WO2023143322A1 - p95HER2抗体及其应用 - Google Patents

p95HER2抗体及其应用 Download PDF

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Publication number
WO2023143322A1
WO2023143322A1 PCT/CN2023/072966 CN2023072966W WO2023143322A1 WO 2023143322 A1 WO2023143322 A1 WO 2023143322A1 CN 2023072966 W CN2023072966 W CN 2023072966W WO 2023143322 A1 WO2023143322 A1 WO 2023143322A1
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antibody
seq
antigen
cancer
chain variable
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PCT/CN2023/072966
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English (en)
French (fr)
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马晓丽
马怀远
付雅媛
曹卓晓
唐任宏
任晋生
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山东先声生物制药有限公司
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Priority to CN202380017729.7A priority Critical patent/CN118556082A/zh
Publication of WO2023143322A1 publication Critical patent/WO2023143322A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

Definitions

  • the present disclosure relates to the field of antibodies, in particular p95HER2 antibodies and applications thereof.
  • HER2 is a tumor-associated antigen of high concern, and it is widely present in high-incidence tumor populations such as breast cancer and gastric cancer. For example, HER2-positive patients account for about 20% to 25% of all breast cancers. This type of breast cancer is more aggressive and has a poorer outcome.
  • HER2-targeted therapies such as trastuzumab, pertuzumab, lapatinib, neratinib, etc.
  • trastuzumab pertuzumab
  • lapatinib neratinib
  • neratinib HER2-positive breast cancer patients great hope and more choices.
  • the current first-line standard of care for HER2-positive metastatic breast cancer is double blockade with pertuzumab (Perjeta) and trastuzumab plus chemotherapy.
  • Perjeta pertuzumab
  • trastuzumab plus chemotherapy.
  • multiple drug resistance mechanisms are involved, including the blocking of the binding region of HER2 and trastuzumab by proteins such as MUC4, or the downregulation of HER2 expression.
  • therapies targeting novel tumor-associated antigens deserve attention.
  • p95HER2 A new tumor-associated antigen, p95HER2, has been reported to be present in HER2-positive patients and predicts a worse prognosis.
  • trastuzumab-resistant patients the proportion of p95HER2-positive patients was higher.
  • p95HER2 is specifically expressed in tumor tissues, and its expression is almost undetectable in normal tissues, and the expression profile is tumor-specific. Therefore, p95HER2 becomes an ideal choice for new tumor-associated antigens. It is of great significance to develop p95HER2 antibody.
  • the present disclosure provides antibodies or antigen-binding fragments that specifically bind to human p95HER2, and corresponding multispecific antibodies or antigen-binding fragments, antibody-drug conjugates or pharmaceutically acceptable salts or solvates thereof, nucleic acids, and carriers , host cells, immune effector cells, pharmaceutical compositions, preparation methods, pharmaceutical uses, methods for treating cancer or tumors, methods for detecting p95HER2, applications for preparing p95HER2 detection kits and corresponding kits.
  • the present disclosure provides antibodies or antigen-binding fragments that specifically bind human p95HER2, comprising a heavy chain variable region (VH) and a light chain variable region (VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 18, 78 or 86, and the light chain variable region comprises SEQ ID NO : LCDR1, LCDR2 and LCDR3 contained in the VL shown in 19, 79 or 87;
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 16, 76 or 84, and the light chain variable region includes SEQ ID NO: 17, 77 or 85 LCDR1, LCDR2, and LCDR3 contained in the VL shown;
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 20, 80 or 88, and the light chain variable region includes SEQ ID NO: 21, 81 or 89 LCDR1, LCDR2 included in VL shown and LCDR3;
  • the heavy chain variable region includes HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 22, 82 or 90, and the light chain variable region includes SEQ ID NO: 23, 83 or 91 LCDR1, LCDR2, and LCDR3 contained in the indicated VL; or
  • said heavy chain variable region and said light chain variable region include each CDR in said HCDR1-3 and LCDR1-3 in any one of groups (1)-(4) , a sequence with at least 80% identity, or a sequence with at most 3 insertions, deletions or substitution mutations; preferably, the HCDR1-3 and the LCDR1-3 have the same Each of the HCDR1-3 and LCDR1-3 of any group has at least 80% identity compared to each CDR; preferably, the HCDR1-3 and the LCDR1-3 have the same identity as that of (1)-(4) ) group, compared with each CDR in HCDR1-3 and LCDR1-3 in any group, at most 3 insertions, deletions or substitution mutations occur; preferably, the substitution mutation occurs in the second of "DDD" Each "D” is preferably mutated to "DND", or the substitution mutation occurs in the "N" in "NG”, preferably mutated to "SG".
  • said at least 80% identity is 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the at most 3 insertions, deletions or substitutions are at most 2, 1 or 0 insertions, deletions or substitutions; more preferably, the substitutions are substitutions of conservative amino acid residues.
  • said HCDR1-3 and said LCDR1-3 are determined according to the Kabat numbering system, Chothia numbering system or IMGT numbering system, more preferably, are determined according to the Kabat numbering system, most preferably, said HCDR1-3 and said LCDR1-3 are selected from Table 7 or Table 12.
  • it comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, the light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the HCDR1-3 and the LCDR1-3 are selected from Table 7 or Table 12;
  • the HCDR1 is selected from SEQ ID NO:37, 40 and 42, the HCDR2 is selected from SEQ ID NO:38, 41 and 43, and the HCDR3 is selected from SEQ ID NO:39 and 44;
  • the LCDR1 is selected from From SEQ ID NO:45 and 48, said LCDR2 is selected from SEQ ID NO:46 and 49, said LCDR3 is selected from SEQ ID NO:47;
  • the HCDR1 is selected from SEQ ID NO:24,27 and 29, the HCDR2 is selected from SEQ ID NO:25,28,30 and 92, and the HCDR3 is selected from SEQ ID NO:26 and 31;
  • LCDR1 is selected from SEQ ID NO:32 and 35, said LCDR2 is selected from SEQ ID NO:33 and 36, said LCDR3 is selected from SEQ ID NO:34;
  • the HCDR1 is selected from SEQ ID NO:50, 53, 55 and 93
  • the HCDR2 is selected from SEQ ID NO:51, 54 and 56
  • the HCDR3 is selected from SEQ ID NO:52 and 57
  • LCDR1 is selected from SEQ ID NO:58 and 61
  • said LCDR2 is selected from SEQ ID NO:59 and 62
  • said LCDR3 is selected from SEQ ID NO:60;
  • the HCDR1 is selected from SEQ ID NO:63,66 and 68
  • the HCDR2 is selected from SEQ ID NO:64,67 and 69
  • the HCDR3 is selected from SEQ ID NO:65 and 70
  • the LCDR1 is selected from From SEQ ID NO:71 and 74
  • said LCDR2 is selected from SEQ ID NO:72 and 75
  • said LCDR3 is selected from SEQ ID NO:73; or,
  • said HCDR1-3 and said LCDR1-3 have at least 80% of each CDR in said HCDR1-3 and LCDR1-3 in any one of groups (1)-(4) identity, or up to 3 insertions, deletions, or substitutions Replace mutation; preferably, the HCDR1-3 and the LCDR1-3 have each CDR in any group of the HCDR1-3 and LCDR1-3 in (1)-(4) group, having At least 80% identity; preferably, said HCDR1-3 and said LCDR1-3 have the same CDR as each of said HCDR1-3 and LCDR1-3 in any one of groups (1)-(4) Ratio, at most three insertion, deletion or substitution mutations occur; preferably, the substitution mutation occurs in the second "D" in "DDD", preferably the mutation is "DND", or the substitution mutation occurs in "NG The "N” in " is preferably mutated to "SG".
  • said at least 80% identity is 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the at most 3 insertions, deletions or substitutions are at most 2, 1 or 0 insertions, deletions or substitutions; more preferably, the substitutions are substitutions of conservative amino acid residues.
  • the antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody;
  • the antibody or antigen-binding fragment is a humanized antibody
  • the heavy chain variable region and the light chain variable region include a human framework region and the HCDR1-3 and LCDR1-3 implanted therein;
  • the framework region of the heavy chain variable region includes framework region 1-3 (HFR1-3) of IGHV2-70*04 and framework region 4 (HFR4) of IGHJ6*01, and the framework region of the light chain variable region
  • the framework region includes framework region 1-3 (LFR1-3) of IGKV1-39*01 and framework region 4 (HFR4) of IGKJ4*01;
  • the framework region of the heavy chain variable region includes framework region 1-3 (HFR1-3) of IGHV2-70*01 and framework region 4 (HFR4) of IGHJ6*01, and the framework region of the light chain variable region
  • the framework region includes framework region 1-3 (LFR1-3) of IGKV3-11*01 and framework region 4 (LFR4) of IGKJ2*01;
  • the framework region of the heavy chain variable region includes framework region 1-3 (HFR1-3) of IGHV2-70*10 and framework region 4 (HFR4) of IGHJ6*01, and the framework region of the light chain variable region
  • the framework region includes framework region 1-3 (LFR1-3) of IGKV4-1*01 and framework region 4 (LFR4) of IGKJ2*01;
  • the framework region of the heavy chain variable region includes framework region 1-3 (HFR1-3) of IGHV33-7*01 and framework region 4 (HFR4) of IGHJ1*01, and the framework region of the light chain variable region
  • the framework regions include framework regions 1-3 (LFR1-3) of IGKV2-29*02 and framework region 4 (LFR4) of IGKJ2*01; or,
  • the framework region of the heavy chain variable region and the framework region of the light chain variable region comprise sequences that are at least 80% identical to each of the FRs in HFR1-4 and LFR1-4, or Sequences with at most 10 insertion, deletion or substitution mutations; preferably, numbered in natural order, (i) the heavy chain variable region includes one or more of the following mutations: I71V and A98V mutations, the light The chain variable region includes a D1L mutation; (ii) the heavy chain variable region includes a S30Y and/or K77G mutation; (iii) the heavy chain variable region includes one or more of the following mutations: L4F, G27F , I29L, S30I, V71R, N76S, F78V, S79F and A96T, the framework region of the light chain variable region includes a P43S mutation; (iv) the heavy chain variable region includes one or more of the following mutations: S30N, K76Q and A97T, the light chain variable region includes one or more of the following mutation
  • said at least 80% identity is 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the at most 10 insertions, deletions or substitution mutations are at most 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 insertions, deletions Or a substitution mutation; more preferably, the substitution mutation is a substitution of a conservative amino acid residue.
  • the antibody or antigen-binding fragment comprises:
  • the heavy chain variable region includes the sequence shown in SEQ ID NO: 18, 78 or 86, and the light chain variable region includes the sequence shown in SEQ ID NO: 19, 79 or 87;
  • the heavy chain variable region includes the sequence shown in SEQ ID NO: 16, 76 or 84, and the light chain variable region includes the sequence shown in SEQ ID NO: 17, 77 or 85;
  • the heavy chain variable region includes the sequence shown in SEQ ID NO: 20, 80 or 88, and the light chain variable region includes the sequence shown in SEQ ID NO: 21, 81 or 89;
  • the heavy chain variable region includes a sequence shown in SEQ ID NO: 22, 82 or 90, and the light chain variable region includes a sequence shown in SEQ ID NO: 23, 83 or 91; or
  • said heavy chain variable region and said light chain variable region comprise compared to said heavy chain variable region and light chain variable region in any one of groups (1)-(4), Sequences with at least 80% identity, or up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 occurrences , 2, 1 or 0 insertions, deletions or substitution mutations.
  • said at least 80% identity is 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the at most 15 insertions, deletions or substitutions are at most 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 , 3, 2, 1 or 0 insertion, deletion or substitution mutations; more preferably, the substitution mutations are substitutions of conservative amino acid residues.
  • the antibody or antigen-binding fragment also binds monkey p95HER2, preferably with a KD of less than 10 nM, 3 nM, 1 nM, 0.5 nM or 0.1 nM.
  • it comprises a heavy chain constant region sequence and/or a light chain constant region sequence selected from a complete constant region sequence or a fragment thereof, the constant region Fragments include CH1, hinge region, CH2, CH3 or Fc;
  • the heavy chain constant region is selected from human or murine IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or murine kappa constant region or lamda constant region;
  • said antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, said heavy chain consisting of said VH and a heavy chain constant region, said heavy chain constant region preferably having the following expression as shown in SEQ ID NO:9 Sequence, said light chain is made up of said VL and light chain constant region, and said light chain constant region preferably has the sequence shown in SEQ ID NO:10.
  • the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, (Fab')2, scFv or diabody.
  • the present disclosure also provides an antibody or an antigen-binding fragment specifically binding to human p95HER2, which is combined with the aforementioned antibody or antigen
  • the binding fragments compete for binding to p95HER2, or bind to the same or overlapping epitopes as the preceding antibodies or antigen-binding fragments.
