 supplemented with 0.3% of the culture volume of CDF16 feed medium, and maintained the glucose content at 2-6g / L.
  • the viability of the cells is lower than 60% or after 15 days of culture, the culture is terminated, and the antibody expression level reaches 4-5g / L. Then, it was purified by Mabselect Sure Protein A affinity medium to obtain humanized antibody, and the purified antibody was concentrated to 60 mg / mL.
  • the GLM chip labeled 6 goat anti-human antibodies in the horizontal direction rotated the channel by 90 degrees to capture the chimeric or humanized antibody expressed by 293F cells in the vertical direction, and the channel was turned to the horizontal direction.
  • Channels 1-5 were injected with 20nM, 10nM, 5nM, 2.5nM, 1.25nM HER3 extracellular domain protein, channel 6 injection buffer, to obtain 5 kinetic response curves, respectively representing the reaction results of different concentrations of antigen and capture antibodies, using Langmuir model to calculate the embedding Affinity constant KD (M) of the binding antibody and each humanized antibody mutant.
  • FB1 solution 90g sodium chloride, 73.5g trisodium citrate, 7g polysorbate 80 in 1L water for injection, pH adjusted to 6.5
  • FB2 buffer 0.3g histidine, 0.5g histidine hydrochloride 0.08 g of polysorbate 20 and 18.9 g of trehalose were dissolved in 1 L of water for injection and the pH was adjusted to 6.0).
  • Deionized water was used to configure two 40% PEG 8000 stock solutions, the pH was adjusted to 6.5 and 6.0, and then the 40% PEG 8000 stock solutions were diluted to different concentrations with FB1 and FB2, respectively.
  • HER3 humanized antibody 60 mg / mL of HER3 humanized antibody was replaced by ultrafiltration into FB1 and FB1 buffers with ultrafiltration, the replacement volume was 100 times, and the final concentration was 20 mg / mL. Then dilute rituximab, trastuzumab, HER3 humanized monoclonal antibody (FB1), and HER3 humanized monoclonal antibody (FB1) to their respective buffers to 10 mg / mL, and take 100 ⁇ L of each antibody. Mix 100 ⁇ L of PEG (8000, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%) with different concentrations in a 96-well plate, and observe the result after 10 minutes of reaction.
  • PEG 8000, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%
  • CE-SDS Non-reducing capillary electrophoresis detection
  • Mobile phase A 10 mM sodium phosphate solution, pH 7.4;
  • Mobile phase B 10 mM sodium phosphate solution, 0.25 M sodium chloride, pH 7.4;
  • Chromatographic column Dipac Propac WCX-10 (4.0 ⁇ 250mm);
  • Detector UV Detector, wavelength 280nm; column temperature: 35 ° C; injection volume: 40 ⁇ g; flow rate 0.5mL / min; separation gradient: mobile phase B increased from 0% to 30% in 27min; collection time: 41min.
  • Mobile phase 0.2M sodium phosphate buffer, pH 6.8; Column: TOSOH Bioscience Super SW3000 (4.6 ⁇ 300mm, 4 ⁇ m); Detector: UV detector, wavelength 280nm; Flow rate: 0.35mL / min; Column temperature: 30 °C; injection volume: 30 ⁇ g; collection time: 20 min.
  • the samples were diluted to 1.0 mg / mL with their respective buffers, and added to a cuvette for detection on SpectraMax M5.
  • the excitation wavelength was 295 nm
  • the emission wavelength was scanned from 310 nm to 450 nm
  • the scanning step was 1 nm.
  • citrate buffer containing 25 mM sodium citrate, 140 mM sodium chloride, 0.07% Tween-80, pH 6.5
  • phosphate buffer containing 50 mM phosphate, 140 mM sodium chloride, 0.07 % Tween-80, pH 8.0
  • histidine buffer containing 2 mM histidine, 2 mM histidine hydrochloride, 1.5% trehalose, 0.07% Tween-80, pH 6.0
  • humanized purified The antibodies were replaced with the above three buffers, the final concentration of the antibody was 40mg / mL, and the charge heterogeneous changes were detected by IEX-HPLC method at -70 ° C, 2-8 ° C and 37 ° C for one week.
  • citrate buffer containing 25 mM sodium citrate, 140 mM sodium chloride, 0.07% Tween-80, pH 6.5
  • histidine buffer containing 2 mM histidine, 2 mM histamine hydrochloride
  • Acid 0.07% Tween-80, pH 6.0
  • histidine buffer (containing 2mM histidine, 2mM histidine hydrochloride, 0.07% Tween-80, pH 6.0), add 25, 50, 75, 125, 150mM sodium chloride, and then humanize
  • the antibody was prepared into the above buffers with different salt ions, and the final concentration of the antibody was 40 mg / mL. The effect of salt ions on the turbidity of the antibody was observed.
  • Histidine buffer (containing 2 mM histidine, 2 mM histidine hydrochloride, 0.07% Tween-80, pH 6.0) buffer was used to prepare humanized antibodies to 40 mg / mL, and left at 37 ° C for two weeks.
  • HPLC method, SEC-HPLC method and non-reducing CE-SDS method were used to detect the quality change.
  • human IGKV3-20 * 01 amino acid sequence was selected as the light chain humanized template
  • human IGHV1-69 * 01 amino acid sequence was selected as the heavy chain humanized template.
  • the mouse CDR1104 light and heavy chain CDR regions were transplanted into the human template backbone region.
  • the human J ⁇ 1 sequence replaced the mouse monoclonal antibody J ⁇ region and the human JH4 sequence replaced the mouse monoclonal antibody JH region.
  • the sequence comparison of the mouse monoclonal antibody and human Germline backbone region is shown in Figure 1.
  • sequences of the light chain variable region and the heavy chain variable region of murine mAb 1044 are shown in SEQ ID NOs: 5 and 6, respectively.
  • VL1, VL4, and VH74 retained the amino acid of the original mouse monoclonal antibody to obtain a humanized antibody h1044-0.
  • the light chain variable region and heavy The sequences of the chain variable regions are shown in SEQ ID NOs: 1 and 2, respectively.
  • VL1, VL4 sites retain mouse monoclonal antibody amino acids
  • VH74 site retains mouse monoclonal antibody amino acid
  • the humanized antibody h1044-0 was mutated.
  • the VL69 reverse mutation corresponds to the mutation of Gly to Ser at position 69 in SEQ ID NO.:1, and the affinity is significantly improved, which is about 2 times that of the chimeric antibody.
  • VH38, VH48, or VH68 were mutated (VH38 was mutated from Arg to Lys, VH48 was mutated from Met to Ile, and VH68 was mutated from Val to Ala).
  • the affinity of the humanized antibody was significantly improved, which was basically close to the chimeric antibody, and was restored to less than 2 times the affinity constant of the chimeric antibody.
  • the mutated sequences of VL69 and VH38 met the requirements of humanization.
  • VL63 / 69 back mutants VL63 from Ile to Val, VL69 from Gly to Ser
  • VH38 VH38 from Arg to Lys
  • VH68 VH68 from Val to Ala
  • VH67 / 68 mutant VH67 back-mutated from Arg to Lys, VH68 back-mutated from Val to Ala
  • the VL69 and VH38 back-mutated sequences are used as the final humanized antibody hu1044 (the light chain variable region and heavy chain variable region sequences are shown in SEQ ID NO .: 3 and 4 respectively).
  • VL69 back mutation corresponds to SEQ ID No .: 1 in which the VL69 position is mutated from Gly to Ser (mutated sequence is SEQ ID No .: 3).
  • VL63 / VL69 back mutation is in SEQ ID No .: 1, VL63 is back-mutated from Ile to Val, and VL69 is back-mutated from Gly to Ser (mutated sequence is SEQ ID No .: 7).
  • VH38 back mutation is in SEQ ID No.:2, the VH38 position is back-mutated from Arg to Lys (mutated sequence is SEQ ID No.:4).
  • VH48 back mutation is in SEQ ID No .: 2, the VH48 position is mutated from Met to Ile.
  • VH68 back mutation is in SEQ ID No .: 2, the VH68 position is back-mutated from Val to Ala.
  • VH67 / VH68 backmutation is in SEQ ID No .: 2, VH67 is back-mutated from Arg to Lys, and VH68 is back-mutated from Val to Ala (SEQ ID No .: 8).
  • the dosage of antibody drugs in the human body is relatively large, and it is possible that the antibody molecules have a high degree of solubility to have a certain drug-making property.
  • Solubility is not only related to the physicochemical properties of the antibody, such as isoelectric point, surface hydrophobicity, binding force between light and heavy chains, etc., but also to the buffer system in which the antibody is located.
  • the PEG precipitation method can be used to detect the solubility of the protein.
  • VL69, VH38 back-mutated humanized antibody hu1044 was recombinantly expressed in CHO-K1 cells, and after proteinA affinity chromatography, ultrafiltration was substituted into different buffer systems.
  • FB1 is a rituximab preparation buffer
  • FB2 is a trastuzumab buffer.
  • the PEG precipitation method showed that in FB1 buffer, the humanized antibody hu1044 began to precipitate under the condition of 5% PEG, and in FB2 buffer, the humanized antibody hu1044 began to precipitate under the condition of 10% PEG, as shown in FIG. 2.
  • HER3 humanized antibody hu1044 had significantly better solubility in FB2 buffer.
  • HER3 humanized antibody hu1044 has better solubility in FB2 buffer than rituximab control and lower solubility than trastuzumab control.