  • the present disclosure also provides a multispecific antibody or antigen-binding fragment, which includes the aforementioned antibody or antigen-binding fragment that specifically binds to human p95HER2, and also includes a binding unit that binds to other antigens, preferably also includes an antibody or antigen-binding fragment that binds to HER2.
  • the disclosure also provides a multispecific antibody or antigen-binding fragment comprising a first peptide chain and a second peptide chain, and a third peptide chain and a fourth peptide chain, wherein:
  • the first peptide chain and the third peptide chain include VH1-(X1)n-VH2-CH1-Fc, the X1 is a linker 1, and n is 0 or 1;
  • the second peptide chain and the fourth peptide chain include VL1-(X2)n-VL2-CL, the X2 is linker 2, n is 0 or 1;
  • the VH1 and VL1 form a HER2 binding domain
  • the VH2 and VL2 form a p95 HER2 binding domain
  • the VH1 and VL1 form a p95 HER2 binding domain
  • the VH2 and VL2 form a HER2 binding domain.
  • the p95Her2 binding domain comprises the foregoing antibody or antigen-binding fragment.
  • the HER2 binding domain binds domain II or domain IV of the extracellular domain of HER2; preferably, the HER2 binding domain has one of the following characteristics:
  • the HER2 binding domain includes HCDR1-3 in the VH shown in SEQ ID NO: 5 and LCDR1-3 in the VL shown in SEQ ID NO: 6; preferably, the HCDR1-3 has a sequence as shown in SEQ ID The sequence shown in NO: 108-110, the LCDR1-3 has the sequence shown in SEQ ID NO: 111-113; more preferably, the HER2 binding domain includes SEQ ID NO: 5 and SEQ ID NO: 6 display sequence;
  • the HER2 binding domain includes HCDR1-3 in the VH shown in SEQ ID NO: 7 and LCDR1-3 in the VL shown in SEQ ID NO: 8; preferably, the HCDR1-3 has a sequence as shown in SEQ ID The sequence shown in NO: 102-104, the LCDR1-3 has the sequence shown in SEQ ID NO: 105-107; more preferably, the HER2 binding domain includes SEQ ID NO: 7 and SEQ ID NO: 8 display sequence;
  • the linker 1 and linker 2 are (G4S)n, where n is selected from 1, 2, 3, 4, 5 or 6;
  • the CH1-Fc has a sequence that is identical to or at least 80% identical to SEQ ID NO: 9 or has at most 20 deletion, insertion or substitution mutations;
  • the CL has a sequence identical to or at least 80% identical to SEQ ID NO: 10 or having at most 20 deletions, insertions or substitution mutations.
  • said at least 80% identity is 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the at most 20 insertions, deletions or substitutions are at most 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9 , 8, 7, 6, 5, 4, 3, 2, 1 or 0 insertion, deletion or substitution mutations; more preferably, the substitution mutations are substitutions of conservative amino acid residues.
  • the first peptide chain and the third peptide chain have the same or at least 80% identity with SEQ ID NO: 94, 96, 98 or there are at most 30 deletions, insertions or substitution mutations Sequence, the second peptide chain and the fourth peptide chain have the same or at least 80% identity with 95, 97, 99 or a sequence with at most 30 deletions, insertions or substitution mutations.
  • said at least 80% identity is 85% identity, 90% identity, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the at most 30 insertions, deletions or substitutions are at most 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19 , 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
  • the substitution mutations are substitutions of conservative amino acid residues
  • the present disclosure also provides an antibody-drug conjugate as shown in A-(L-D)n or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • A represents the aforementioned antibody or antigen-binding fragment
  • L represents a linker for connecting A and D, preferably mc-vc-pAB or mc;
  • D represents a drug, preferably dolastatin or its derivatives, more preferably MMAE or MMAF;
  • n represents the drug-to-antibody ratio (DAR), which is optionally 1-10, preferably 2 or 4.
  • DAR drug-to-antibody ratio
  • the LD used to conjugate the antibody has the structure shown below:
  • the present disclosure also provides isolated nucleic acids, wherein the nucleic acid fragments encode the aforementioned antibodies or antigen-binding fragments.
  • the present disclosure also provides a nucleic acid encoding a chimeric antigen receptor (chimeric antigen receptor, CAR), wherein the CAR includes a signal peptide, an antigen binding domain, a hinge region, a transmembrane region and an intracellular cell conduction region, and the antigen
  • the binding domain comprises the aforementioned antibody or antigen binding domain.
  • the present disclosure also provides a vector comprising the aforementioned nucleic acid.
  • the present disclosure also provides a host cell comprising the aforementioned nucleic acid or vector or expressing the aforementioned antibody or antigen-binding fragment;
  • the host cell is a prokaryotic cell or a eukaryotic cell
  • the host cell is selected from Escherichia coli, yeast, mammalian cells or other cells suitable for producing antibodies or antigen-binding fragments, such as HEK293 cells or CHO cells.
  • the present disclosure also provides an immune effector cell, which comprises the aforementioned nucleic acid or a chimeric antigen receptor whose carrier is encoded by the aforementioned nucleic acid; preferably, the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cell), monocytes, macrophages, dendritic cells, or mast cells.
  • the immune effector cell is selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cell), monocytes, macrophages, dendritic cells, or mast cells.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof or immune effector cells, and optionally a pharmaceutically acceptable carrier (carrier), and optionally other therapeutic agents.
  • the present disclosure also provides a method for preparing the aforementioned antibody or antigen-binding fragment or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof or immune effector cells, the method comprising:
  • the method further includes isolating the antibody or antigen-binding fragment; or,
  • the present disclosure also provides the aforementioned antibodies or antigen-binding fragments or the antibody-drug conjugates or pharmaceutically acceptable salts or solvates or nucleic acids or vectors or host cells or immune effector cells or pharmaceutical compositions or according to the aforementioned methods
  • the prepared product its use in the preparation of medicines for treating cancer or tumors;
  • the cancer or tumor is selected from solid tumors, for example, breast cancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, head and neck cancer, lung cancer, biliary tract cancer, urothelial cancer , liver cancer, esophageal cancer or osteosarcoma;
  • solid tumors for example, breast cancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, head and neck cancer, lung cancer, biliary tract cancer, urothelial cancer , liver cancer, esophageal cancer or osteosarcoma;
  • said cancer or tumor expresses HER2+ and/or p95HER2+;
  • the cancer or tumor exhibits drug resistance to HER2-targeted therapy, such as Trastuzumab, Pertuzumab, T-DM1, DS-8201 or RC48 drug resistance;
  • HER2-targeted therapy such as Trastuzumab, Pertuzumab, T-DM1, DS-8201 or RC48 drug resistance;
  • the drug also includes other therapeutic agents, such as chemotherapeutic drugs (taxanes, selected from docetaxel or paclitaxel; anthracyclines, selected from doxorubicin, epirubicin, daunorubicin or rubicin), or immune checkpoint inhibitors (PD-1 antibody or PD-L1 antibody).
  • chemotherapeutic drugs such as taxanes, selected from docetaxel or paclitaxel
  • anthracyclines selected from doxorubicin, epirubicin, daunorubicin or rubicin
  • immune checkpoint inhibitors PD-1 antibody or PD-L1 antibody
  • the present disclosure also provides the aforementioned antibodies or antigen-binding fragments or antibody-drug conjugates or pharmaceutically acceptable salts or solvates or nucleic acids or vectors or host cells or immune effector cells or pharmaceutical compositions or prepared according to the aforementioned methods products for the treatment of cancer or tumors;
  • the cancer or tumor is selected from solid tumors, for example, breast cancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, head and neck cancer, lung cancer, biliary tract cancer, urothelial cancer , liver cancer, esophageal cancer or osteosarcoma;
  • solid tumors for example, breast cancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, head and neck cancer, lung cancer, biliary tract cancer, urothelial cancer , liver cancer, esophageal cancer or osteosarcoma;
  • said cancer or tumor expresses HER2+ and/or p95HER2+;
  • the cancer or tumor exhibits drug resistance to HER2-targeted therapy, such as Trastuzumab, Pertuzumab, T-DM1, DS-8201 or RC48 drug resistance;
  • HER2-targeted therapy such as Trastuzumab, Pertuzumab, T-DM1, DS-8201 or RC48 drug resistance;
  • the treatment also includes other therapeutic agents, such as chemotherapeutic agents (taxanes, selected from docetaxel or paclitaxel; anthracyclines, selected from doxorubicin, epirubicin, daunorubicin or rubicin), or immune checkpoint inhibitors (PD-1 antibody or PD-L1 antibody).
  • chemotherapeutic agents such as taxanes, selected from docetaxel or paclitaxel; anthracyclines, selected from doxorubicin, epirubicin, daunorubicin or rubicin
  • immune checkpoint inhibitors PD-1 antibody or PD-L1 antibody
  • the present disclosure also provides a method for treating cancer or tumor, which comprises administering to a subject an effective amount of the aforementioned antibody or antigen Binding fragments or antibody-drug conjugates or pharmaceutically acceptable salts or solvates or nucleic acids or vectors or host cells or immune effector cells or pharmaceutical compositions or products prepared by the method;
  • the cancer or tumor is selected from solid tumors, for example, breast cancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, head and neck cancer, lung cancer, biliary tract cancer, urothelial cancer , liver cancer, esophageal cancer or osteosarcoma;
  • solid tumors for example, breast cancer, gastric cancer, bladder cancer, ovarian cancer, lung cancer, prostate cancer, pancreatic cancer, colorectal cancer, head and neck cancer, lung cancer, biliary tract cancer, urothelial cancer , liver cancer, esophageal cancer or osteosarcoma;
  • said cancer or tumor expresses HER2+ and/or p95HER2+;
  • the cancer or tumor exhibits drug resistance to HER2-targeted therapy, such as Trastuzumab, Pertuzumab, T-DM1, DS-8201 or RC48 drug resistance;
  • HER2-targeted therapy such as Trastuzumab, Pertuzumab, T-DM1, DS-8201 or RC48 drug resistance;
  • the drug also includes other therapeutic agents, such as chemotherapeutic drugs (taxanes, selected from docetaxel or paclitaxel; anthracyclines, selected from doxorubicin, epirubicin, daunorubicin or rubicin), or immune checkpoint inhibitors (PD-1 antibody or PD-L1 antibody).
  • chemotherapeutic drugs such as taxanes, selected from docetaxel or paclitaxel
  • anthracyclines selected from doxorubicin, epirubicin, daunorubicin or rubicin
  • immune checkpoint inhibitors PD-1 antibody or PD-L1 antibody
  • the present disclosure also provides a method for detecting p95HER2, which includes contacting the test sample with the antibody or antigen-binding fragment under the condition that the aforementioned antibody or antigen-binding fragment can form a complex with p95HER2;
  • detecting the formation of said complex is indicative of the presence or expression level of p95HER2 in the sample
  • the sample is a sample of a cancer or tumor patient, and according to the presence or expression level of p95HER2 in the sample, it is identified whether the patient can benefit from p95HER-specific treatment;
  • the method further includes contacting the test sample with the HER2 antibody or antigen-binding fragment under the condition that the HER2 antibody or its antigen-binding fragment can form a complex with HER2, and detecting the formation of the complex, Indicating the presence or expression level of HER2 in a sample, and optionally, said sample being a sample of a cancer or tumor patient, based on said presence or expression level of HER2, it is identified whether said patient would benefit from HER2-specific therapy.
  • the treatment includes the aforementioned methods of treating cancer or tumors
  • said detection is by immunohistochemistry (ICH).
  • the present disclosure also provides the application of the aforementioned antibody or antigen-binding fragment in the preparation of a kit for detecting p95HER2.
  • the present disclosure provides an antibody or an antigen-binding fragment thereof with high affinity for the p95HER2 target, and has good endocytic ability, and the ADC drug prepared therefrom has a good tumor killing effect, especially for trastuzumab drug-resistant patient, with good prospects for treatment.
  • FIG. 1A FACS detection results of CHO-K1 stably transfected cell line expressing human p95HER2 protein
  • FIG. 1B FACS detection results of CHO-K1 stably transfected cell line expressing human HER2 protein
  • FIG. 1C FACS detection results of HEK293T stably transfected cell line expressing monkey p95HER2 protein
  • FIG. 1D FACS detection results of SKBR3 stably transfected cell line expressing human p95HER2 protein
  • Figure 1E FACS detection results of a cell line (OE19-Tet/on-p95HER2.73#) expressing human p95HER2 protein;
  • Figure 1F FACS detection results of a cell line (T47D-Tet/on-p95HER2.3H6) expressing human p95HER2 protein;
  • FIG. 2A-2D FACS detection results of p95HER2 mouse antibody binding to CHO-K1-hu p95HER2 cells
  • FIG. 3A to Figure 3D FACS detection results of p95HER2 humanized antibody or chimeric antibody binding to CHO-K1-hu p95HER2 cells;
  • FIG. 6A-6B The killing results of p95HER2 humanized antibody on SKBR3-p95HER2 cells
  • Figure 10A- Figure 10C Schematic diagram of p95HER2/HER2 double antibody
  • Figure 11A- Figure 11B FACS detection results of p95HER2/HER2 double antibody binding to CHO-K1-hu HER2 cells
  • Fig. 12A-Fig. 12B Results of endocytosis test of p95HER2/HER2 double antibody on SKBR3-p95HER2 cells;
  • FIG. 13A-13B FACS detection results of p95HER2/HER2 double antibody ADC binding to CHO-K1-hu p95HER2 cells/CHO-K1-hu HER2 cells;
  • Figure 14A- Figure 14B The results of the killing experiment of p95HER2/HER2 double antibody ADC on SKBR3-p95HER2 cells;
  • Figure 15A- Figure 15B Results of killing experiments on OE19-p95HER2 cells
  • Figures 16A-16B are the results of killing experiments on T47D-p95HER2 cells.
  • compositions including A and B should be understood as the following technical scheme: a composition composed of A and B , and a composition containing other components in addition to A and B, all fall into Into the scope of the aforementioned "a composition”.
  • p95HER2 refers to 611-CTF or 100-115kDa p95HER2, see Cancer Res 71, 1515-1519 (2011) for details), which has the ability to form homodimers maintained by intermolecular disulfide bonds (Pedersen et al., Mol Cell Biol 29, 3319-31 (2009)).
  • p95HER2 appears to be expressed in a homogenous subset of HER2-positive breast cancers (Parra-Palau et al., JNatl Cancer Inst 106, dju291 (2014)), but is not recognized by e.g. The epitope recognized by the antibody (Pedersen et al., Mol Cell Biol 29, 3319-31 (2009)).
  • "p95HER2" herein includes human or non-human mammalian p95HER2.
  • p95HER2 can be selected from the sequence shown in SEQ ID NO:11 or SEQ ID NO:12.
  • HER2 also known as ErbB-2, NEU, HER-2, and CD340
  • HER2 includes human or non-human mammalian HER2, and when used herein refers to human epidermal growth factor receptor 2 (SwissProt P04626) and includes variants, isoforms and species homologues of any HER2 that are naturally expressed by cells (including tumor cells) or expressed on cells transfected with the HER2 gene. Unless otherwise specified, "HER2" herein does not include p95HER2.
  • an antigen-binding molecule eg, an antibody
  • an antigen-binding molecule specifically binds an antigen and substantially the same antigen with high affinity, typically, but does not bind an unrelated antigen with high affinity.
  • Affinity is usually reflected in an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity.
  • KD equilibrium dissociation constant
  • high affinity usually refers to having about 10 -7 M or lower, about 10 -8 M or lower, about 1 ⁇ 10 -9 M or lower, about 1 ⁇ 10 -10 M or lower, KD of 1 ⁇ 10 -11 M or lower or 1 ⁇ 10 -12 M or lower.
  • KD KD/Ka, where Kd represents the dissociation rate and Ka represents the on-rate.
  • the equilibrium dissociation constant KD can be measured by methods known in the art, such as surface plasmon resonance (eg, Biacore) or equilibrium dialysis. For example, refer to the method for obtaining the KD value shown in the examples herein.
  • antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations.
  • Antibody herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical "four-chain antibody”, which belongs to the immunoglobulins composed of two heavy chains (HC) and two light chains (LC); In the N-terminal to C-terminal direction, it consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain a heavy chain constant region CH2 domain
  • CH3 domain heavy chain constant region
  • the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain
  • the light chain is
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the ⁇ chain and the delta chain , ⁇ chain, ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA.
  • IgA2 Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa chain or a lambda chain.
  • Antibody herein also includes antibodies that do not comprise light chains, for example, antibodies produced from camels (Camelus dromedarius), Bactrianus (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe ) and alpacas ( Heavy-chain antibodies (HCAbs) produced by Vicugna pacos) and immunoglobulins found in cartilaginous fishes such as sharks Leukin new antigen receptor (Ig new antigen receptor, IgNAR).
  • camels Camelus dromedarius
  • Bactrianus Bactrianus
  • Camelus bactrianus Bactrianus
  • llamas Liama glama
  • guanacos Liama guanicoe
  • alpacas alpacas
  • an “antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).
  • Antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibody, chimeric antibody, humanized antibody or fully human antibody.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variants (such as containing naturally occurring mutations or arising during the manufacture of a formulation, such variants typically appear as In addition to being present in small amounts), the individual antibodies comprising the population are identical and/or bind the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), in monoclonal antibody preparations each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies can be produced by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology and the use of transgenic animals containing all or part of the human immunoglobulin loci methods and other methods known in the art.
  • natural antibody herein refers to antibodies produced and paired by the immune system of a multicellular organism.
  • engineered antibody herein refers to a non-natural antibody obtained through genetic engineering, antibody engineering and other techniques.
  • engineered antibody includes humanized antibodies, small molecule antibodies (such as scFv, etc.), specific antibodies, etc.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.
  • multispecific antibody herein refers to an antibody having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen. bit binding.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.
  • full-length antibody intact antibody
  • intact antibody intact antibody
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein, and do not possess the full structure of an intact antibody, but only include partial or partial variants of an intact antibody that possess the ability to bind Antigen capacity.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, and scFv.
  • Papain digestion of intact antibodies yields two identical antigen-binding fragments, termed "Fab” fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy-terminus of the CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region.
  • Fab'-SH is one in which the cysteine residues of the constant domains carry a free thiol group Fab' fragments. Pepsin treatment yields an F(ab')2 fragment with two antigen-combining sites (two Fab fragments) and part of the Fc region.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al, Proc. New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur.J. Immunol.31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001 ), Cancer Immunol.
  • diabody herein, whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thus forcing the domains to The complementary domains of the other chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • Chimeric antibody herein refers to an antibody whose light chain and/or heavy chain are partly derived from an antibody (which may be derived from a specific species or belong to a specific class or subclass of antibodies). class), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the Binding activity to target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).
  • a primary antibody e.g., a murine antibody
  • the light chain constant region is from a second antibody (eg, a human antibody).
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) is derived from a human Immunoglobulin (receptor antibody).
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.
  • Fully human antibody refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • another mammalian species eg, mouse
  • variable region herein refers to the region of an antibody heavy or light chain that is involved in enabling the antibody to bind antigen
  • VH heavy chain variable region
  • HCVR heavy chain variable region
  • VL light chain variable region
  • the variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p.91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). It can form a precise complementarity with the antigen epitope, so it is also called complementarity determining region.
  • the CDR of the variable region of the heavy chain can be abbreviated as HCDR
  • the CDR of the variable region of the light chain can be abbreviated as LCDR.
  • framework region or “FR region” are used interchangeably and refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs.
  • CDRs For further descriptions of CDRs, refer to Kabat et al., J.Biol.Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest” (1991); People such as Chothia, J.Mol.Biol.196:901-917 (1987); People such as Al-Lazikani B., J.Mol.Biol., 273:927-948 (1997); People such as MacCallum, J.Mol .Biol.262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P.
  • CDR herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but not limited to AbRSA website (http://cao.labshare.
  • CDRs herein include overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
  • IMGT numbering system herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol. 27:55-77, 2003.
  • IMGT ImMunoGeneTics information system
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • the "heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • Heavy chain constant region includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region”part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • a typical "heavy chain constant region segment" can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxy-terminal part of the antibody obtained by papain hydrolysis of the whole antibody, which typically includes the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, thus the Fc region can comprise or excluding Lys447.
  • the numbering of amino acid residues of "antibody” or “antigen-binding fragment” herein is determined by the Kabat numbering system, see, for details, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • numbering in natural order refers to numbering in natural order directly according to the position of the amino acid residues in the sequence.
  • the 30th amino acid residue numbered according to the natural order refers to the amino acid residue at the 30th position in the sequence according to the natural order
  • S30Y means that, according to the natural order, the S mutation position at the 30th position in the sequence Y.
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • identity may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between two sequences will vary with the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6, determines the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described in the present disclosure may further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402.
  • the default parameters of the respective programs eg, XBLAST and NBLAST
  • XBLAST and NBLAST the default parameters of the respective programs. See www.ncbi.nlm.nih.gov.
  • antigen chimeric receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically binds an antigen, comprising at least (1) an extracellular antigen binding domain, such as an antibody (2) the hinge region; (3) the transmembrane domain that anchors the CAR into immune effector cells; and (4) the intracellular signaling domain.
  • CARs are able to redirect T cells and other immune effector cells to a target of choice, such as cancer cells, in a non-MHC-restricted manner using an extracellular antigen-binding domain.
  • nucleic acid includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of antibodies of the present disclosure in vitro and/or in vivo, eg, in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified.
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce antibodies in vivo (see e.g.
  • isolated nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or in a location other than its natural chromosomal location at the chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.
  • Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) unwanted physiological changes or pathological changes in the subject of treatment, such as cancer, autoimmune diseases and viral infections. progress.
  • Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented.
  • slow down lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.
  • subject herein refers to an organism receiving treatment for a particular disease or condition as described in this disclosure.
  • subjects and patients include mammals, such as humans, primate (eg, monkeys) or non-primate mammals, receiving treatment for a disease or disorder.
  • an effective amount herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition.
  • a therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • autoimmune disease refers to a condition in which cells, tissues and/or organs are damaged by a subject's immune response to its own cells, tissues and/or organs.
  • cancer refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.
  • the p95HER2 extracellular region fusion protein with tag was designed, respectively Cloned to the pTT5 vector, transiently expressed in HEK293 cells, and purified to obtain proteins for immunizing animals, screening antibodies and/or detecting antibody functions: hu p95HER2.ECD-Fc and cyno p95HER2.ECD-Fc. See Table 5 for the specific sequence information of the fusion protein.
  • the cell culture supernatant expressing the protein was collected by high-speed centrifugation.
  • the Protein A affinity column was washed with 6M guanidine hydrochloride for 3-5 times the column volume, and then with pure water for 3-5 times the column volume.
  • the cell supernatant is loaded and combined at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer.
  • solution replacement can be performed by methods well known to those skilled in the art, such as using ultrafiltration tubes for ultrafiltration concentration and solution replacement to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required A buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove aggregate components in the eluted product to improve sample purity.
  • the nucleic acid sequences encoding antibody VH and VL were recombined into the expression vector pTT5 with signal peptide and heavy chain constant region/light chain constant region sequences to obtain a recombinant plasmid expressing VH-CH1-Fc/VL-CL.
  • 611mab-hIgG1, trastuzumab-hIgG1 and pertuzumab-hIgG1 recombined the corresponding VH and VL into human CH1-Fc and human CL
  • 611mab-mIgG2a recombined VH and VL into mouse CH1-Fc and mouse CL.
  • the plasmid was extracted and transfected into host cells. After culturing, cell culture supernatants secreting antibodies were obtained. Referring to the method described in Example 1, the antibody was purified from the supernatant by using Protein A affinity chromatography.
  • Protein A affinity chromatography For details of the molecular biology operations involved in the above method, see “Molecular Cloning Experiment Guide (Third Edition)", (USA) J. Sambrook et al.
  • the VH and VL sequences of the positive control antibodies 611mab-hIgG1 and 611mab-mIgG2a are respectively from Chinese patent application publication CN109843926A, and 611mab-hIgG1 and 611mab-mIgG2a can specifically bind human p95HER2 and monkey p95HER2.
  • the VH and VL sequences of Trastuzumab-hIgG1 were obtained from U.S. published patent documents This US5821337A is able to bind domain IV of human HER2 extracellular domain.
  • the VH and VL of Pertuzumab-IgG1 are from the Chinese patent authorization text CN100340575C, which can bind to the domain II of the extracellular domain of human HER2. See Table 5 for specific sequence information.
  • Nucleotide sequences encoding human p95HER2, human HER2 or monkey p95HER2 protein were cloned into pcDNA3.1 vector and plasmids were prepared. Plasmid transfection ( 3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015) to the cell line, afterward, in the DMEM/F12 medium containing 10 ⁇ g/ml puromycin and 10% (v/v) fetal bovine serum selective culture 2 weeks, Use the primary and secondary antibodies to enrich the cell population expressing the target protein at a high level on the flow cytometer FACS Aria III (BD Biosciences) to a 6-well plate, and culture it at 37°C, 5% (v/v) CO 2 , digest the 6-well plate cells for expansion after about 1 week.
  • Plasmid transfection 3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015
  • the amplified cells were detected by flow cytometry (FACS), and the positive cell population with good growth, high fluorescence intensity and good uniformity was selected to continue to expand and culture and cryopreserved in liquid nitrogen. See Table 1 for materials and reagents, and see Figures 1A-1C and Table 2 for FACS results.
  • the nucleotide sequence encoding the amino acid sequence of human p95HER2 was cloned into the pLVX lentiviral vector, and virus particles were prepared in HEK293T cells. After SKBR3 cell line (ATCC, HTB-30) was infected with lentivirus, it was selectively cultured in McCoy's 5A Medium medium containing 2 ⁇ g/ml puromycin and 10% (v/v) fetal bovine serum for 2 weeks.