  • the HER3 humanized antibody hu1044 was significantly reduced in purity and polymer increased after ultrafiltration replacement in FB1 buffer, and the purity did not change significantly after ultrafiltration replacement in FB2 buffer. It was further confirmed in FB2 HER3 humanized antibody hu1044 has better solubility, see Table 3.
  • the HER3 humanized antibody hu1044 showed different solubility in different buffer systems.
  • the solubility in FB2 buffer was better than FB1, and better than rituximab.
  • Tryptophan and tyrosine in protein molecules emit fluorescence under appropriate excitation light, especially tryptophan has strong autofluorescence. Tryptophan and tyrosine are relatively sensitive to changes in the microenvironment of the protein. When tryptophan and tyrosine are exposed to the protein surface from the hydrophobic center of the protein, the red shift of the fluorescence spectrum occurs. Detection of protein endogenous fluorescence can determine the stability of the protein structure. The maximum emission wavelength ⁇ max of the endogenous fluorescence of the antibody can reflect the spatial distribution of tryptophan and tyrosine in the antibody molecule. Tryptophan and tyrosine at the positions of VL35W, VL86Y, VH36W, VH90Y, and VH103W in the backbone region of the antibody molecule are very conserved and embedded in the hydrophobic center.
  • HER3 humanized antibody hu1044 ⁇ max is similar to trastuzumab, which is a typical structure with good tryptophan and tyrosine embedment. It fits well to the CDR regions of the mouse monoclonal antibody, and the light and heavy chain variable regions can be paired together well.
  • the humanized antibody hu1044 significantly inhibited the growth of A549 tumors, and the inhibitory effect was related to the therapeutic dose (see Figure 4).
  • the highest inhibitory rate was 30mg / kg, and the tumor growth inhibitory rate was 63%, confirming that the humanized antibody still has good biological activity and has the potential to treat tumors in the body.
  • Human Pharyngeal Squamous Carcinoma Fadu cells were cultured to logarithmic growth phase, and cells were collected after trypsin digestion and counted. Nude mice were subcutaneously inoculated with 5 ⁇ 10 6 human pharyngeal squamous cell carcinoma Fadu cells. After the average tumor volume grew to 100 mm 3 , the animals were grouped according to tumor volume (D0), with 6 animals in each group and 12 in the solvent control group. Mice were injected with HER3 humanized antibody (hu1044-7) intravenously, twice a week for a total of 4 times; the administration volume was 10 mL / kg. HER3 humanized antibody was administered at doses of 0.2, 0.6, and 2 mg / kg.
  • Erbitux (Erbitux, manufactured by Merck, monoclonal antibody for first-line treatment of recurrent metastatic head and neck squamous cell carcinoma) was administered at a dose of 0.5 mg / kg.
  • the solvent group was given the same volume of saline.
  • the tumor volume was measured twice a week, the weight of the mice was weighed, and the data was recorded.
  • Nude tumor volume: V 0.5 ⁇ a ⁇ b 2 , where a is the long diameter of the tumor entity, and b is the short diameter of the tumor entity.
  • Human breast cancer BT-474 cells were cultured to the logarithmic growth phase, and cells were collected after trypsin digestion and counted. Nude mice were subcutaneously inoculated with 8 ⁇ 10 6 human breast cancer BT-474 cells. After the average tumor volume grew to 100-150 mm 3 , the animals were grouped according to tumor volume (D0). There were 6 mice in each group and a solvent control group. Twelve. Mice were injected with HER3 humanized antibody (hu1044-7) intravenously 2 times a week for a total of 6 times; the administration volume was 10 mL / kg. HER3 humanized antibody was administered at doses of 2, 6, and 20 mg / kg.
  • Herceptin Herceptin, manufactured by Roche, monoclonal antibody for first-line treatment of Her2-positive breast cancer
  • the solvent group was given the same volume of saline.
  • the tumor volume was measured twice a week, the weight of the mice was weighed, and the data was recorded.
  • Nude tumor volume: V 0.5 ⁇ a ⁇ b 2 , where a is the long diameter of the tumor entity, and b is the short diameter of the tumor entity.
  • the humanized antibody hu1044 was relatively stable regardless of whether it was placed at 37 ° C or 2-8 ° C for one week, but it was stable in phosphate In the buffer solution, the charge isomers significantly changed after being placed at 37 ° C and 2-8 ° C for one week (see Table 4), indicating that the antibody is relatively stable in 25mM citrate buffer and 2mM histidine buffer.