  • the p95HER2 antibody (611mab-hIgG1, self-produced, see Example 2) and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088) were sorted on the flow cytometer FACS Aria III (BD Biosciences) to sort the positive pool Cells were put into 6-well plates and cultured at 37°C with 5% (v/v) CO 2 . After about 1 week, some cells were selected for expansion. The expanded cells were screened by flow cytometry. Select the positive cell population with good growth, high fluorescence intensity and good uniformity to continue to expand the culture and freeze in liquid nitrogen. FACS results are shown in Table 3 and Figure 1D.
  • nucleotide sequence encoding the amino acid sequence of human p95HER2 was cloned into the pTRIPZ empty lentiviral vector, and virus particles were prepared in HEK293T cells.
  • OE19 cell line After lentiviral infection of OE19 cell line (Kebai, CBP60495), in 1640 Medium+ containing 1 ⁇ g/ml puromycin and 10% (v/v) fetal bovine serum (to Tetracycline) Selectively culture in 1% PS medium for 2 weeks, take part of the cells and add 1 ⁇ g/ml DOX (Tetracycline) to induce culture for 48 hours, use anti-human p95HER2 antibody (611mab-hIgG1, self-produced, see Example 2) and goat anti-human IgG (H+L) antibody (Jackson, catalog number: 109605088) was used to detect the expression of p95HER2 on OE19 pool cells on a flow cytometer FACS Cantol II (purchased from BD Biosciences).
  • the limited dilution method was used to spread the plate, and the 96-well plate was spread at a density of 1-2 cells per well, and placed at 37° C., 5% (v/v) CO 2 for culture. Two weeks later, the grown clones were selected and transferred to a 48-well plate, and about 1 week later, cell wells with a confluence of about 70% of the cell wells in the 48-well plate were selected to be expanded to a 24-well plate. Part of the cells were induced and cultured with 1 ⁇ g/ml DOX (Tetracycline) for 48 hours, and detected and screened by flow cytometry.
  • DOX Tetracycline
  • T47D cells After lentivirus infection of T47D cell line (purchased from ATCC), in DMEM Medium+10ug/ml containing 1 ⁇ g/ml puromycin and 10% (v/v) fetal bovine serum (to Tetracycline) Selectively culture in bovine insulin medium for 2 weeks, take some cells and add 1 ⁇ g/ml DOX (Tetracycline) to induce culture overnight, and use anti-human p95HER2 antibody (611mab-hIgG1, self-produced, see Example 2) and goat anti-human IgG ( H+L) antibody (Jackson, catalog number: 109605088) sorted p95HER2 positive single cells on the flow cytometer FACS Aria III (purchased from BD Biosciences) to a 96-well plate, and placed at 37 ° C, 5% (v/v ) CO 2 culture, after about 2 weeks, some clones with better growth were selected for expansion. The amplified clones were screened by
  • Anti-human p95HER2 monoclonal antibody was produced by immunizing mice.
  • Experimental balb/c mice female, 6-8 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Breeding environment SPF grade. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12/12 hour light/dark cycle adjustment, a temperature of 20-25° C., and a humidity of 40-60%. The acclimatized mice were immunized according to the following scheme.
  • Immunogen hup95HER2.ECD-Fc protein, 25 ⁇ g/mouse/time, injected subcutaneously, plantar, or intraperitoneally.
  • Immunization method Immunogen mixed with Titermax, Alum or CPG, injected subcutaneously, plantarally or intraperitoneally on the 0th day, and boosted immunization every 7 days after that, subcutaneously or subcutaneously and plantarly injected in turn. Blood was collected on the 21st, 35th, 49th, and 63rd day, and the antibody titer in the mouse serum was determined by ELISA method.
  • Splenic lymphocytes were fused with myeloma cells Sp2/0( CRL-8287 TM ) were fused to obtain hybridoma cells.
  • Fused hybridoma cells were mixed with complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ bovine insulin, 1 ⁇ non-essential amino acid, 1 ⁇ double antibody, 1 ⁇ IL) at a density of 0.5-1 ⁇ 10 5 /ml -6 DMEM medium) resuspended, 200 ⁇ l/well seeded in a 96-well plate, and incubated at 37° C., 5% CO 2 for 7-11 days until the formation of pinpoint-like clones.
  • DMEM medium containing 10% FBS, 1 ⁇ HT and 1 ⁇ bovine insulin, 1 ⁇ non-essential amino acid, 1 ⁇ double antibody
  • 37°C 5% CO 2
  • ELISA or FACS detection After culturing for 1 day, perform ELISA or FACS detection.
  • hybridoma culture supernatant and hup95HER2.ECD-Fc protein was detected by ELISA method.
  • the combination of the supernatant of positive wells detected by ELISA with CHO-K1-hu p95HER2 and CHO-K1 cells was further detected by FACS.
  • FACS FACS-activated cell sorting
  • Subcloned cells also need to carry out hu p95HER2.ECD-Fc protein ELISA, CHO-K1-hu p95HER2 cell binding experiment and CHO-K1 cell binding experiment.
  • the hybridoma clones were screened through the above experiments, and the antibodies were further prepared by the serum-free cell culture method, and the antibodies were purified according to the purification method described in Example 1 for subsequent use.
  • the procedure for cloning sequences from positive hybridomas was as follows. Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, Cat No. 15596-018) according to the instructions of the kit, and reverse transcribed with PrimeScript TM Reverse Transcriptase Kit (Takara, Cat No. 2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and then sent to a sequencing company for sequencing. The amino acid sequences of the murine anti-human p95HER2 antibodies Mab01, Mab02, Mab03 and Mab04 are shown in the table below.
  • the CDR region of the above-mentioned p95HER2 monoclonal antibody was analyzed and analyzed using bioinformatics methods, wherein the CDR region was determined and annotated using the Kabat numbering system, the Chothia numbering system and the IMGT numbering system (Kabat and Chothia numbering systems: http:// www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi; IMGT numbering system: http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results), the specific results are shown in Table 7.
  • Example 2 the nucleic acid sequences encoding the murine antibody VH and VL shown in Example 4 were recombined into the expression vector pTT5 with signal peptide and heavy chain constant region/light chain constant region to construct and express the full-length chimeric antibody Recombinant plasmid of VH-CH1-Fc/VL-CL, and prepare corresponding chimeric antibodies ChAb01, ChAb02, ChAb03 and ChAb04, the constant regions of chimeric antibodies are human CH1-Fc and human CL, the preparation method and constant region sequence are detailed See Example 2 and Table 5.
  • amino acid residues of the antibodies in this example are numbered in natural order, and the CDR regions are determined by the Kabat numbering system .
  • the humanized templates of Mab01, Mab02, Mab03, and Mab04 are shown in Table 8; the sequences of the humanized templates transplanted with mouse antibody CDRs are shown in Table 9; back mutations or hotspots were performed on the basis of the sequences shown in Table 9 Mutations, the mutation information is shown in Table 10, the final optimized humanized antibody and the corresponding sequence are shown in Table 11, and the corresponding CDRs are shown in Table 12.
  • Example 2 express and purify anti-p95HER2 humanized full-length antibodies: Hab01.11a, Hab02.11, Hab03.11a and Hab04.11, wherein the constant region of the full-length humanized antibody is human CH1-Fc and human CL, the preparation method and the constant region sequence are detailed in Example 2 and Table 5.
  • the binding ability of the antibody to human p95HER2 was detected by FACS experiment between the antibody and the CHO-K1-hu p95HER2 cell line.
  • the specific experimental method is as follows:
  • Collect CHO-K1-hu p95HER2 cells adjust the cell density to 5 ⁇ 10 5 cells/ml, add to 96-well culture plate (Corning, Cat No.3799) at a volume of 100 ⁇ l/well, centrifuge at 1500 rpm at 4°C for 5 min . Discard the supernatant, add 250 ⁇ l/well PBS (HyClone, Cat No. SH30256.01), centrifuge at 1500 rpm at 4°C for 5 min. After discarding the supernatant, add 100 ⁇ l/well of different concentrations of the antibody to be tested diluted with sample diluent (2% BSA-PBS), and incubate in a 4°C refrigerator for 1 hour.
  • sample diluent 2% BSA-PBS
  • the binding ability of antibody to monkey p95HER2 was detected by FACS experiment of antibody and 293T-cyno p95HER2 cells.
  • the specific experimental method is as follows:
  • 293T-cyno p95HER2 cells were collected, and the cell density was adjusted to 5 ⁇ 10 5 cells/ml, added to a 96-well culture plate (Corning, Cat No. 3799) at a volume of 100 ⁇ l/well, and centrifuged at 1500 rpm at 4°C for 5 min. Discard the supernatant, add 50 ⁇ l/well of 4% FBS-PBS buffer, and incubate in a refrigerator at 4°C for 1 hour. After the incubation, centrifuge at 4°C, 1500rpm, for 5min.
  • Biacore 8K (GE) instrument was used to determine the affinity of p95HER2 antibody to be tested with human p95HER2 (hup95HER2.ECD-Fc) and monkey P95HER2 (cyno p95HER2.ECD-Fc) by multi-cycle kinetics.
  • the experimental running buffer is 1 ⁇ HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (Cat.#BR-1006-69, GE), the temperature of the flow-through cell is set to 25°C, and the sample The chamber temperature was set at 16°C. Both were pretreated with running buffer.
  • Use the ProteinA biosensor chip (Cat.#29127556, GE) to capture a certain amount of the antibody to be tested, and then flow a certain concentration of human and monkey p95HER2 antigens on the surface of the chip, and use the Biacore 8K instrument (GE) to detect the reaction signal in real time
  • association and dissociation curves are obtained.
  • the pH 1.5 glycine-hydrochloric acid regeneration solution (Cat.#BR-1003-54, GE) washes and regenerates the antigen-antibody complex. Specifically, the binding process was detected by injecting different concentrations of human p95HER2 and monkey p95HER2 antigens in the solution for 240s. The flow rate was 30 ⁇ L/min, starting from 50 nM, diluting 1:1, and setting a series of concentration gradients; the dissociation time was long Up to 900s, and finally washed with 10mM glycine-hydrochloric acid solution (pH 1.5) at a flow rate of 30 ⁇ L/min for 30s to complete the regeneration of the chip surface.
  • 10mM glycine-hydrochloric acid solution pH 1.5
  • MAb04 can be used as a p95HER2-specific IHC staining antibody.
  • the specific detection method is as follows: respectively use SKBR3 cell slices, SKBR3-p95HER2 cell slices, SKBR3 tumor slices, SKBR3-p95HER2 tumor slices, and PDX (patient-drivenxenotransplantation, PDX) slices IHC staining.
  • the staining steps are as follows: paraffin sections were dewaxed and hydrated by xylene and graded alcohol in an automatic tissue dehydrator.
  • Use EDTA repair solution (PH9.0) Fluzhou Maixin Biotechnology, dilution ratio 1:50) to carry out antigen retrieval by high-pressure heat restoration.
  • the repaired slices were naturally cooled to room temperature, and the slices were washed with PBS three times for 5 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide solution (abcam, ab64218) and incubated for 15 min. Sections were washed with PBS, 3 times for 5 min. The sections were blocked with 10% normal goat serum for 20 minutes to block non-specific staining. Shake off the serum, add the primary antibody (Dako antibody diluent) dropwise directly, and incubate overnight at 4°C in a wet box. Take out and rewarm to room temperature, wash the slices with PBST (0.05% Tween 20), 10 min 3 times.
  • PBST 0.05% Tween 20
  • the HRP-labeled secondary antibody corresponding to the primary antibody was added dropwise, incubated at room temperature for 60 min, and the sections were washed with PBST for 3 times for 10 min. Configure DAB chromogenic solution for color development under the microscope, and uniform color development time for each slice. After fully washing with water, counterstain with hematoxylin on an automatic tissue staining machine, and seal the slides after dehydration.
  • the detection of the endocytic ability of the anti-human p95HER2 antibody was performed by combining the antibody with DT3C protein (M.Yamaguchi et al, Biochem.Biophys.Res.Commun., 2014 Nov 28; 454(4):600-3.doi:10.1016/ j.bbrc.2014.10.133) to simulate a DAR2 ADC, and detect the killing effect of the antibody-DT3C complex on cells to characterize.
  • DT3C protein M.Yamaguchi et al, Biochem.Biophys.Res.Commun., 2014 Nov 28; 454(4):600-3.doi:10.1016/ j.bbrc.2014.10.133
  • the SKBR3-p95HER2 overexpression cell line was digested with trypsin, resuspended in McCoy's 5A medium containing 10% fetal bovine serum, seeded into a 96-well flat bottom plate at a density of 5000 cells/well, and placed at 37°C for 5 Incubate for 24 hours in a % CO 2 incubator.
  • the antibody was serially diluted with a culture medium containing 10% fetal bovine serum, then mixed with equal volumes of DT3C solution with twice the mass concentration, and allowed to stand at room temperature for 30 minutes.