  • the opalescence of the humanized antibody hu1044 increased significantly, and the higher the salt concentration, the more obvious the opalescence, indicating that the humanized antibody does not contain More stable with salt or low salt.
  • the humanized antibody hu1044 in histidine buffer was subjected to an accelerated stability test at 37 ° C for two weeks, and the quality changes of IEX-HPLC, SEC-HPLC, and non-reducing CE-SDS were detected.
  • the results showed that the SEC-HPLC purity slowly decreased with time, but at 14 days, the SEC purity was still above 98%, the acid peak of IEX-HPLC increased slightly, the basic peak remained almost unchanged, and the non-reduced CE-SDS showed a slight initial Reduced, see Table 6.
  • the humanized antibody hu1044 showed different stability in different buffers, and the charge heterogeneity of the antibody was easily changed in the phosphate buffer system, and was relatively stable in citrate buffer and histidine buffer.
  • the increase in salt ions significantly increased the opalescence of the humanized antibody, confirming that the antibody was not suitable for use in higher saline buffers.
  • Excipients such as trehalose and sorbitol have no significant effect on the stability of the antibody.
  • the above screening results confirmed that the quality of the humanized antibody was the most stable in 2 mM histidine buffer (containing 0.07% Tween-80, pH 6.0).

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Abstract

提供了一种抗HER3人源化单克隆抗体，所述抗体结合HER3抗原，具有较高的亲和力和生物活性，以及低的免疫原性，结构稳定，可用于制备预防或治疗HER3相关的疾病的药物。

Description

抗HER3人源化单克隆抗体及其制剂 技术领域
本发明涉及医药领域，具体地涉及抗HER3人源化单克隆抗体及其制剂。
背景技术
越来越多的研究发现人表皮生长因子3(human epidermal growth factor receptor 3，HER3)在肿瘤的发生、进展等方面发生着重要的作用。HER3通常与EGFR或HER2分子形成异二聚体而发挥作用，在癌症病理组织，EGFR、HER2过表达常常伴随着HER3的过表达，并且认为HER3过表达对EGFR、HER2靶向治疗产生耐药性起到重要作用。HER3与配体Heregulin(HRG)结合后构象发生变化，暴露出与EGFR、HER2结合的位点，进而与EGFR、HER2分子形成异二聚体，激活细胞内信号，促进肿瘤细胞增殖。
由于鼠单抗在临床治疗时会引起人抗鼠抗体反应(human anti-mouse antibody，HAMA)，因此在临床治疗上受到限制。抗体人源化技术可以很大程度的降低鼠单抗的免疫原性。
因此，鉴于HER3在各类相关疾病中作用和功能，本领域仍然需要开发适于治疗患者的抗HER3人源化抗体。
发明内容
本发明目的是提供了一种高亲和力高生物活性的HER3人源化抗体及其应用。
本发明的第一方面，提供了一种抗体的轻链可变区，所述轻链可变区选自下组：
(1)序列如SEQ ID NO.:1所示的轻链可变区；或
(2)将SEQ ID NO.:1所示的氨基酸序列经过至少一个(如1-20个、较佳地1-15个、更佳地1-10个、更佳地1-8个、更佳地1-3个、最佳地1或2个)氨基酸残基的取代、缺失、修饰和/或添加而形成的，具有(1)所述轻链可变区功能的由SEQ ID NO.:1所示序列衍生的轻链可变区。
在另一优选例中，所述的氨基酸残基的取代、缺失、修饰和/或添加不发生CDR区，而发生框架区。
在另一优选例中，所述轻链可变区对应于SEQ ID NO.:1所示序列中第69位甘氨酸(G)发生突变。
在另一优选例中，所述轻链可变区对应于SEQ ID NO.:1所示序列中第69位甘氨酸(G)突变为丝氨酸(S)(即G69S)。
在另一优选例中，所述轻链可变区对应于SEQ ID NO.:1所示序列中第63位异亮氨酸(I)发生突变。
在另一优选例中，所述轻链可变区对应于SEQ ID NO.:1所示序列中第63位Ile突变为Val(即Ile→Val)。
在另一优选例中，所述轻链可变区序列如SEQ ID NO.:1或3或7所示。
本发明的第二方面，提供了一种抗体的轻链，所述的轻链具有如本发明第一方面所述的轻链可变区。
在另一优选例中，所述抗体的轻链还包括重链恒定区。
在另一优选例中，所述的轻链恒定区为人源、鼠源或兔源的，较佳地为人源的。
本发明的第三方面，提供了一种抗体的重链可变区，所述重链可变区选自下组：
(i)序列如SEQ ID NO.:2所示的重链可变区；或
(ii)将SEQ ID NO.:2所示的氨基酸序列经过至少一个(如1-20个、较佳地1-15个、更佳地1-10个、更佳地1-8个、更佳地1-3个、最佳地1或2个)氨基酸残基的取代、缺失、修饰和/或添加而形成的，具有(i)所述重链可变区功能的由SEQ ID NO.:2所示序列衍生的重链可变区。
在另一优选例中，所述的氨基酸残基的取代、缺失、修饰和/或添加不发生CDR区，而发生框架区。
在另一优选例中，所述重链可变区对应于SEQ ID NO.:2所示序列存在选自下组的氨基酸突变：第38位精氨酸(R)、第48位甲硫氨酸(M)、第67位精氨酸(R)、第68位缬氨酸(V)、或其组合。
在另一优选例中，所述重链可变区对应于SEQ ID NO.:2所示序列的突变选自下组：R38K、M48I、V68A、R67K、或其组合；较佳地，所述突变为R38K、或R67K+V68A。
在另一优选例中，所述重链可变区对应于SEQ ID NO.:2所示序列的第67位精氨酸(R)发生突变。
在另一优选例中，所述重链可变区对应于SEQ ID NO.:2所示序列的突变选自下组：R67K。
在另一优选例中，所述重链可变区序列如SEQ ID NO.:2或4或8所示。
在另一优选例中，所述的重链可变区为SEQ ID No.:2且具有选自下组的突变：
VH38位Arg→Lys(R38K)、VH48位Met→Ile(M48I)、VH68位Val→Ala(V68A)、VH67位Arg→Lys(R67K)且VH68位Val→Ala(V68A)。
本发明的第四方面，提供了一种抗体的重链，所述的重链具有如本发明第三方面所述的重链可变区。
在另一优选例中，所述的抗体的重链还包括重链恒定区。
在另一优选例中，所述的重链恒定区为人源、鼠源或兔源的，较佳地为人源的。
本发明的第五方面，提供了一种抗体，所述抗体具有：
(1)如本发明第一方面所述的轻链可变区；和/或
(2)如本发明第三方面所述的重链可变区；
或者，所述抗体具有：如本发明第二方面所述的轻链；和/或如本发明第四方面所述的重链。
在另一优选例中，所述抗体具有如SEQ ID NO.:1或3所示的轻链可变区；和/或如SEQ ID NO.:2或4所示的重链可变区。
在另一优选例中，所述抗体具有选自下组的突变：
(a)所述抗体的轻链可变区对应于SEQ ID NO.:1所示序列中第69位甘氨酸(G)发生突变；和/或
(b)所述抗体的重链可变区对应于SEQ ID NO.:2所示序列存在选自下组的氨基酸突变：第38位精氨酸(R)、第48位甲硫氨酸(M)、第68位缬氨酸(V)、或其组合。
在另一优选例中，所述的抗体的轻链可变区对应于SEQ ID NO.:1所示序列中第69位甘氨酸(G)发生突变。
在另一优选例中，所述的抗体还包括对应于SEQ ID NO.:2所示的重链可变区序列存在选自下组的氨基酸突变：第38位精氨酸(R)、第48位甲硫氨酸(M)、第68位缬氨酸(V)、或其组合。
在另一优选例中，所述抗体的轻链可变区对应于SEQ ID NO.:1所示序列中第69位甘氨酸(G)突变为丝氨酸(S)，且所述抗体的重链可变区对应于SEQ ID NO.:2所示序列中第38位精氨酸(R)突变为赖氨酸(K)。
在另一优选例中，所述抗体的轻链可变区序列如SEQ ID NO.:1或3所示；和/或所述抗体的重链可变区序列如SEQ ID NO.:2或4所示。
在另一优选例中，所述抗体的轻链可变区序列如SEQ ID NO.:3所示；并且所述抗体的重链可变区序列如SEQ ID NO.:2或4所示。
在另一优选例中，所述抗体具有序列如SEQ ID NO.:3所示的轻链可变区；并且所述抗体具有序列如SEQ ID NO.:4所示的重链可变区。
在另一优选例中，所述抗体为人源化抗体。
在另一优选例中，所述抗体为特异性结合HER3。
在另一优选例中，所述抗体对人HER3的亲和力的KD值(M)为1.0E-8～2.0E-10。
在另一优选例中，所述的抗体为双链抗体、或单链抗体。
在另一优选例中，所述的抗体为单克隆抗体。
在另一优选例中，所述的抗体为双特异性抗体。
在另一优选例中，所述的抗体为药物偶联物形式。