  • the conjugate of the antibody and DT3C was added to the cells and cultured in a 37°C, 5% CO 2 incubator for 72 hours.
  • the SKBR3-p95HER2 overexpression cell line was digested with trypsin, resuspended in McCoy's 5A medium containing 10% fetal bovine serum, seeded into a 96-well flat bottom plate at a density of 5000 cells/well, and placed at 37°C for 5 Incubate for 24 hours in a % CO 2 incubator.
  • the antibody was serially diluted with medium containing 10% fetal bovine serum, then Hab02.11-MMAE ADC molecule (DAR4) or its control molecule was added to the cells, and placed in a 37°C, 5% CO 2 incubator for 72 Hour.
  • the tumor cell line OE19 cell line stably expressing the p95HER2 tet-on system or the tumor cell line T47D cell line stably expressing the p95HER2 tet-on system were induced with 2 ⁇ g/mL doxycycline for 3 days, digested and collected with trypsin, and treated with 2 ⁇ g/mL doxycycline medium was resuspended, seeded into 96-well flat-bottom plates at a density of 3500 cells/well, and incubated in a 37°C, 5% CO 2 incubator for 24 hours.
  • the experimental results are shown in Figures 8-9. The results show that Hab02.11-MMAE ADC has a good killing effect on OE19-p95HER2 cells and T47D-P95HER2 cells, indicating that it has a good ability to kill cells expressing target antigens .
  • the antibody shown in Figure 10A is a DVD-Ig antibody composed of VH1-linker-VH2-CH1-Fc and VL1-linker-VL2-CL, wherein VH1 and VL1 target HER2, and VH2 and VL2 target p95HER2 , linker can be omitted.
  • the antibody shown in Figure 10B is a DVD-Ig antibody composed of VH1-linker-VH2-CH1-Fc and VL1-linker-VL2-CL, wherein VH1 and VL1 target p95HER2, and VH2 and VL2 target HER2 , linker can be omitted.
  • the antibody shown in Figure 10C is a four-chain antibody consisting of VH1-CH1-Fc and VL1-CL-VH2-linker-VL2, wherein VH1 and VL1 target p95HER2, VH2 and VL2 target HER2, and the linker can omitted.
  • the expression plasmids for the light and heavy chains of the bispecific antibody were respectively constructed on the pTT5 vector, expressed in HEK293 cells, and then purified by the method of Protein A affinity chromatography described in Example 1. After purification, the protein that meets the purity requirements is dialyzed to change the medium and undergo subsequent testing to evaluate different forms of p95HER2/HER2 double antibody forms.
  • the binding ability of the p95HER2/HER2 double antibody to HER2 was detected by FACS experiments between the antibody and the CHO-K1-hu HER2 cell line.
  • the specific experimental method is as follows:
  • Collect CHO-K1-hu HER2 cells adjust the cell density to 5 ⁇ 10 5 cells/ml, add to 96-well culture plate (Corning, Cat No.3799) at a volume of 100 ⁇ l/well, centrifuge at 1500 rpm at 4°C for 5 min . Discard the supernatant, add 250 ⁇ l/well PBS (HyClone, Cat No. SH30256.01), centrifuge at 1500 rpm at 4°C for 5 min. After discarding the supernatant, add 100 ⁇ l/well of different concentrations of the antibody to be tested diluted with sample diluent (2% BSA-PBS), and incubate in a 4°C refrigerator for 1 hour.
  • sample diluent 2% BSA-PBS
  • BsAb02-P (10A shows the double antibody structure, and the HER2 antibody partially targets the domain II of the extracellular domain of HER2 ) is better than BsAb02-P-B (double antibody domain shown in Figure 10B, HER2 antibody partially targets domain II of HER2 extracellular domain), and is also better than BsAb02-T (double antibody shown in Figure 10A domain, the HER2 antibody portion targets Domain IV of the HER2 extracellular domain).
  • a Biacore 8K (GE) instrument was used to determine the affinity of the antibody BsAb02-P to be tested to human p95HER2 (hup95HER2.ECD-Fc) and HER2 by using multi-cycle kinetics.
  • the experimental running buffer is 1 ⁇ HBS-EP+ buffer solution (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (Cat.#BR-1006-69, GE), the temperature of the flow-through cell is set to 25°C, and the sample The chamber temperature was set at 16°C. Both were pretreated with running buffer.
  • Use ProteinA biosensor chip (Cat.#29127556, GE) to affinity capture a certain amount of antibody to be tested, and then flow a certain concentration of human p95HER2 antigen or human HER2 on the surface of the chip
  • the Biacore 8K instrument (GE) was used to detect the reaction signal in real time to obtain the association and dissociation curves.
  • the pH 1.5 glycine-hydrochloric acid regeneration solution (Cat.#BR-1003-54, GE) washes and regenerates the antigen-antibody complex. Specifically, the binding process was detected by injecting different concentrations of human p95HER2 and human HER2 antigen in the solution for 240 s, the flow rate was 30 ⁇ L/min, starting from 50 nM, diluting 1:1, and setting a series of concentration gradients; the dissociation time was long Up to 900s, and finally washed with 10mM glycine-hydrochloric acid solution (pH 1.5) at a flow rate of 30 ⁇ L/min for 30s to complete the regeneration of the chip surface.
  • 10mM glycine-hydrochloric acid solution pH 1.5
  • the binding ability of p95HER2/HER2 double antibody BsAb02-P and corresponding double antibody ADC to p95HER2 and HER2 was detected by FACS experiment.
  • the cells were collected into a 96-well cell plate, and the strength of the signal after the antibody was added was used to determine the binding activity of the antibody to CHO-K1-hup95HER2 and CHO-K1-hu HER2.
  • the specific experimental method is as follows:
  • Collect CHO-K1-hu p95HER2 or CHO-K1-hu HER2 cells adjust the cell density to 5 ⁇ 10 5 cells/ml, add to 96-well culture plate (Corning, Cat No.3799) at a volume of 100 ⁇ l/well, 4°C, 1500rpm, 5min centrifugation. Discard the supernatant, add 250 ⁇ l/well PBS (HyClone, CatNo.SH30256.01), and centrifuge at 1500 rpm at 4°C for 5 minutes.
  • the experimental results are shown in Figs. 13A to 13B.
  • the experimental results show that BsAb02-P and its ADC have a good binding effect on CHO-K1-hu p95HER2 cells and CHO-K1-hu HER2, and the coupling of MMAE toxin does not affect the binding of the antibody to the target protein.
  • SKBR3 cell line and SKBR3-p95HER2 overexpression cell line were digested with trypsin, resuspended in McCoy's 5A medium containing 10% fetal bovine serum, seeded into 96-well flat-bottom plate at a density of 5000 cells/well, and placed in Incubate in a 37°C, 5% CO 2 incubator for 24 hours.
  • the antibody was serially diluted with a medium containing 10% fetal bovine serum, and then BsAb02-P-MMAE or its control molecule was added to the cells, and cultured in a 37°C, 5% CO 2 incubator for 72 hours.
  • the OE19 cell line a tumor cell line stably expressing the p95tet-on system, was induced with 2 ⁇ g/mL doxycycline for 3 days, digested with trypsin and collected, resuspended in medium containing 2 ⁇ g/mL doxycycline, and incubated at 3500 Cells/well were seeded into 96-well flat-bottom plates and incubated in a 37°C, 5% CO 2 incubator for 24 hours.
  • the antibody was serially diluted with a medium containing 2 ⁇ g/mL doxycycline, then BsAb02-P-MMAE or its control molecule was added to the cells, and placed in a 37°C, 5% CO 2 incubator for 7 days.
  • the tumor cell line T47D cell line stably expressing the p95tet-on system was induced with 2 ⁇ g/mL doxycycline for 3 days, digested with trypsin and collected, resuspended in medium containing 2 ⁇ g/mL doxycycline, and incubated at 3500 Cells/well were seeded into 96-well flat-bottom plates and incubated in a 37°C, 5% CO 2 incubator for 24 hours.

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Abstract

提供了p95HER2抗体及其应用，具体提供了特异性结合人p95HER2的抗体或抗原结合片段、相应的多特异性抗体或抗原结合片段、抗体-药物偶联物、核酸、载体、细胞、组合物、制备方法、制药用途以及治疗方法、检测方法和相应试剂盒，对p95HER2相关治疗或检测具有重要意义。

Description

p95HER2抗体及其应用
相关专利申请的交叉引用
本申请要求2022年01月26日向中国国家知识产权局提交的，专利申请号为202210094806.6，发明名称为《p95HER2抗体及其应用》的中国专利申请的优先权。上述在先申请的全文通过引用的方式并入本申请中。
技术领域
本公开涉及抗体领域，具体而言，涉及p95HER2抗体及其应用。
背景技术
HER2是关注度极高的一个肿瘤相关抗原，在乳腺癌、胃癌等高发病率肿瘤人群中均广泛存在。譬如，HER2阳性病人约占全部乳腺癌的20％～25％，该类型乳腺癌侵袭性较高、结局差。
HER2靶向疗法，比如曲妥珠单抗、帕妥珠单抗、拉帕替尼、来那替尼等，给了HER2阳性乳腺癌患者巨大的希望和更多的选择。目前对HER2阳性转移性乳腺癌的一线护理标准是使用帕妥珠单抗(Perjeta)和曲妥珠单抗加化疗的双重阻断。但是仍有一部分患者对药物没有响应，或是在治疗一段时间后对药物产生了耐药性。在这些患者中，有多种耐药机制参与，包括HER2和曲妥珠单抗的结合区域被MUC4等蛋白遮挡，或者HER2的表达下调。在这种情况下，针对新的肿瘤相关抗原的疗法值得关注。据报道，HER2阳性患者中存在的新肿瘤相关抗原p95HER2，且预示着更差的预后。曲妥珠耐药的病人中，p95HER2阳性的病人比例更高。而且，p95HER2特异地在肿瘤组织表达，在正常组织几乎检测不到其表达，表达谱具有肿瘤特异性。因此，p95HER2成为理想的新肿瘤相关抗原的选择。开发p95HER2抗体具有重要意义。
发明内容
本公开提供特异性结合人p95HER2的抗体或抗原结合片段，和相应的多特异性抗体或抗原结合片段，抗体-药物偶联物或其在药学上可接受的盐或溶剂合物，核酸，载体，宿主细胞，免疫效应细胞，药物组合物，制备方法，制药用途，治疗癌症或肿瘤的方法，检测p95HER2的方法，制备p95HER2检测试剂盒的应用和相应的试剂盒。
本公开提供特异性结合人p95HER2的抗体或抗原结合片段，其包括重链可变区(VH)和轻链可变区(VL)。
在一些实施方式中，(1)所述重链可变区包括SEQ ID NO:18，78或86所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:19，79或87所示VL中所含的LCDR1、LCDR2和LCDR3；
(2)所述重链可变区包括SEQ ID NO:16，76或84所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:17，77或85所示VL中所含的LCDR1、LCDR2和LCDR3；
(3)所述重链可变区包括SEQ ID NO:20，80或88所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:21，81或89所示VL中所含的LCDR1、LCDR2 和LCDR3；
(4)所述重链可变区包括SEQ ID NO:22，82或90所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:23，83或91所示VL中所含的LCDR1、LCDR2和LCDR3；或
(5)所述重链可变区和所述轻链可变区包括与第(1)-(4)组的任一组中所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性的序列，或至多发生3个插入、缺失或替换突变的序列；优选地，所述HCDR1-3和所述LCDR1-3具有与第(1)-(4)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性；优选地，所述HCDR1-3和所述LCDR1-3具有与第(1)-(4)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，至多发生3个插入、缺失或替换突变；优选地，所述替换突变发生在“DDD”中的第二个“D”，优选突变为“DND”，或所述替换突变发生在“NG”中的“N”，优选突变为“SG。
优选地，所述至少80％同一性为85％同一性，90％同一性，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性。
优选地，所述至多发生3个插入、缺失或替换突变为至多发生2个、1个或0个插入、缺失或替换突变；更优选地，所述替换突变为保守氨基酸残基的替换。
优选地，所述HCDR1-3和所述LCDR1-3根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定，更优选地，根据Kabat编号系统确定，最优选地，所述HCDR1-3和所述LCDR1-3选自表7或表12。
在一些实施方式中，其包括重链可变区(VH)和轻链可变区(VL)，所述重链可变区包括HCDR1、HCDR2和HCDR3，所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中，所述HCDR1-3和所述LCDR1-3选自表7或表12；
优选地，
(1)所述HCDR1选自SEQ ID NO:37，40和42，所述HCDR2选自SEQ ID NO:38，41和43，所述HCDR3选自SEQ ID NO:39和44；所述LCDR1选自SEQ ID NO:45和48，所述LCDR2选自SEQ ID NO:46和49，所述LCDR3选自SEQ ID NO:47；
(2)所述HCDR1选自SEQ ID NO:24，27和29，所述HCDR2选自SEQ ID NO:25，28，30和92，所述HCDR3选自SEQ ID NO:26和31；所述LCDR1选自SEQ ID NO:32和35，所述LCDR2选自SEQ ID NO:33和36，所述LCDR3选自SEQ ID NO:34；
(3)所述HCDR1选自SEQ ID NO:50，53，55和93，所述HCDR2选自SEQ ID NO:51，54和56，所述HCDR3选自SEQ ID NO:52和57；所述LCDR1选自SEQ ID NO:58和61，所述LCDR2选自SEQ ID NO:59和62，所述LCDR3选自SEQ ID NO:60；
(4)所述HCDR1选自SEQ ID NO:63，66和68，所述HCDR2选自SEQ ID NO:64，67和69，所述HCDR3选自SEQ ID NO:65和70；所述LCDR1选自SEQ ID NO:71和74，所述LCDR2选自SEQ ID NO:72和75，所述LCDR3选自SEQ ID NO:73；或，
(5)所述HCDR1-3和所述LCDR1-3具有与第(1)-(4)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性，或至多发生3个插入、缺失或替 换突变；优选地，所述HCDR1-3和所述LCDR1-3具有与第(1)-(4)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性；优选地，所述HCDR1-3和所述LCDR1-3具有与第(1)-(4)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，至多发生3个插入、缺失或替换突变；优选地，所述替换突变发生在“DDD”中的第二个“D”，优选突变为“DND”，或所述替换突变发生在“NG”中的“N”，优选突变为“SG”。
优选地，所述至少80％同一性为85％同一性，90％同一性，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性。
优选地，所述至多发生3个插入、缺失或替换突变为至多发生2个、1个或0个插入、缺失或替换突变；更优选地，所述替换突变为保守氨基酸残基的替换。
在一些实施方式中，所述抗体或抗原结合片段为鼠源抗体，嵌合抗体，人源化抗体或全人抗体；
优选地，所述抗体或抗原结合片段为人源化抗体，所述重链可变区和轻链可变区包括人源框架区和植入其中的所述HCDR1-3和LCDR1-3；
更优选地，
(1)所述重链可变区的框架区包括IGHV2-70*04的框架区1-3(HFR1-3)和IGHJ6*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV1-39*01的框架区1-3(LFR1-3)和IGKJ4*01的框架区4(HFR4)；
(2)所述重链可变区的框架区包括IGHV2-70*01的框架区1-3(HFR1-3)和IGHJ6*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV3-11*01的框架区1-3(LFR1-3)和IGKJ2*01的框架区4(LFR4)；
(3)所述重链可变区的框架区包括IGHV2-70*10的框架区1-3(HFR1-3)和IGHJ6*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV4-1*01的框架区1-3(LFR1-3)和IGKJ2*01的框架区4(LFR4)；
(4)所述重链可变区的框架区包括IGHV33-7*01的框架区1-3(HFR1-3)和IGHJ1*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV2-29*02的框架区1-3(LFR1-3)和IGKJ2*01的框架区4(LFR4)；或，
所述重链可变区的框架区和所述轻链可变区的框架区包括与所述HFR1-4和LFR1-4中的每个FR相比，具有至少80％同一性的序列，或至多发生10个插入、缺失或替换突变的序列；优选地，按自然顺序编号，(i)所述重链可变区包括下述突变中的一个或多个：I71V和A98V突变，所述轻链可变区包括D1L突变；(ii)所述重链可变区包括S30Y和/或K77G突变；(iii)所述重链可变区包括下述突变中的一个或多个：L4F、G27F、I29L、S30I、V71R、N76S、F78V、S79F和A96T，所述轻链可变区的框架区包括P43S突变；(iv)所述重链可变区包括下述突变中的一个或多个：S30N、K76Q和A97T，所述轻链可变区包括下述突变中的一个或多个：Y41L、A48S和G73E。
优选地，所述至少80％同一性为85％同一性，90％同一性，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性。
优选地，所述至多发生10个插入、缺失或替换突变为至多发生9个、8个、7个、6个、5个、4个、3个、2个、1个或0个插入、缺失或替换突变；更优选地，所述替换突变为保守氨基酸残基的替换。
在一些实施方式中，所述抗体或抗原结合片段包括：
(1)所述重链可变区包括SEQ ID NO:18，78或86所示序列，所述轻链可变区包括SEQ ID NO:19，79或87所示序列；
(2)所述重链可变区包括SEQ ID NO:16，76或84所示序列，所述轻链可变区包括SEQ ID NO:17，77或85所示序列；
(3)所述重链可变区包括SEQ ID NO:20，80或88所示序列，所述轻链可变区包括SEQ ID NO:21，81或89所示序列；
(4)所述重链可变区包括SEQ ID NO:22，82或90所示序列，所述轻链可变区包括SEQ ID NO:23，83或91所示序列；或
(5)所述重链可变区和所述轻链可变区包括与第(1)-(4)组的任一组中所述重链可变区和轻链可变区相比，具有至少80％同一性的序列，或至多发生15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个插入、缺失或替换突变的序列。
优选地，所述至少80％同一性为85％同一性，90％同一性，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性。
优选地，所述至多发生15个插入、缺失或替换突变为至多发生14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个插入、缺失或替换突变；更优选地，所述替换突变为保守氨基酸残基的替换。
在一些实施方式中，其结合人p95HER2的KD小于10nM，2nM，1nM，0.5nM，0.2nM，0.01nM或0.06nM；
任选地，所述抗体或抗原结合片段还结合猴p95HER2，优选KD小于10nM，3nM，1nM，0.5nM或0.1nM。
在一些实施方式中，其包含重链恒定区序列和/或轻链恒定区序列，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区片段包括CH1，铰链区，CH2，CH3或Fc；
优选地，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；
优选地，所述抗体或抗原结合片段包括完整的重链和轻链，所述重链由所述VH和重链恒定区组成，所述重链恒定区优选具有如SEQ ID NO:9所示序列，所述轻链由所述VL和轻链恒定区组成，所述轻链恒定区优选具有如SEQ ID NO:10所示序列。
在一些实施方式中，所述抗原结合片段选自Fab，Fab’，Fab’-SH，(Fab’)2、scFv或双抗体(diabody)。
本公开还提供特异性结合人p95HER2的抗体或抗原结合片段，其与前述抗体或抗原 结合片段竞争结合p95HER2，或与前述抗体或抗原结合片段结合相同或重叠的表位。
本公开还提供多特性抗体或抗原结合片段，其包括前述特异性结合人p95HER2的抗体或抗原结合片段，还包括结合其他抗原的结合单元，优选还包括结合HER2的抗体或抗原结合片段。
本公开还提供多特异性抗体或抗原结合片段，其包括第一肽链和第二肽链，以及第三肽链和第四肽链，其中：
所述第一肽链和第三肽链包括VH1-(X1)n-VH2-CH1-Fc，所述X1为接头1，n是0或1；
所述第二肽链和第四肽链包括VL1-(X2)n-VL2-CL，所述X2为接头2，n是0或1；
所述VH1和VL1形成HER2结合结构域，所述VH2和VL2形成p95HER2结合结构域，或所述VH1和VL1形成p95HER2结合结构域，所述VH2和VL2形成HER2结合结构域。
在一些实施方式中，所述p95Her2结合结构域包括前述抗体或抗原结合片段。
在一些实施方式中，所述HER2结合结构域结合HER2胞外结构域的结构域II或结构域IV；优选地，所述HER2结合结构域具有以下特征之一：
(1)所述HER2结合结构域包括SEQ ID NO:5所示VH中的HCDR1-3和SEQ ID NO:6所示VL中的LCDR1-3；优选地，所述HCDR1-3具有如SEQ ID NO:108-110所示的序列，所述LCDR1-3具有如SEQ IDNO:111-113所示序列；更优选地，所述HER2结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示序列；
(2)所述HER2结合结构域包括SEQ ID NO:7所示VH中的HCDR1-3和SEQ ID NO:8所示VL中的LCDR1-3；优选地，所述HCDR1-3具有如SEQ ID NO:102-104所示的序列，所述LCDR1-3具有如SEQ IDNO:105-107所示序列；更优选地，所述HER2结合结构域包括SEQ ID NO:7和SEQ ID NO:8所示序列；
(3)与第(1)或(2)组所述HER2抗原结合结构域竞争性结合HER2，或结合表位相同或重叠。
在一些实施方式中，所述接头1和接头2为(G4S)n，n选自1，2，3，4，5或6；
任选地，所述CH1-Fc具有具有与SEQ ID NO:9相同或至少具有80％同一性或至多存在20个缺失、插入或替换突变的序列；
任选地，所述CL具有与SEQ ID NO:10相同或至少具有80％同一性或至多存在20个缺失、插入或替换突变的序列。
优选地，所述至少80％同一性为85％同一性，90％同一性，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性。
优选地，所述至多发生20个插入、缺失或替换突变为至多发生19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个插入、缺失或替换突变；更优选地，所述替换突变为保守氨基酸残基的替换。
在一些实施方式中，所述第一肽链和所述第三肽链具有与SEQ ID NO:94、96、98相同或至少具有80％同一性或至多存在30个缺失、插入或替换突变的序列，所述第二肽链和所述第四肽链具有与95、97、99相同或至少具有80％同一性或至多存在30个缺失、插入或替换突变的序列。
优选地，所述至少80％同一性为85％同一性，90％同一性，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％同一性。
优选地，所述至多发生30个插入、缺失或替换突变为至多发生29个、28个、27个、26个、25个、24个、23个、22个、21个、20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个、1个或0个插入、缺失或替换突变；更优选地，所述替换突变为保守氨基酸残基的替换