本发明的第六方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明第一方面所述的轻链可变区、如本发明第二方面所述的轻链、如本发明第三方面所述的重链可变区、如本发明第四方面所述的重链、或如本发明第五方面所述的抗体；以及
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括6His标签。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体、或多聚体。
本发明的第七方面，提供了一种抗体制剂，所述的抗体制剂包括：
(a)如本发明第五方面所述的抗体；以及
(b)载体，所述的载体包括：缓冲剂、无菌水，任选的表面活性剂。
在另一优选例中，在所述的制剂中，所述抗体的浓度为5-100mg/mL；优选为10-70mg/mL，更优选为20-60mg/mL。
在另一优选例中，所述缓冲剂选自下组：柠檬酸缓冲体系、组氨酸缓冲体系、或其组合。
在另一优选例中，所述缓冲剂为组氨酸缓冲体系。
在另一优选例中，在所述的制剂中，所述组氨酸缓冲体系的浓度为0.5-20mM，较佳地为1-10mM。
在另一优选例中，所述柠檬酸缓冲体系中含有组氨酸和盐酸组氨酸，较佳地含有1-10mM组氨酸和1-10mM盐酸组氨酸，以所述抗体制剂的总重量计。
在另一优选例中，所述缓冲剂为柠檬酸缓冲体系。
在另一优选例中，在所述的制剂中，所述柠檬酸缓冲体系的浓度为5-100mM，较佳地为10-50mM。
在另一优选例中，所述柠檬酸缓冲体系中含有柠檬酸钠和氯化钠，较佳地含有10-50mM柠檬酸钠和50-200mM氯化钠，以所述抗体制剂的总重量计。
在另一优选例中，所述的表面活性剂选自下组：吐温80、吐温20、或其组合。
在另一优选例中，在所述的制剂中，所述的表面活性剂含量为0.005-0.2wt％，以所述抗体制剂的总重量计。
在另一优选例中，所述表面活性剂为吐温80，所述浓度为0.02-0.1wt％。
在另一优选例中，所述的制剂pH范围为5.0-7.5，较佳地为5.5-7。
在另一优选例中，所述的制剂为注射制剂。
在另一优选例中，所述的制剂包括：本发明第五方面所述的抗体、组氨酸缓冲体系、表面活性剂，和余量的无菌注射用水。
在另一优选例中，所述的制剂包括：
本发明第五方面所述的抗体         20-60mg/mL；
组氨酸缓冲体系                                1–10mM；
吐温80                                    0.02-0.1wt％；
以及作为余量的无菌注射用水，且所述的制剂pH范围为优选5.5-6.5。
本发明的第八方面，提供了一种试剂盒，所述的试剂盒含有本发明第七方面所述的抗体制剂，以及盛装所述抗体制剂的容器。
本发明的第九方面，提供了一种CAR构建物，所述的CAR构建物的抗原结合区域的scFv段为特异性结合于HER3的结合区，并且所述scFv具有如本发明第一方面所述的轻链可变区和如本发明第三方面所述的重链可变区。
本发明的第十方面，提供了一种重组的免疫细胞，所述的免疫细胞表达外源的如本发明第九方面所述的CAR构建物。
在另一优选例中，所述的免疫细胞选自下组：NK细胞、T细胞。
在另一优选例中，所述的免疫细胞来自人或非人哺乳动物(如鼠)。
本发明的第十一方面，提供了一种抗体药物偶联物，所述的抗体药物偶联物含有：
(a)抗体部分，所述抗体部分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、或其组合；和
(b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
在另一优选例中，所述的抗体部分与所述的偶联部分通过化学键或接头进行偶联。
本发明的第十二方面，提供了一种活性成分的用途，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗体药物偶联物、或其组合，所述活性成分用于
(a)制备检测试剂或试剂盒；
(b)制备预防和/或治疗HER3相关疾病的药物或制剂；和/或
(c)制备预防和/或治疗癌症或肿瘤的药物或制剂。
在另一优选例中，所述肿瘤选自下组：血液肿瘤、实体瘤、或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤 (DLBCL)、霍奇金淋巴瘤、或其组合。
在另一优选例中，所述实体瘤选自下组：胃癌、胃癌腹膜转移、肝癌、白血病、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、宫颈癌、卵巢癌、淋巴癌、鼻咽癌、肾上腺肿瘤、膀胱肿瘤、非小细胞肺癌(NSCLC)、脑胶质瘤、子宫内膜癌、或其组合。
在另一优选例中，所述肿瘤为高表达HER3的肿瘤。
在另一优选例中，所述药物或制剂用于制备预防和/或治疗与HER3(表达阳性的)相关的疾病的药物或制剂。
在另一优选例中，所述的抗体为药物偶联物(ADC)形式。
在另一优选例中，所述的检测试剂或试剂盒用于诊断HER3相关疾病。
在另一优选例中，所述检测试剂或试剂盒用于检测样品中HER3蛋白。
在另一优选例中，所述的检测试剂为检测片。
本发明的第十三方面，提供了一种药物组合物，所述的药物组合物含有：
(i)活性成分，所述活性成分选自下组：如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体、如本发明第六方面所述的重组蛋白、如本发明第十方面所述的免疫细胞、如本发明第十一方面所述的抗体药物偶联物、或其组合；以及
(ii)药学上可接受的载体。
在另一优选例中，所述的药物组合物为液态制剂。
在另一优选例中，所述的药物组合物为注射剂。
在另一优选例中，所述的药物组合物用于治疗肿瘤。
在另一优选例中，所述肿瘤为高表达HER3的肿瘤。
本发明的第十四方面，提供了一种多核苷酸，所述的多核苷酸编码选自下组的多肽：
(1)如本发明第一方面所述的重链可变区、如本发明第二方面所述的重链、如本发明第三方面所述的轻链可变区、如本发明第四方面所述的轻链、或本发明第五方面所述的抗体；或
(2)如本发明第六方面所述的重组蛋白；
(3)如本发明第九方面所述的CAR构建物。
本发明的第十五方面，提供了一种载体，所述的载体含有如本发明第十四方面所述的多核苷酸。
在另一优选例中，所述的载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
本发明的第十六方面，提供了一种遗传工程化的宿主细胞，所述的宿主细胞含有如本发明第十五方面所述的载体或基因组中整合有如本发明第十四方面所述的多核苷酸。
本发明的第十七方面，提供了一种体外检测(包括诊断性或非诊断性)样品中HER3蛋白的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明第五方面所述的抗体接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在HER3蛋白。
本发明的第十八方面，提供了一种检测板，所述的检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明第五方面所述的抗体或如本发明第十一方面所述的抗体药物偶联物。
本发明的第十九方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明第五方面所述的抗体；和/或
(2)第二容器，所述第二容器中含有抗如本发明第五方面所述的抗体的二抗；
或者，所述试剂盒含有如本发明第十八方面所述的检测板。
本发明的第二十方面，提供了一种重组多肽的制备方法，所述方法包括：
(a)在适合表达的条件下，培养如本发明第十四方面所述的宿主细胞；
(b)从培养物中分离出重组多肽，所述的重组多肽是如本发明第五方面所述的抗体或如本发明第六方面所述的重组蛋白。
本发明的第二十一方面，提供了一种HER3相关疾病的方法，所述方法包括：给需要的对象施用如本发明第五方面所述的抗体、所述抗体的抗体-药物偶联物、或表达所述抗体的CAR-T细胞、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了HER3鼠单抗及人Germline模板可变区序列。其中，a为轻链可变区，b为重链可变区。上行为鼠单抗可变区序列(SEQ ID No.:5或6)，下行为CDR移植后可变区序列(SEQ ID No.:9或10)，划线部分为CDR区，灰色标记部分氨基酸为鼠单抗及人 Germline模板骨架区不同的氨基酸。
图2显示了PEG沉淀法检测溶解度结果，箭头所示为开始出现沉淀的孔。
图3显示了本发明人源化抗体内源荧光检测图谱。
图4显示了本发明人源化抗体动物体内肿瘤(A549)抑制效果。
图5显示了本发明人源化抗体hu1044-7对动物体内肿瘤(Fadu)的抑制效果。
图6显示了本发明人源化抗体hu1044-7对动物体内肿瘤(BT-474)的抑制效果。
具体实施方式
本发明人通过广泛而深入的研究，经过大量筛选，意外地获得一种具有亲和力优异的和良好的结构稳定性的抗HER3人源化抗体。具体地，本发明在选择人源化骨架区时兼顾相似性及人体使用频率，选择了IGKV3-20*01和IGHV1-69*01两个序列的骨架区进行人源化，并对获得的人源化抗体进行突变筛选。人源化后的抗体，尤其是突变后的人源化抗体达到与嵌合抗体相近的亲和力，通过对该人源化抗体溶解度及内源荧光的初步研究，证实该人源化已具备初步的成药性，未来有进一步开发成靶向治疗的人源化单抗药物。并对人源化后的抗体进行了初步成药性研究，筛选并确定了该抗HER3人源化抗体的制剂配方。在此基础上完成了本发明。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
如本文所用，术语“给予”和“处理”是指外源性药物、治疗剂、诊断剂或组合物应用于动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断；包括抗HER3抗体与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。
如本文所用，术语“治疗”指给予患者内用或外用治疗剂，包含本发明的任何一种抗HER3抗体及其组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，以有效缓解一种或多种疾病症状的治疗剂的量(治疗有效量)给予患者。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。例如，“任选包含1-3个抗体重链可变区”是指特定序列的抗体重链可变区可以 有但不是必须有，可以是1个、2个或3个。
本发明所述的“序列同一性”表示当具有适当的替换、插入或缺失等突变的情况下最佳比对和比较时，两个核酸或两个氨基酸序列之间的同一性程度。本发明中所述的序列和其具有同一性的序列之间的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％,86％,87％,88％,89％,90％,91％,92％,93％,94％,95％,96％,97％,98％,99％,100％。
HER3
人表皮生长因子3(human epidermal growth factor receptor 3，HER3)在肿瘤的发生、进展等方面发生着重要的作用。HER3通常与EGFR或HER2分子形成异二聚体而发挥作用，在癌症病理组织，EGFR、HER2过表达常常伴随着HER3的过表达，并且认为HER3过表达对EGFR、HER2靶向治疗产生耐药性起到重要作用。HER3与配体Heregulin(HRG)结合后构象发生变化，暴露出与EGFR、HER2结合的位点，进而与EGFR、HER2分子形成异二聚体，激活细胞内信号，促进肿瘤细胞增殖。
抗HER3鼠单克隆抗体1044(申请号为2014104015453)可阻断HER3分子与其配体HRG的结合，从而抑制其与HER家族其它分子形成异二聚体，该抗体可以和肿瘤表面HER3分子结合，并抑制人表皮鳞状细胞癌A431细胞的增值，具有治疗多种HER3过表达肿瘤的潜力。
1044鼠单抗轻链可变区(SEQ ID NO.:5)
Figure PCTCN2019096418-appb-000001
1044鼠单抗重链可变区(SEQ ID NO.:6)
Figure PCTCN2019096418-appb-000002
抗体
如本文所用，术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其较链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2、IgG3，IgG4。轻链根据恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。不同类免疫球蛋白的亚单位结构和三维构型是本领域人员所熟知的。
本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成，从氨基端到竣基端依次排列的顺序序为:FR1，CDR1，FR2，CDR2，FR3，CDR3和FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的抗HER3的单克隆抗体。制备时用HER3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。在本发明一个优选的实施方案中，所述的鼠源HER3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，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。
术语“抗体的抗原结合片段”(或简称“抗体片段”)是指抗体的保持特异性结合抗原(例如，HER3)的能力的一个或多个片段。己显示可利用全长抗体的片段来进行抗体的抗原结合功能。术语“抗体的抗原结合片段”中包含的结合片段的实例包括
(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；
(ii)F(ab') 2片段，包含通过较链区上的二硫桥连接的两个Fab片段的二价片段；
(iii)由VH和CH1结构域组成的Fd片段；
(iv)由抗体的单臂的VH和VL结构域组成的Fv片段。
Fv抗体含有抗体重链可变区、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。一般的，Fv抗体还包含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。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A等人，(1991)Sequences of proteins of  immunological interest.NIH Publication91-3242)提供。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，HER3分子上的特定部位)。表位通常以独特的空间构象包括至少3,4,5,6,7,8,9,10,11,12,13,14或15个连续或非连续的氨基酸。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于1O -8M、1O -9M或lO -10M或更小的亲和力(KD)结合。
术语“竞争结合”是指与本发明的单克隆抗体识别HER3的胞外区上的相同表位(也称为抗原决定簇)或相同表位的一部分并与所述抗原结合的抗体。与本发明的单克隆抗体结合相同表位的抗体是指识别并结合于本发明的单克隆抗体所识别的HER3的氨基酸序列的抗体。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本发明的抗体以小于大约10 -7M，例如小于大约1O -8M、1O -9M或lO -10M或更小的解离平衡常数(KD)结合HER3。
如本文所用，术语“抗原决定簇”指抗原上不连续的，由本发明抗体或抗原结合片段识别的三维空间位点。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
在本发明中，抗体包括用本领域技术人员熟知技术所制备的鼠的、嵌合的、人源化的或者全人的抗体。重组抗体，例如嵌合的和人源化的单克隆抗体，包括人的和非人的部分，可以采用本领域熟知的DNA重组技术制备。
如本文所用，术语“单克隆抗体”指得自单个细胞来源的克隆分泌的抗体。单克隆抗体是高度特异性的，针对单个抗原表位。所述的细胞可能是真核的、原核的或噬菌体的克隆细胞株。
在本发明中，抗体可以是单特异性、双特异性、三特异性、或者更多的多重特异性。
在本发明中，本发明的抗体还包括其保守性变异体，指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
抗HER3人源化抗体
本发明提供抗HER3人源化抗体(以下简称HER3抗体)。具体地，本发明提供一种针对HER3的高特异性和高亲和力的人源化抗体，其包括重链和轻链，所述重链含有重链可变区(VH)氨基酸序列，所述轻链含有轻链可变区(VL)氨基酸序列。
1986年Jones等人首次将鼠单抗重链CDR移植到人抗体重链骨架区，然后与鼠单抗轻链组装成完整抗体并保持了与原鼠单抗相似的亲和力，为抗体人源化技术的发展提供了思路。1989年Queen等人通过CDR移植的方法，成功构建抗CD25人源化抗体，该方法使用的是人抗体Eu骨架区进行人源化，在骨架区部分位点保留了鼠源抗体氨基酸以保持亲和力。1992年Presta等人报道了以人抗体亚群共有序列(consensus sequence)为模板进行CDR移植成功构建人源化的方法。1994年Pedersen等人报道了用表面重塑(resurfacing)的方法对抗体人源化。1994年Hsiao等人报道了以人抗体Germline序列骨架区进行CDR移植的人源化方法。1994年Jespers等人用噬菌体库(shuffling library)的方法成功构建人源化方法。
抗体人源化中人骨架区的选择通常有两种，一种是已知的成熟抗体，一种是人Germline序列。已知的成熟抗体骨架区通常含有体细胞突变位点，可能带来潜在的免疫原性。相比已成熟抗体，人Germline序列骨架区理论上免疫原性更低，而且结构更灵活，可塑性强，容易接受不同的CDR区。人抗体Germline基因在人体中的使用频率具有一定的偏向性，选择使用频率高的Germline骨架区人源化后的抗体具有免疫原性低、表达量高、结构稳定等优点，因此本发明在人源化时并未选择与鼠源抗体相似度最高的Germline序列，而是兼顾相似性及人体使用频率，经过大量实验筛选，选择了IGKV3-20*01和IGHV1-69*01两个序列的骨架区进行人源化。本发明选用人抗体Germline骨架区进行CDR移植，这样构建的人源化抗体结构更加稳定、表达量高、免疫原性低、成药性更高。
具体地，如本发明第一方面至第五方面所述。
1044CDR移植后轻链可变区(VL1、VL4位点保留鼠单抗氨基酸)(SEQ ID NO.:1)
Figure PCTCN2019096418-appb-000003
Figure PCTCN2019096418-appb-000004
1044CDR移植后重链可变区(VH74位点保留鼠单抗氨基酸)(SEQ ID NO.:2)
Figure PCTCN2019096418-appb-000005
1044人源化抗体轻链可变区(VL1、VL4、VL69位点保留鼠单抗氨基酸)(SEQ ID NO.:3)
Figure PCTCN2019096418-appb-000006
1044人源化抗体重链可变区(VH38、VH74位点保留鼠单抗氨基酸)(SEQ ID NO.:4)
Figure PCTCN2019096418-appb-000007
在另一优选例中，所述人源化抗体的恒定区同抗HER3鼠单克隆抗体1044(申请号为2014104015453)的恒定区相同。
在另一优选例中，所述经过添加、缺失、修饰和/或取代至少一个氨基酸序列所形成的序列优选为同源性为至少80％，较佳地至少85％，更佳地至少为90％，最佳地至少95％的氨基酸序列。
本发明的抗体可以是双链或单链抗体，并且可以优选为全人源化抗体。
本发明所述抗体衍生物可以是单链抗体、和/或抗体片段，如：Fab、Fab'、(Fab') 2、或该领域内其他已知的抗体衍生物等，以及IgA、IgD、IgE、IgG以及IgM抗体或其他亚型的抗体中的任意一种或几种。
本发明抗体可以是靶向HER3的人源化抗体、CDR嫁接和/或修饰的抗体。
本发明上述内容中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
本发明对HER3鼠单抗成功进行了人源化改造，人源化后的抗体达到与嵌合抗体相近的亲和力，通过对该人源化抗体溶解度及内源荧光的初步研究，证实该人源化已具备初步的成药性，未来有进一步开发成靶向治疗的人源化单抗药物。
抗体的制备
任何适于产生单克隆抗体的方法都可用于产生本发明的HER3抗体。例如，可以用连接或天然存在的HER3蛋白或其片段免疫动物。可以使用合适的免疫接种方法，包括佐剂、免疫刺激剂、重复加强免疫接种，可以使用一种或多种途径。