本公开还提供如A-(L-D)n所示的抗体-药物偶联物或其药学上可接受的盐或溶剂合物，其中：
A表示前述抗体或抗原结合片段；
L表示用于连接A与D的接头，优选为mc-vc-pAB或mc；
D表示药物，优选为海兔毒素肽或其衍生物，更优选为MMAE或MMAF；
n表示药物抗体比(drug-to-antibody ratio,DAR)，任选为1～10，优选为2或4。
在一些实施方式中，用于偶联抗体的L-D具有如下所示的结构：
本公开还提供分离的核酸，其中，所述核酸片段编码前述抗体或抗原结合片段。
本公开还提供编码嵌合抗原受体(chimeric antigen receptor,CAR)的核酸，其中，所述CAR包括信号肽、抗原结合结构域、铰链区、跨膜区和胞内细胞传导区，所述抗原结合结构域包含前述抗体或抗原结合结构域。
本公开还提供载体(vector)，其包含前述的核酸。
本公开还提供宿主细胞，其包含前述核酸或载体或表达前述抗体或抗原结合片段；
优选地，所述宿主细胞是原核细胞或真核细胞；
更优选地，所述宿主细胞选自大肠杆菌、酵母、哺乳动物细胞或适合制备抗体或抗原结合片段地其他细胞，例如HEK293细胞或CHO细胞。
本公开还提供免疫效应细胞，其包含前述核酸或载体由前述核酸编码的嵌合抗原受体；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞。
本公开还提供药物组合物，其包含前述抗体或抗原结合片段或抗体-药物偶联物或其药学上可接受的盐或溶剂合物或免疫效应细胞，以及任选的药学上接受的运载体(carrier)，以及任选的其他治疗剂。
本公开还提供制备前述抗体或抗原结合片段或抗体-药物偶联物或其药学上可接受的盐或溶剂合物或免疫效应细胞的方法，所述方法包括：
(1)培养所述宿主细胞表达前述抗体或抗原结合片段，任选地，所述方法还包括分离所述抗体或抗原结合片段；或，
(2)在前述抗体或抗原结合片段上偶联药物，获得前述抗体-药物偶联物或其药学上可接受的盐或溶剂合物；或，
(3)在免疫效应细胞中转入前述核酸或载体，获得包含所述核酸或载体或获得表达前述核酸编码的嵌合抗原受体的免疫细胞。
本公开还提供前述抗体或抗原结合片段或所述抗体-药物偶联物或其药学上可接受的盐或溶剂合物或核酸或载体或宿主细胞或免疫效应细胞或药物组合物或根据前述方法制备的产物，在制备用于治疗癌症或肿瘤中药物中的用途；
优选地，所述癌症或肿瘤选自实体瘤，例如，乳腺癌，胃癌，膀胱癌，卵巢癌，肺癌，前列腺癌，胰腺癌，结直肠癌，头颈癌，肺癌，胆道癌，尿路上皮癌，肝癌，食道癌或骨肉瘤；
优选地，所述癌症或肿瘤表现为HER2+和/或p95HER2+；
优选地，所述癌症或肿瘤表现出HER2靶向疗法耐药性，例如曲妥珠、帕妥珠、T-DM1、DS-8201或RC48耐药性；
优选地，所述药物还包括其他治疗剂，例如化疗药(紫杉烷，选自多西他赛或紫杉醇；蒽环类，选自阿霉素，表柔比星，柔红霉素或戊柔比星)，或免疫检查点抑制剂(PD-1抗体或PD-L1抗体)。
本公开还提供前述抗体或抗原结合片段或抗体-药物偶联物或其药学上可接受的盐或溶剂合物或核酸或载体或宿主细胞或免疫效应细胞或药物组合物或根据前述方法制备的产物，用于治疗癌症或肿瘤；
优选地，所述癌症或肿瘤选自实体瘤，例如，乳腺癌，胃癌，膀胱癌，卵巢癌，肺癌，前列腺癌，胰腺癌，结直肠癌，头颈癌，肺癌，胆道癌，尿路上皮癌，肝癌，食道癌或骨肉瘤；
优选地，所述癌症或肿瘤表现为HER2+和/或p95HER2+；
优选地，所述癌症或肿瘤表现出HER2靶向疗法耐药性，例如曲妥珠、帕妥珠、T-DM1、DS-8201或RC48耐药性；
优选地，所述治疗还包括其他治疗剂，例如化疗药(紫杉烷，选自多西他赛或紫杉醇；蒽环类，选自阿霉素，表柔比星，柔红霉素或戊柔比星)，或免疫检查点抑制剂(PD-1抗体或PD-L1抗体)。
本公开还提供治疗癌症或肿瘤的方法，其包括向受试者给予有效量的前述抗体或抗原 结合片段或抗体-药物偶联物或其药学上可接受的盐或溶剂合物或核酸或载体或宿主细胞或免疫效应细胞或药物组合物或所述方法制备的产物；
优选地，所述癌症或肿瘤选自实体瘤，例如，乳腺癌，胃癌，膀胱癌，卵巢癌，肺癌，前列腺癌，胰腺癌，结直肠癌，头颈癌，肺癌，胆道癌，尿路上皮癌，肝癌，食道癌或骨肉瘤；
优选地，所述癌症或肿瘤表现为HER2+和/或p95HER2+；
优选地，所述癌症或肿瘤表现出HER2靶向疗法耐药性，例如曲妥珠、帕妥珠、T-DM1、DS-8201或RC48耐药性；
优选地，所述药物还包括其他治疗剂，例如化疗药(紫杉烷，选自多西他赛或紫杉醇；蒽环类，选自阿霉素，表柔比星，柔红霉素或戊柔比星)，或免疫检查点抑制剂(PD-1抗体或PD-L1抗体)。
本公开还提供检测p95HER2的方法，其包括在前述抗体或抗原结合片段能与p95HER2形成复合物的条件下，使待测样品与所述抗体或抗原结合片段接触；
任选地，检测所述复合物的形成，指示样品中p95HER2的存在或表达水平；
任选地，所述样品为癌症或肿瘤患者的样品，根据样品中p95HER2的存在或表达水平，鉴定所述患者是否能从p95HER特异性治疗中受益；
任选地，所述方法还包括在HER2抗体或其抗原结合片段能与HER2形成复合物的条件下，使待测样品与所述HER2抗体或抗原结合片段接触，检测所述复合物的形成，指示样品中HER2的存在或表达水平，和任选地，所述样品为癌症或肿瘤患者的样品，根据所述HER2的存在或表达水平，鉴定所述患者是否能从HER2特异性治疗中受益。
任选地，所述治疗包括前述治疗癌症或肿瘤的方法；
任选地，所述检测通过免疫组织化学(ICH)进行。
本公开还提供前述抗体或抗原结合片段在制备用于检测p95HER2的试剂盒中的应用。
本公开提供了对p95HER2靶点具有较高亲和力的抗体或其抗原结合片段，且具有良好的内吞能力，由其制备的ADC药物具有良好的肿瘤杀伤效果，特别是对于曲妥珠耐药的病人，有良好的治疗前景。
附图说明
图1A.表达人p95HER2蛋白的CHO-K1稳转细胞系FACS检测结果；
图1B.表达人HER2蛋白的CHO-K1稳转细胞系FACS检测结果；
图1C.表达猴p95HER2蛋白的HEK293T稳转细胞系FACS检测结果；
图1D.表达人p95HER2蛋白的SKBR3稳转细胞系FACS检测结果；
图1E.表达人p95HER2蛋白的细胞系(OE19-Tet/on-p95HER2.73#)FACS检测结果；
图1F.表达人p95HER2蛋白的细胞系(T47D-Tet/on-p95HER2.3H6)FACS检测结果；
图2A～图2D.p95HER2鼠抗和CHO-K1-hu p95HER2细胞结合的FACS检测结果；
图3A～图3D.p95HER2人源化抗体或嵌合抗体和CHO-K1-hu p95HER2细胞结合的FACS检测结果；
图4.p95HER2人源化抗体和293T-cyno p95HER2细胞结合的FACS检测结果；
图5.p95HER2抗体的IHC染色结果；
图6A～图6B.p95HER2人源化抗体对SKBR3-p95HER2细胞的杀伤结果；
图7.p95HER2-ADC(单抗)在SKBR3-p95HER2细胞上的杀伤结果；
图8.p95HER2-ADC(单抗)在OE19-p95HER2细胞上的杀伤结果；
图9.p95HER2-ADC(单抗)在T47D-p95HER2细胞上的杀伤结果；
图10A～图10C.p95HER2/HER2双抗的示意图；
图11A～图11B.p95HER2/HER2双抗与CHO-K1-hu HER2细胞结合的FACS检测结果；
图12A～图12B.p95HER2/HER2双抗在SKBR3-p95HER2细胞上的杀伤内吞试验结果；
图13A～13B.p95HER2/HER2双抗ADC与CHO-K1-hu p95HER2细胞/CHO-K1-hu HER2细胞结合的FACS检测结果；
图14A～图14B.p95HER2/HER2双抗ADC在SKBR3-p95HER2细胞上的杀伤实验结果；
图15A～图15B.在OE19-p95HER2细胞上的杀伤实验结果；
图16A～16B在T47D-p95HER2细胞上的杀伤实验结果。
发明的详细描述
术语定义和说明
除非本文另外定义，本文所有术语均具有本领域普通技术人员通常理解的含义。
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的 组合物，均落入前述“一种组合物”的范围内。
术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。
本文p95HER2是指611-CTF或100-115kDa p95HER2，详见Cancer Res 71,1515-1519(2011))，它具有形成通过分子间二硫键维持的同二聚体的能力(Pedersen等,Mol Cell Biol 29,3319-31(2009))。p95HER2似乎在HER2阳性乳腺癌的一个同质亚组中表达(Parra-Palau等,JNatl Cancer Inst 106,dju291(2014))，但是不受例如曲妥单抗识别，因为该片段缺少受到 该抗体识别的表位(Pedersen等,Mol Cell Biol 29,3319-31(2009))。本文的“p95HER2”包括人或非人哺乳动物的p95HER2.，示例性地，p95HER2可选自SEQ ID NO:11或SEQ ID NO:12所示序列。
本文术语“HER2”(也被称为ErbB-2、NEU、HER-2和CD340)，包括人或非人哺乳动物的HER2，当用于本文时指的是人表皮生长因子受体2(SwissProt P04626)且包括由细胞(包括肿瘤细胞)天然表达或在HER2基因转染的细胞上表达的任何HER2的变体、同种型和种同源物。如无特别指出，本文“HER2”不包括p95HER2。
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约10-7M或更低、约10-8M或更低、约1×10-9M或更低、约1×10-10M或更低、1×10-11M或更低或1×10-12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定，示例性地，可参见本文实施例所示KD值获得方法。
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于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，可将本文“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根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋 白新抗原受体(Ig new antigen receptor,IgNAR)。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体或全人抗体。
本文术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体，即，除了可能的变异体(例如含有天然存在的突变或在制剂的生产过程中产生，此类变体通常以少量存在)之外，包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。本文修饰语“单克隆”不应解释为需要通过任何特定方法产生所述抗体或抗原结合分子。举例来说，单克隆抗体可通过多种技术制得，包括(但不限于)杂交瘤技术、重组DNA方法、噬菌体库展示技术和利用含有全部或部分人免疫球蛋白基因座的转殖基因动物的方法和其它本领域已知的方法。
本文术语“天然抗体”是指通过多细胞生物体的免疫系统制造和配对的抗体。本文术语“工程化抗体”的抗体是指通过基因工程、抗体工程等技术获得的非天然抗体，示例性地，“工程化抗体”包括人源化抗体、小分子抗体(例如scFv等)、双特异性抗体等等。
本文术语“单特异性”是指表示具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
本文术语“多特异性抗体”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2和scFv、。
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此,本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段,和重链的VH域和第一恒定域(CH1)的抗体片段。Fab’片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同,包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团 的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’)2片段。
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。
本文术语“双抗体(diabody)”，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
本文术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
本文术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
本文术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区” 与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“Kabat编号系统”通常是指由ElvinA.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“Chothia编号系统”通常是指由Chothia等人提出的免疫球蛋白编号系统，其是基于结构环区的位置鉴定CDR区边界的经典规则(参见，例如Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)。
本文术语“IMGT编号系统”通常是指基于由Lefranc等人发起的国际免疫遗传学信息系统(The international ImMunoGeneTics information system(IMGT))的编号系统，可参阅Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白酶水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据Kabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
如无其他说明，本文所述“抗体”或“抗原结合片段”氨基酸残基编号由Kabat编号系统确定，详见，Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
本文术语“自然顺序编号”，是指直接按找氨基酸残基在序列中的位置，按自然顺序进行编号。示例性地，按自然顺序编号的第30位氨基酸残基指的就是，按自然顺序处于序列的第30位的氨基酸残基，S30Y表示，按照自然顺序，处于序列的第30位的S突变位Y。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；
2)天冬氨酸(D)、谷氨酸(E)；
3)天冬酰胺(N)、谷氨酰胺(Q)；
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用 NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本公开所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本公开核酸(SEQ ID NO:1)分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本公开蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。
本文术语“抗原嵌合受体(CAR)”是指经改造以在免疫效应细胞上表达并且特异性结合抗原的人工细胞表面受体，其包含至少(1)细胞外抗原结合结构域，例如抗体的可变重链或轻链，(2)铰链区；(3)锚定CAR进入免疫效应细胞的跨膜结构域，和(4)胞内信号传导结构域。CAR能够利用细胞外抗原结合结构域以非MHC限制性的方式将T细胞和其它免疫效应细胞重定向至所选择的靶标，例如癌细胞。