任何合适形式的HER3都可以作为免疫原(抗原)，用于产生对HER3特异的非人抗体，筛选所述抗体的生物学活性。免疫原可以单独使用，或与本领域已知的一种或多种免疫原 性增强剂组合使用。免疫原可以由天然来源纯化，或者在遗传修饰的细胞中产生。编码免疫原的DNA在来源上可以是基因组或非基因组的(例如cDNA)。可以使用合适的遗传载体表达编码免疫原的DNA，所述载体包括但不限于腺病毒载体、杆状病毒载体、质粒和非病毒载体。
人源化抗体可以选自任何种类的免疫球蛋白，包括IgM、IgD、IgG、IgA和IgE。同样，任一类轻链都可以在本文的化合物和方法中使用。具体地说，κ、λ链或其变体在本发明的化合物和方法中是可以用的。
人源化本发明HER3抗体的示例性方法描述于实施例1。
本发明抗体或其片段的DNA分子的序列可以用常规技术，比如利用PCR扩增或基因组文库筛选等方法获得。此外，还可将轻链和重链的编码序列融合在一起，形成单链抗体。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输己与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物或动物细胞(如哺乳动物细胞)。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域熟知的技术进行。获得的转化子可用常规方法培养，转化子表达本发明的基因所编码的多肽。根据所用的宿主细胞，用常规培养基在合适的条件下培养。
通常，在适合本发明抗体表达的条件下，培养转化所得的宿主细胞。然后用常规的免疫球蛋白纯化步骤，如蛋白A-Sepharose、羟基磷灰石层析、凝胶电泳、透析、离子交换层析、疏水层析、分子筛层析或亲和层析等本领域技术人员熟知的常规分离纯化手段纯化得到本发明的抗体。
所得单克隆抗体可用常规手段来鉴定。比如，单克隆抗体的结合特异性可用免疫沉淀 或体外结合试验(如放射性免疫测定(RIA)或酶联免疫吸附测定(ELISA))来测定。
抗体制剂
抗体在不同的制剂缓冲液中具有不同的稳定性，表现为电荷异质性的变化、抗体分子的降解、聚合等，这些质量性质的变化与抗体本身的理化性质相关，因此，在抗体药物开发过程，需根据不同抗体的理化性质筛选适合其自身的制剂缓冲液。目前常用的抗体制剂缓冲体系有磷酸盐缓冲液、柠檬酸缓冲液、组氨酸缓冲液等，同时根据抗体性质会添加不同浓度的盐离子或山梨醇、海藻糖、蔗糖等赋形剂，以及适量的吐温20或吐温80等表面活性剂，以维持抗体的稳定性。
本发明抗体制剂如本发明第七方面所述。
本发明的抗体药物组合制剂可有效抑制本发明人源化抗体的聚集沉淀、水解、氧化及脱酰胺等副反应，同时能有效提高产品在加压(高温、强光照射及冻融等)、加速及长期冷藏条件下的稳定性。
药物组合物
本发明还提供了一种组合物。在优选例中，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白或其ADC或相应的CAR-T细胞，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明所述抗体也可以是由核苷酸序列在细胞内表达用于的细胞治疗，比如，所述抗体用于嵌合抗原受体T细胞免疫疗法(CAR-T)等。
本发明的药物组合物可直接用于结合HER3蛋白分子，因而可用于预防和治疗HER3相关的疾病。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的单克隆抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克/千克体重-约5毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的药物组合物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约20毫克/千克体重。当然，具体剂量还应考虑给药途径、 病人健康状况等因素，这些都是熟练医师技能范围之内。
检测用途和试剂盒
本发明的抗体可用于检测应用，例如用于检测样本，从而提供诊断信息。
本发明中，所采用的样本(样品)包括细胞、组织样本和活检标本。本发明使用的术语“活检”应包括本领域技术人员已知的所有种类的活检。因此本发明中使用的活检可以包括例如通过内窥镜方法或器官的穿刺或针刺活检制备的组织样本。
本发明中使用的样本包括固定的或保存的细胞或组织样本。
本发明还提供了一种指含有本发明的抗体(或其片段)的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。在优选例中，本发明的抗体可以固定于检测板。
本发明的主要优点
(a)本发明人源化抗体为典型的色氨酸、酪氨酸包埋较好的结构，所筛选的人VH、VL骨架区模板结构稳定，且与鼠单抗CDR区适配较好，轻重链可变区能较好的配对在一起，亲和力高、结构稳定。
(b)与嵌合抗体相比，本发明人源化抗体具有优异的生物活性、特异性，在保留与HER3相当的亲合力的同时，具有更低的免疫原性和更高的表达量。
(c)本发明人源化抗体具有较高的溶解度，具备良好的成药性。
(d)本发明人源化抗体在A549肿瘤模型中显示有明显的抑制肿瘤生长的特性，且对肿瘤的生长抑制活性与治疗剂量相关，证明人源化后的抗HER3单抗仍然具有良好的生物活性。
(e)相比已成熟抗体，本发明在选择人源化骨架区时兼顾相似性及人体使用频率，人源化后的抗体免疫原性更低、表达量更高(尤其在哺乳动物细胞，如CHO-K1细胞中)、结构更稳定、成药性更好。
(f)本发明人源化抗体在本发明抗体制剂中的稳定性非常好。
(g)本发明人源化抗体在CHO-K1细胞中进行稳定表达细胞株构建，筛选的稳定细胞株在摇瓶进行Fed Batch培养，抗体表达量达4-5g/L。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本发明实施例或测试例中未注明具体条件的实验，通常按常规条件进行，或按照原料 /商品制造商建议的条件；未注明具体来源的试剂，为市场购买的常规试剂。
材料与方法
1.菌株、细胞、质粒及试验动物
E.coli DH5α感受态细胞由本研究室制备；FreeStyle 293-F细胞及表达培养基购自Gibco公司；pSGHV0真核表达质粒由美国约翰霍普金斯大学医学院生物物理系Dr.D.J.Leahy惠赠；Babl/c裸鼠购自上海斯莱克实验动物有限责任公司。
2.主要试剂及设备
定点突变试剂盒购买自北京赛百盛基因技术有限公司；Mabselect Sure亲和介质购自GE公司；利妥昔单抗(批号H0709)、曲妥珠单抗(批号N3674)购自上海罗氏制药有限公司；HER3胞外区重组蛋白由CHO细胞重组表达；羊抗人抗体购自Jackson ImmunoResearch公司；Polyethylenimine(PEI MW 25000)购自Polysciences公司；聚乙二醇8000(PEG 8000)购自西格玛奥德里奇公司；SDS-MW Analysis Kit购自贝克曼库尔特公司；OPM-CHO CD07、PFF05、CDF16培养基购自上海奥浦迈生物科技有限公司；1044鼠单抗为2014104015453中的抗体1044。
3.人源化抗体构建
1044鼠单抗CDR移植到人抗体骨架区后，由上海博尚生物技术有限公司合成人源化可变区基因序列，然后将重链、轻链可变区基因分别构建到含人IgG1重链恒定区及κ链恒定区的pSGHV0载体上。用定点突变试剂盒对人源化后的抗体骨架区VH38、VH48、VH68、VL69等位点进行回复突变并经测序确认。1044人鼠嵌合抗体及各回复突变人源化抗体在293F细胞中用PEI进行瞬时转染，瞬时转染的细胞培养上清2000rpm离心后用ProteOn XPR36测定亲和力。最终选择的人源化抗体序列由上海奥浦迈生物科技有限公司进行CHO-K1稳定细胞株的构建，用稳定细胞株表达该人源化抗体hu1044，筛选的稳定细胞株在摇瓶进行Fed Batch培养，按5×10 5/ml的细胞密度将稳定细胞株接种到培养瓶中，基础培养基为OPM-CHO CD07，在培养第3、5、7、9、11天补加培养体积3％、5％、7％、5％、3％的PFF05补料培养基，及培养第5、7、9、11天补加培养体积0.3％的CDF16补料培养基，并维持葡萄糖含量在2-6g/L，待细胞活率低于60％或培养至15天即结束培养，抗体表达量达4-5g/L。然后经Mabselect Sure Protein A亲和介质纯化得到人源化抗体，纯化后的抗体浓缩到60mg/mL。
4.ProteOn XPR36蛋白质相互作用阵列系统测定抗体亲和力
GLM芯片横向6个通道标记羊抗人抗体，将通道旋转90度变成纵向捕获293F细胞表达的嵌合抗体或人源化抗体，通道再转到横向，1-5号通道分别进样20nM、10nM、5nM、2.5nM、1.25nM的HER3胞外区蛋白，6号通道进样缓冲液，得到5个动力学反应曲线，分别代表不同浓度的抗原与捕获抗体的反应结果，用Langmuir模式计算嵌合抗体及各人源化抗体突变体的亲和力常数KD(M)。
5.PEG沉淀法测抗体溶解度
配置FB1溶液(90g氯化钠、73.5g柠檬酸三钠、7g聚山梨酯80溶于1L注射用水中，pH调至6.5)和FB2缓冲液(0.3g组氨酸、0.5g盐酸组氨酸、0.08g聚山梨酯20、18.9g海藻糖溶于1L注射用水中，pH调至6.0)。用去离子水配置两份40％的PEG 8000母液，pH分别调至6.5和6.0，然后分别用FB1和FB2将40％PEG 8000母液稀释至不同的浓度。将60mg/mL的HER3人源化抗体用超滤管分别超滤置换到FB1和FB1缓冲液中，置换体积100倍，终浓度均为20mg/mL。然后将利妥昔单抗、曲妥珠单抗、HER3人源化单抗(FB1)、HER3人源化单抗(FB1)分别用各自对应的缓冲液稀释至10mg/mL，各取100μL抗体与100μL不同浓度的PEG 8000(5％、10％、15％、20％、25％、30％、35％、40％)在96孔板中混匀，反应10分钟后观察结果。
6.非还原毛细管电泳检测(CE-SDS)
取100μg样品，加入70μL SDS样品缓冲液，使最终体积为95μL，再加入250mM的碘乙酰胺5μL混匀，5000rpm离心1分钟，在65℃下水浴4分钟，冷却到室温后，5000rpm离心1分钟，取100μL加入到毛细管电泳进样瓶中进行分析。采用非涂层-熔融石英毛细管(内径50μm)，有效长度为24.5cm。5kV反相极性电动进样20s，分离电压15kV，分离时间40min，检测波长220nm。
7.离子交换高效液相色谱法(IEX-HPLC)
流动相A：10mM磷酸钠溶液，pH 7.4；流动相B：10mM磷酸钠溶液，0.25M氯化钠，pH 7.4；色谱柱：戴安公司Propac WCX-10(4.0×250mm)；检测器：紫外检测器，波长280nm；柱温：35℃；进样量：40μg；流速0.5mL/min；分离梯度：27min内流动相B由0％增至30％；采集时间：41min。
8.分子排阻高效液相色谱法(SEC-HPLC)
流动相：0.2M磷酸钠缓冲液，pH 6.8；色谱柱：TOSOH Bioscience公司Super SW3000(4.6×300mm，4μm)；检测器：紫外检测器，波长280nm；流速：0.35mL/min；柱温：30℃；进样量：30μg；采集时间：20min。
9.内源荧光检测
将样品分别用各自对应缓冲液稀释至1.0mg/mL，加入比色皿中在SpectraMax M5上进行检测，激发波长295nm，发射波长从310nm扫描到450nm，扫描步进1nm。
10.人源化抗体体内药效实验
人非小细胞肺癌细胞A549培养至对数生长期，用胰酶消化，用无血清培养基进行洗涤，按1×10 7细胞量接种至3-4周龄Babl/c裸鼠背部皮下，两周后看到明显的肿瘤长出时将裸鼠随机分组，每组6只。HER3人源化单抗给药浓度分别为：30mg/kg、15mg/kg、7.5mg/kg，以PBS作为对照，腹腔注射治疗，每周治疗两次，每周测量两次肿瘤体积。裸鼠肿瘤体积大小：V＝0.5×a×b 2，a为肿瘤实体长直径，b为肿瘤实体短直径。
11.人源化抗体制剂配方筛选
11.1制剂配方筛选Ⅰ
配制以下三种缓冲液：柠檬酸缓冲液(含25mM柠檬酸钠、140mM氯化钠、0.07％Tween-80，pH6.5)、磷酸盐缓冲液(含50mM磷酸盐、140mM氯化钠、0.07％Tween-80，pH8.0)、组氨酸缓冲液(含2mM组氨酸、2mM盐酸组氨酸、1.5％海藻糖、0.07％Tween-80，pH6.0)，将纯化的人源化抗体分别置换到以上三种缓冲液，抗体终浓度40mg/mL，在-70℃、2-8℃和37℃放置一周用IEX-HPLC法检测其电荷异质体变化。
11.2制剂配方筛选Ⅱ
配制以下两种缓冲液：柠檬酸缓冲液(含25mM柠檬酸钠、140mM氯化钠、0.07％Tween-80，pH6.5)、组氨酸缓冲液(含2mM组氨酸、2mM盐酸组氨酸、0.07％Tween-80，pH6.0)，在这两种缓冲液中分别加入1.5％海藻糖或2％山梨醇，再将纯化的人源化抗体分别置换到以上缓冲液，抗体终浓度40mg/mL，在37℃放置一周用IEX-HPLC法、SEC-HPLC法检测其质量变化。
11.3制剂配方筛选Ⅲ
配制组氨酸缓冲液(含2mM组氨酸、2mM盐酸组氨酸、0.07％Tween-80，pH6.0)，分别加入25、50、75、125、150mM氯化钠，然后将人源化抗体配制到以上不同盐离子的缓冲液中，抗体终浓度40mg/mL，观察盐离子对抗体浊度的影响。
用组氨酸缓冲液(含2mM组氨酸、2mM盐酸组氨酸、0.07％Tween-80，pH6.0)缓冲液将人源化抗体配制到40mg/mL，37℃放置两周用IEX-HPLC法、SEC-HPLC法及非还原CE-SDS法检测其质量变化。
实施例1抗体人源化
经过大量的实验筛选，最终选择人IGKV3-20*01氨基酸序列作为轻链人源化模板，选择人IGHV1-69*01氨基酸序列作为重链人源化模板。将鼠单抗1044轻重链CDR区移植到人模板骨架区，人Jκ1序列替换鼠单抗Jκ区，人JH4序列替换鼠单抗JH区。鼠单抗及人Germline骨架区序列对比见图1。在SEQ ID No.:9基础上保留鼠源VL1和VL4氨基酸，得到SEQ ID No.:1；在SEQ ID No.:10基础上保留鼠源VH74氨基酸，得到SEQ ID No.:2。
其中，鼠单抗1044的轻链可变区和重链可变区的序列分别如SEQ ID NO.:5和6所示。
1044鼠单抗轻链可变区(SEQ ID NO.:5)
Figure PCTCN2019096418-appb-000008
1044鼠单抗重链可变区(SEQ ID NO.:6)
Figure PCTCN2019096418-appb-000009
Figure PCTCN2019096418-appb-000010
如图1所示，CDR移植后h_VL和h_VH的序列如SEQ ID No.:9和10所示。
1044CDR移植后轻链可变区(SEQ ID NO.:9)
Figure PCTCN2019096418-appb-000011
1044CDR移植后重链可变区(SEQ ID NO.:10)
Figure PCTCN2019096418-appb-000012
同时，在CDR移植过程中将这3个位点(VL1位点、VL4位点、VH74位点)保留原鼠单抗氨基酸，得到人源化抗体h1044-0，其轻链可变区和重链可变区的序列分别如SEQ ID NO.:1和2所示。
1044CDR移植后轻链可变区(VL1、VL4位点保留鼠单抗氨基酸)(SEQ ID NO.:1)
Figure PCTCN2019096418-appb-000013
1044CDR移植后重链可变区(VH74位点保留鼠单抗氨基酸)(SEQ ID NO.:2)
Figure PCTCN2019096418-appb-000014
结果如表1所示，CDR移植后的人源化抗体h1044-0与HER3蛋白亲和力比嵌合抗体约降低32倍。此外对骨架区一些位点进行了回复突变研究，包括：VH1位由Gln突变为Glu、VH25位由Ser突变为Thr、VH76位由Thr突变为Ser、VH77位由Ser突变为Asn。未发现对人源化抗体亲和力有明显提升。
表1 嵌合抗体及CDR移植后人源化抗体与HER3亲和力
抗体 Ka(1/Ms) Kd(1/s) KD(M)
嵌合抗体 8.74E+06 2.29E-04 2.62E-10
h1044-0 5.03E+05 4.27E-03 8.48E-9
VH1回复突变 4.18E+05 3.82E-03 9.14E-9
VH25回复突变 5.25E+05 3.17E-03 6.02E-9
VH76回复突变 5.31E+05 3.44E-03 6.49E-9
VH77回复突变 4.63E+05 3.27E-03 7.07E-9
进一步地，对人源化抗体h1044-0进行突变。VL69回复突变为对应于SEQ ID NO.:1中第69位由Gly突变为Ser，亲和力明显提升，与嵌合抗体相差约2倍。
在此基础上对VH38、VH48、或VH68位点突变(VH38由Arg突变为Lys，VH48由Met突变为Ile，VH68由Val突变为Ala)。突变后，人源化抗体亲和力均明显提升，基本接近嵌合抗体，恢复到嵌合抗体亲和力常数2倍以内，VL69、VH38回复突变后的序列符合人源化要求。此外，VL63/69回复突变体(VL63由Ile回复突变为Val，VL69由Gly回复突变为Ser)与VH38突变体(VH38由Arg回复突变为Lys)、VH68突变体(VH68由Val回复突变为Ala)、VH67/68突变体(VH67由Arg回复突变为Lys，VH68由Val回复突变为Ala)组合后，亲和力亦接近嵌合抗体，见表2。VL69、VH38回复突变后的序列作为最终的人源化抗体hu1044(轻链可变区和重链可变区序列分别如SEQ ID NO.:3和4所示)。
表2 嵌合抗体及回复突变后人源化抗体与HER3亲和力
抗体 Ka(1/Ms) Kd(1/s) KD(M)
嵌合抗体 1.05E+06 2.93E-04 2.78E-10
VL69回复突变(hu1044-1) 8.43E+05 5.02E-04 5.95E-10
VL69、VH38回复突变(hu1044) 8.75E+05 3.86E-04 4.42E-10
VL69、VH48回复突变(hu1044-3) 8.82E+05 4.94E-04 5.54E-10
VL69、VH68回复突变(hu1044-4) 8.41E+05 3.88E-04 4.61E-10
VL63/69、VH38回复突变(hu1044-5) 8.44E+05 3.53E-04 4.19E-10
VL63/69、VH68回复突变(hu1044-6) 9.04E+05 3.80E-04 4.20E-10
VL63/69、VH67/68回复突变(hu1044-7) 1.00E+06 3.76E-04 3.75E-10
“VL69回复突变”是对应于SEQ ID NO.:1中，VL69位由Gly突变为Ser(突变后的序列为SEQ ID No.:3)。
“VL63/VL69回复突变”是在SEQ ID No.:1中，VL63由Ile回复突变为Val，且VL69由Gly回复突变为Ser(突变后的序列为SEQ ID No.:7)。
Figure PCTCN2019096418-appb-000015
“VH38回复突变”是在SEQ ID No.:2中，VH38位从Arg回复突变为Lys(突变后的序列为SEQ ID No.:4)。
“VH48回复突变”是在SEQ ID No.:2中，VH48位由Met突变为Ile。
“VH68回复突变”是在SEQ ID No.:2中，VH68位由Val回复突变为Ala。
“VH67/VH68回复突变”是在SEQ ID No.:2中，VH67位由Arg回复突变为Lys，且VH68由Val回复突变为Ala(SEQ ID No.:8)。
Figure PCTCN2019096418-appb-000016
实施例2人源化抗体溶解度分析
一般抗体药物在人体使用的剂量相对较大，抗体分子具有较高的溶解度才可能具备一定的成药性。溶解度既与抗体的理化性质相关，如等电点、表面疏水性、轻重链之间的结合力等，又与抗体所在的缓冲液体系有关。
PEG沉淀法可用来检测蛋白的溶解度。VL69、VH38回复突变后的人源化抗体hu1044在CHO-K1细胞进行重组表达，经ProteinA亲和层析后，超滤置换入不同的缓冲体系中。FB1为利妥昔单抗制剂缓冲液，FB2为曲妥珠单抗缓冲液。PEG沉淀法看到，在FB1缓冲液中，5％PEG条件下人源化抗体hu1044开始出现沉淀，在FB2缓冲液中，10％PEG条件下人源化抗体hu1044开始出现沉淀，见图2。
相比FB1缓冲液，在FB2缓冲液中，HER3人源化抗体hu1044溶解度明显较好。HER3人源化抗体hu1044在FB2缓冲液中溶解度好于利妥昔单抗对照，比曲妥珠单抗对照溶解度低。在非还原毛细管电泳中，HER3人源化抗体hu1044在FB1缓冲液中超滤置换后明显纯度降低，聚合体增加，而在FB2缓冲液超滤置换后纯度未发生明显变化，进一步证实在FB2中HER3人源化抗体hu1044溶解度较好，见表3。HER3人源化抗体hu1044在不同的缓冲体系中表现出不同的溶解度，在FB2缓冲液中溶解度优于FB1，且优于利妥昔单抗。
表3 HER3人源化抗体hu1044非还原毛细管电泳检测结果
人源化抗体hu1044 低分子量杂质 IgG主峰 高分子量杂质
未超滤置换缓冲液对照 2.84％ 97.16％ 0
超滤置换到FB1 2.69％ 73.22％ 24.10％
超滤置换到FB2 2.28％ 97.73％ 0
实施例3人源化抗体hu1044内源荧光分析
蛋白分子中的色氨酸、酪氨酸在合适的激发光下会发射荧光，尤其是色氨酸具有强的自发荧光。色氨酸、酪氨酸对蛋白微环境的变化比较敏感，当色氨酸、酪氨酸由蛋白疏水中心暴露于蛋白表面时，荧光图谱会发生红移现象。检测蛋白内源荧光可以判断蛋白结构的稳定性。抗体内源荧光最大发射波长λmax可以反映抗体分子中色氨酸、酪氨酸的空间分布情况。在抗体分子骨架区中VL35W、VL86Y、VH36W、VH90Y、VH103W这些位点的色氨酸、酪氨酸非常保守，包埋于疏水中心。
结果如图3所示，通过比较HER3人源化抗体hu1044及曲妥珠、利妥珠单抗的内源荧光，可以看到HER3人源化单抗hu1044最大发射波长λ max为335nm，曲妥珠单抗λ max为337nm，利妥昔单抗λ max为348nm，HER3人源化抗体hu1044与曲妥珠单抗为λ max相近，为典型的色氨酸包埋较好的结构，利妥昔单抗稍差。HER3人源化抗体hu1044λmax值与曲妥珠单抗相似，为典型的色氨酸、酪氨酸包埋较好的结构，证实人源化过程所选的 人VH、VL骨架区模板结构稳定，与鼠单抗CDR区适配较好，且轻重链可变区能较好的配对在一起。
实施例4人源化抗体动物体内药效
1.非小细胞肺癌细胞A549模型
人非小细胞肺癌细胞A549培养至对数生长期，用胰酶消化，用无血清培养基进行洗涤，按1×10 7细胞量接种至3-4周龄Babl/c裸鼠背部皮下，两周后看到明显的肿瘤长出时将裸鼠随机分组，每组6只。HER3人源化单抗hu1044给药浓度分别为：30mg/kg、15mg/kg、7.5mg/kg，以PBS作为对照，腹腔注射治疗，每周治疗两次，每周测量两次肿瘤体积。裸鼠肿瘤体积大小：V＝0.5×a×b 2，a为肿瘤实体长直径，b为肿瘤实体短直径。
在A549裸鼠肿瘤模型中，人源化抗体hu1044对A549肿瘤生长具有明显的抑制作用，且抑制效果与治疗剂量相关(见图4)，在30mg/kg剂量抑制率最高，肿瘤生长抑制率为63％，证实该人源化抗体仍然具有良好的生物活性，在体内有治疗肿瘤的潜力。
2.人咽鳞癌Fadu模型
将人咽鳞癌Fadu细胞培养至对数生长期，胰酶消化后收集细胞，计数。裸小鼠皮下接种5×10 6人咽鳞癌Fadu细胞，待平均肿瘤体积生长至100mm 3后，根据肿瘤体积将动物分组(D0)，给药组每组6只，溶剂对照组12只。小鼠静脉注射HER3人源化抗体(hu1044-7)，每周2次，共4次；给药体积10mL/kg。HER3人源化抗体给药剂量0.2、0.6、2mg/kg。Erbitux(爱必妥，Merck公司生产，复发转移性头颈部鳞癌一线治疗用单抗)对照组给药剂量0.5mg/kg。溶剂组给予相同体积的生理盐水。每周测2次肿瘤体积，称小鼠体重，记录数据。裸鼠肿瘤体积大小：V＝0.5×a×b 2，a为肿瘤实体长直径，b为肿瘤实体短直径。
结果如图5所示，与溶剂对照组和Erbitux治疗组相比，HER3人源化抗体给药组均表现出显著的肿瘤抑制效果，且在HER3人源化抗体治疗组均有小鼠肿瘤出现部分消退(治疗后肿瘤体积小于给药前肿瘤体积)，2mg/kg组有5只小鼠肿瘤出现部分消退。HER3人源化抗体和Erbitux联合治疗组6只小鼠肿瘤均发生部分消退(P<0.05，与Erbitux单药治疗相比)。治疗组小鼠对药物耐受良好，没有体重减轻等症状发生。
3.乳腺癌BT-474模型
人乳腺癌BT-474细胞培养至对数生长期，胰酶消化后收集细胞，计数。裸小鼠皮下接种8×10 6人乳腺癌BT-474细胞，待平均肿瘤体积生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)，给药组每组6只，溶剂对照组12只。小鼠静脉注射HER3人源化抗体(hu1044-7)，每周2次，共6次；给药体积10mL/kg。HER3人源化抗体给药剂量2、6、20mg/kg。Herceptin(赫赛汀，罗氏公司生产，Her2阳性乳腺癌一线治疗用单抗)对照组给药剂量7.5mg/kg。溶剂组给予相同体积的生理盐水。每周测2次肿瘤体积，称小鼠体重，记录数据。裸鼠肿瘤体积大小：V＝0.5×a×b 2，a为肿瘤实体长直径，b为肿瘤实体短直径。
结果如图6所示，与溶剂对照组和赫赛汀治疗组相比，HER3人源化抗体和赫赛汀联合治疗组显著抑制了小鼠肿瘤的生长(P<0.01，与赫赛汀单药治疗相比)，与赫赛汀单抗具有明显协同治疗效果。治疗组小鼠对药物耐受良好，没有体重减轻等症状发生。
实施例5人源化抗体hu1044制剂配方筛选
在25mM柠檬酸缓冲液及2mM组氨酸盐酸组氨酸缓冲液中，人源化抗体hu1044无论是37℃还是2-8℃放置一周，其电荷异构体均相对稳定，但在磷酸盐缓冲液中，37℃和2-8℃放置一周电荷异构体均发生明显变化(见表4)，说明该抗体在25mM柠檬酸缓冲液及2mM组氨酸缓冲液中相对稳定。
表4 人源化抗体hu1044制剂缓冲液筛选Ⅰ
Figure PCTCN2019096418-appb-000017
在柠檬酸缓冲液及组氨酸盐酸组氨酸缓冲液中加入海藻糖或山梨醇后，与不加海藻糖或山梨醇试验组相比，抗体电荷异构体均无明显差别，SEC-HPLC纯度亦无明显差别，但在组氨酸缓冲液体系中，SEC-HPLC纯度比柠檬酸缓冲体系略好，见表5。
表5 人源化抗体hu1044制剂缓冲液筛选Ⅱ
Figure PCTCN2019096418-appb-000018
在组氨酸缓冲体系中加入氯化钠之后，人源化抗体hu1044乳光明显增加，且盐浓度 越高，乳光越明显，说明该人源化抗体在组氨酸缓冲体系中，不含盐或低盐时更稳定。
将组氨酸缓冲液中的人源化抗体hu1044在37℃进行两周加速稳定性试验，检测IEX-HPLC、SEC-HPLC、非还原CE-SDS质量变化。结果显示，随着时间增加，SEC-HPLC纯度缓慢降低，但14天时，SEC纯度仍在98％以上，IEX-HPLC酸性峰有轻微增加，碱性峰基本未变，非还原CE-SDS初度略微降低，见表6。
表6 人源化抗体hu1044制剂缓冲液筛选Ⅲ
Figure PCTCN2019096418-appb-000019
该人源化抗体hu1044在不同缓冲体中表现出不同的稳定性，在磷酸盐缓冲体系中抗体的电荷异质性易发生变化，在柠檬酸缓冲液及组氨酸缓冲液中相对稳定。盐离子的增加明显导致人源化抗体乳光的增加，证实该抗体不适合在较高的含盐缓冲液中。海藻糖、山梨醇等赋形剂对该抗体的稳定性无明显影响。以上筛选结果证实该人源化抗体在2mM组氨酸缓冲液(含0.07％Tween-80，pH6.0)中质量最稳定。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (17)

  1. 一种抗体的轻链可变区，其特征在于，所述轻链可变区选自下组：
    (1)序列如SEQ ID NO.:1所示的轻链可变区；或
    (2)将SEQ ID NO.:1所示的氨基酸序列经过至少一个(如1-8个、更佳地1-3个、最佳地1或2个)氨基酸残基的取代、缺失、修饰和/或添加而形成的，具有(1)所述轻链可变区功能的由SEQ ID NO.:1所示序列衍生的轻链可变区。
  2. 如权利要求1所述的轻链可变区，其特征在于，所述轻链可变区对应于SEQ ID NO.:1所示序列中第69位甘氨酸(G)突变为丝氨酸(S)。
  3. 如权利要求2所述的轻链可变区，其特征在于，所述轻链可变区对应于SEQ ID NO.:1所示序列中第63位异亮氨酸(I)发生突变。
  4. 如权利要求3所述的轻链可变区，其特征在于，所述轻链可变区对应于SEQ ID NO.:1所示序列中第63位Ile突变为Val。
  5. 如权利要求1所述的轻链可变区，其特征在于，所述的轻链可变区序列如SEQ ID NO.:3或7所示。
  6. 一种抗体的轻链，其特征在于，所述的轻链具有如权利要求1所述的轻链可变区。
  7. 一种抗体的重链可变区，其特征在于，所述重链可变区选自下组：
    (i)序列如SEQ ID NO.:2所示的重链可变区；或
    (ii)将SEQ ID NO.:2所示的氨基酸序列经过至少一个(如1-8个、更佳地1-3个、最佳地1或2个)氨基酸残基的取代、缺失、修饰和/或添加而形成的，具有(i)所述重链可变区功能的由SEQ ID NO.:2所示序列衍生的重链可变区。
  8. 如权利要求7所述的重链可变区，其特征在于，所述重链可变区对应于SEQ ID NO.:2所示序列存在选自下组的氨基酸突变：第38位精氨酸(R)、第48位甲硫氨酸(M)、第67位精氨酸(R)、第68位缬氨酸(V)、或其组合。
  9. 如权利要求7所述的重链可变区，其特征在于，所述重链可变区对应于SEQ ID NO.:2所示序列的突变选自下组：R38K、M48I、V68A、R67K、或其组合；较佳地，所述突变为R38K、或R67K+V68A。
  10. 如权利要求7所述的重链可变区，其特征在于，所述重链可变区序列如SEQ ID NO.:2或4或8所示。
  11. 一种抗体的重链，其特征在于，所述的重链具有如权利要求3所述的重链可变区。
  12. 一种抗体，其特征在于，所述抗体具有：
    (1)如权利要求1所述的轻链可变区；和/或
    (2)如权利要求7所述的重链可变区；
    或者，所述抗体具有：如权利要求6所述的轻链；和/或如权利要求11所述的重链。
  13. 一种重组蛋白，其特征在于，所述的重组蛋白具有：
    (i)如权利要求1所述的轻链可变区、如权利要求6所述的轻链、如权利要求7所述的重链可变区、如权利要求11所述的重链、或如权利要求12所述的抗体；以及
    (ii)任选的协助表达和/或纯化的标签序列。
  14. 一种抗体制剂，其特征在于，所述的抗体制剂包括：
    (a)如权利要求12所述的抗体；以及
    (b)载体，所述的载体包括：缓冲剂、无菌水，任选的表面活性剂。
  15. 一种试剂盒，其特征在于，所述的试剂盒含有权利要求14所述的抗体制剂，以及盛装所述抗体制剂的容器。
  16. 一种CAR构建物，其特征在于，所述的CAR构建物的抗原结合区域的scFv段为特异性结合于HER3的结合区，并且所述scFv具有如权利要求1所述的轻链可变区和如权利要求7所述的重链可变区。
  17. 一种抗体药物偶联物，其特征在于，所述的抗体药物偶联物含有：
    (a)抗体部分，所述抗体部分选自下组：如权利要求1所述的轻链可变区、如权利要求6所述的轻链、如权利要求7所述的重链可变区、如权利要求11所述的重链、或如权利要求12所述的抗体、或其组合；和
    (b)与所述抗体部分偶联的偶联部分，所述偶联部分选自下组：可检测标记物、药物、毒素、细胞因子、放射性核素、酶、或其组合。
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