本文术语“核酸”包括包含核苷酸的聚合物的任何化合物和/或物质。每个核苷酸由碱基，特别是嘌呤或嘧啶碱基(即胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)或尿嘧啶(U))、糖(即脱氧核糖或核糖)和磷酸基团组成。通常，核酸分子由碱基的序列描述，由此所述碱基代表核酸分子的一级结构(线性结构)。碱基的序列通常表示为5′至3′。在本文中，术语核酸分子涵盖脱氧核糖核酸(DNA)，包括例如互补DNA(cDNA)和基因组DNA、核糖核酸(RNA)，特别是信使RNA(mRNA)、DNA或RNA的合成形式，以及包含两种或更多种这些分子的混合的聚合物。核酸分子可以是线性的或环状的。此外，术语核酸分子包括有义链和反义链二者，以及单链和双链形式。而且，本文所述的核酸分子可含有天然存在的或非天然存在的核苷酸。非天然存在的核苷酸的例子包括具有衍生的糖或磷酸骨架键合或化学修饰的残基的修饰的核苷酸碱基。核酸分子还涵盖DNA和RNA分子，其适合作为载体用于在体外和/或体内，例如在宿主或患者中，直接表达本公开的抗体。此类DNA(例如cDNA)或RNA(例如mRNA)载体可以是未修饰的或修饰的。例如，可以对mRNA进行化学修饰以增强RNA载体的稳定性和/或被编码分子的表达，从而可以将mRNA注入到受试者内以在体内产生抗体(参见例如Stadler等人，Nature Medicine 2017，published online 2017年6月12日，doi：10.1038/nm.4356或EP 2 101 823 B1)。本文“分离的”核酸指已经与其天然环境的组分分开的核酸分子。分离的核酸包括在下述细胞中含有的核酸分子，所述细胞通常含有该核酸分子，但该核酸分子存在于染色体外或存在于不同于其天然染色体位置的 染色体位置处。
本文术语“载体”是指能够扩增与其连接的另一个核酸的核酸分子。该术语包括作为自我复制型核酸结构的载体以及整合入已引入该载体的宿主细胞的基因组中的载体。某些载体能够指导与它们可操作连接的核酸的表达。这样的载体在本文中称为“表达载体”。
本文术语“宿主细胞”是指细胞中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转化体”和“经转化的细胞”，其包括原代的经转化的细胞和来源于其的后代，而不考虑传代的次数。后代在核酸内容物上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括具有与在初始转化的细胞中筛选或选择的相同功能或生物学活性的突变体后代。
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症、自身免疫性疾病和病毒感染的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
本文术语“受试者”是指接受对如本公开所述的特定疾病或病症的治疗的生物体。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
本文术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
本文术语“自身免疫性疾病”是指对象对其自身的细胞、组织和/或器官产生免疫反应而引起的细胞、组织和/或器官损伤的病症。
本文术语“癌症”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性癌症。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。
本文术语“EC50”是指半最大有效浓度，其包括在指定暴露时间之后诱导基线与最大值之间的半途响应的抗体浓度。EC50本质上代表其中观察到其最大作用的50％的抗体浓度，可通过本领域已知方法测量。
具体实施方式
下面结合具体实施例来进一步描述本公开，本公开的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件进行，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
本公开实施例仅是范例性的，并不对本公开的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本公开的精神和范围下可以对本公开技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本公开的保护范围内。
实施例1 p95HER2融合蛋白的设计，表达和纯化
1.p95HER2融合蛋白的设计和表达
以人HER2蛋白(Pubmed号：P04626-1)和猴HER2蛋白(Pubmed号：XP_005584091.2)作为p95HER2模板，从胞外段第611位起始，设计带标签的p95HER2胞外区融合蛋白，分别克隆到pTT5载体上，在HEK293细胞中瞬转表达，经纯化获得用于免疫动物、筛选抗体和/或检测抗体功能用蛋白：hu p95HER2.ECD-Fc和cyno p95HER2.ECD-Fc。融合蛋白的具体序列信息参见表5。
2.p95HER2融合蛋白的纯化
首先高速离心收取表达蛋白的细胞培养上清。ProteinA亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外监测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
实施例2阳性对照抗体611mab-hIgG1、611mab-mIgG2a、trastuzumab-hIgG1和pertuzumab-hIgG1的制备
将编码抗体VH和VL的核酸序列重组至带有信号肽和重链恒定区/轻链恒定区序列的表达载体pTT5上，得到表达VH-CH1-Fc/VL-CL的重组质粒。其中，611mab-hIgG1、trastuzumab-hIgG1和pertuzumab-hIgG1是将相应VH和VL重组至human CH1-Fc和human CL，611mab-mIgG2a是将VH和VL重组至mouse CH1-Fc和mouse CL。经测序验证后，提取质粒，并转染宿主细胞。经培养，获得分泌抗体的细胞培养上清。参照实施例1所述方法，利用ProteinA亲和层析，从上清中纯化所述抗体。上述方法涉及的分子生物学操作详见《分子克隆实验指南(第三版)》，(美)J.萨姆布鲁克等著。
阳性对照抗体611mab-hIgG1和611mab-mIgG2a的VH和VL序列分别来自中国专利申请公开文本CN109843926A，611mab-hIgG1和611mab-mIgG2a能够特异性结合人p95HER2和猴p95HER2。Trastuzumab-hIgG1的VH和VL序列分别来自美国公开专利文 本US5821337A，能够结合人HER2胞外结构域的结构域IV。Pertuzumab-IgG1的VH和VL来自中国专利授权文本CN100340575C，能够结合人HER2胞外结构域的结构域II。具体序列信息参见表5。
实施例3细胞系的构建
1.表达人p95HER2、人HER2或猴p95HER2的稳转细胞的构建
编码人p95HER2、人HER2或猴p95HER2蛋白的核苷酸序列被克隆到pcDNA3.1载体并制备质粒。质粒转染(3000 Transfection Kit，购自Invitrogen，货号：L3000-015)至细胞系，之后，在含10μg/ml puromycin和10％(v/v)胎牛血清的DMEM/F12培养基中选择性培养2周，用一抗和二抗在流式细胞仪FACS Aria III(BD Biosciences)上富集高水平表达靶蛋白的细胞群到6孔板，并置于37℃，5％(v/v)CO2培养，大约1周后消化6孔板细胞进行扩增。对扩增后的细胞经流式细胞分析术(FACS)进行检测，选择长势好、荧光强度高、均一性好的阳性细胞群继续扩大培养并液氮冻存。材料与试剂参见表1，FACS检测结果见图1A-图1C和表2。
表1材料与方法
表2稳转细胞系FACS检测结果
2.SKBR3-p95HER2细胞系的构建
编码人p95HER2氨基酸序列(见表5)的核苷酸序列被克隆到pLVX慢病毒载体，并在HEK293T细胞中制备病毒颗粒。对SKBR3细胞系(ATCC,HTB-30)进行慢病毒感染后，在含2μg/ml puromycin和10％(v/v)胎牛血清的McCoy's 5A Medium培养基中选择性培养2周，用抗人p95HER2抗体(611mab-hIgG1，自产，见实施例2)和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)在流式细胞仪FACS Aria III(BD Biosciences)上分选阳性pool细胞群到6孔板，并置于37℃，5％(v/v)CO2培养，大约1周后选择部分细胞进行扩增。对扩增后的细胞经流式细胞分析法进行筛选。选择长势好、荧光强度高、均一性好的阳性细胞群继续扩大培养并液氮冻存。FACS检测结果参见表3和图1D。
表3表达人p95HER2蛋白的SKBR3稳转细胞系FACS检测结果
3.OE19-p95HER2 tet on细胞系或T47D-p95HER2 tet on细胞系的构建
(1)编码人p95HER2氨基酸序列(见表5)的核苷酸序列被克隆到pTRIPZ empty慢病毒载体，并在HEK293T细胞中制备病毒颗粒。
(2)转染和培养细胞
转染和培养OE19细胞系：对OE19细胞系(科佰生物，CBP60495)进行慢病毒感染后，在含1μg/ml puromycin和10％(v/v)胎牛血清(去Tetracycline)的1640 Medium+1％PS培养基中选择性培养2周，取部分细胞加1μg/ml DOX(Tetracycline)诱导培养48h，用抗人p95HER2抗体(611mab-hIgG1，自产，见实施例2)和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)在流式细胞仪FACS Cantol II(购自BD Biosciences)上检测OE19 pool细胞上p95HER2的表达。采用有限稀释法铺板，以每孔1-2个细胞密度铺96孔板，置于37℃，5％(v/v)CO2培养。两周后，挑选生长起来的克隆至48孔板，约1周后选择48孔板中细胞孔汇合度约70％的细胞孔扩增至24孔板。取部分细胞加1μg/ml DOX(Tetracycline)诱导培养48h，经流式细胞分析法进行检测筛选。
转染和培养T47D细胞：对T47D细胞系(购自ATCC)进行慢病毒感染后，在含1μg/ml puromycin和10％(v/v)胎牛血清(去Tetracycline)的DMEM Medium+10ug/ml牛胰岛素培养基中选择性培养2周，取部分细胞加1μg/ml DOX(Tetracycline)诱导培养过夜，用抗人p95HER2抗体(611mab-hIgG1，自产，见实施例2)和山羊抗人IgG(H+L)抗体(Jackson，货号：109605088)在流式细胞仪FACS Aria III(购自BD Biosciences)上分选p95HER2阳性单细胞至96孔板，并置于37℃，5％(v/v)CO2培养，大约2周后选择部分生长较好克隆细胞进行扩增。对扩增后的克隆经流式细胞分析法进行筛选。
(3)选择长势好、荧光强度高、均一性好的阳性克隆继续扩大培养并液氮冻存。FACS检测结果参见表4和图1E-1F。
表4 OE19-p95HER2 tet on细胞系或T47D-p95HER2 tet on细胞系FACS检测结果
实施例1-3涉及的序列信息详见下表5。
表5序列信息
实施例4抗人p95HER2鼠源单克隆抗体的制备
1.免疫
抗人p95HER2单克隆抗体通过免疫小鼠产生。实验用balb/c小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。
免疫原：hup95HER2.ECD-Fc蛋白，25μg/只/次，皮下、足底、腹腔内注射。免疫方式：免疫原混合Titermax、Alum或CPG，第0天进行皮下、足底、腹腔注射，之后每7天进行一次加强免疫，轮流进行皮下或皮下和足底注射。于第21、35、49、63天取血，用ELISA方法确定小鼠血清中的抗体滴度。在7-9免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行淋巴和脾细胞融合。在进行脾细胞融合前3天加强免疫，皮下、足底、腹腔注射蛋白。
2.脾细胞融合
采用电介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0(CRL-8287TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以0.5-1×105/ml的密度用完全培养基(含20％FBS、1×HAT、1×牛胰岛素、1×非必须氨基酸、1×双抗、1×IL-6的DMEM培养基)重悬，200μl/孔种于96孔板中，37℃，5％CO2孵育7-11天至形成针尖般克隆。去除上清，加入200μl/孔的HT完全培养基(含10％FBS、1×HT和1×牛胰岛素、1×非必须氨基酸、1×双抗的DMEM培养基)，37℃，5％CO2培养1天后进行ELISA或者FACS检测。
3.杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用ELISA方法检测杂交瘤培养上清与hup95HER2.ECD-Fc蛋白的结合。进一步通过FACS检测ELISA检测阳性孔的上清与CHO-K1-hu p95HER2、CHO-K1细胞的结合。对hu p95HER2.ECD-Fc蛋白ELISA、CHO-K1-hu p95HER2细胞结合实验均为阳性和CHO-K1细胞结合实验为阴性的孔，及时扩增冻存保种及亚克隆直至获得单细胞克隆。亚克隆细胞也均需进行hu p95HER2.ECD-Fc蛋白ELISA，CHO-K1-hu p95HER2细胞结合实验和CHO-K1细胞结合实验。通过以上实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，按实施例1所述纯化方法纯化抗体，以备后续使用。
4.杂交瘤阳性克隆序列测定
从阳性杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScriptTMReverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326 Rev.B 0503)进行PCR扩增后送测序公司测序。经测序得到鼠源抗人p95HER2抗体Mab01、Mab02、Mab03和Mab04，其重链可变区(VH)和轻链可变区(VL)氨基酸序列如下表所示。
表6鼠源抗人p95HER2抗体可变区序列
采用生物信息学的方法分析分析上述p95HER2单克隆抗体的CDR区，其中，所述CDR区采用Kabat编号系统、Chothia编号系统和IMGT编号系统进行确定和注释(Kabat和Chothia编号系统：http://www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi； IMGT编号系统：http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)，具体结果如表7所示。
表7 CDR分析
实施例5人IgG1嵌合抗体制备
参照实施例2，将编码实施例4所示鼠源抗体VH和VL的核酸序列重组至带有信号肽和重链恒定区/轻链恒定区的表达载体pTT5上，构建表达全长嵌合抗体VH-CH1-Fc/VL-CL的重组质粒，并制备相应的嵌合抗体ChAb01、ChAb02、ChAb03和ChAb04，嵌合抗体的恒定区为human CH1-Fc和human CL，制备方法和恒定区序列详见实施例2和表5。
实施例6鼠源抗人p95HER2抗体的人源化
1.构建人源化的抗人p95HER2抗体可变区
通过比对IMGT(http://imgt.cines.fr)人类抗体重轻链可变区种系基因数据库和MOE(Molecular Operating Environment,分子操作环境)软件，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体Mab01、Mab02、Mab03和Mab04的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列，在此基础上，根据需要，进行回复突变以保证原有的亲和力和/或进行热点突变以消除分子修饰风险。
本实施例抗体的氨基酸残基按自然顺序编号，CDR区由Kabat编号系统确定。
Mab01、Mab02、Mab03和Mab04的人源化模板如表8所示；人源化模板移植鼠源抗体CDR后的序列如表9所示；在表9所示序列的基础上进行回复突变或热点突变，突变信息如表10所示，最终优化的人源化抗体和对应序列如表11所示，其对应CDR参见表12。
表8人源化模板
表9 VH-CDR graft和VL-CDR graft序列信息
表10人源化抗体回复突变和/或热点突变设计
表11优化的人源化抗体及其对应序列信息
表12 CDR分析(Kabat编号系统)
2.抗p95HER2人源化全长抗体的构建和表达
参照实施例2构建、表达和纯化抗p95HER2人源化全长抗体：Hab01.11a、Hab02.11、Hab03.11a和Hab04.11，其中该全长人源化抗体的恒定区为human CH1-Fc和human CL，制备方法和恒定区序列详见实施例2和表5。
实施例7 p95HER2抗体结合活性检测
1.p95HER2抗体与CHO-K1-hu p95HER2细胞的结合实验
通过抗体与CHO-K1-hu p95HER2细胞系的FACS实验检测抗体与人p95HER2的结合力。具体实验方法如下：
收集CHO-K1-hu p95HER2细胞，将细胞密度调整至5×105cells/ml，以100μl/孔的体积加入96孔培养板(Corning，Cat No.3799)中，4℃，1500rpm，5min离心。弃去上清，加入250μl/孔PBS(HyClone，Cat No.SH30256.01)，4℃，1500rpm，5min离心。弃去上清后，加入100μl/孔用样品稀释液(2％BSA-PBS)稀释的不同浓度待测抗体，放于4℃冰箱孵育1小时，孵育结束后，4℃，1500rpm，5min离心。弃去上清，加入250μl/孔PBS(HyClone，Cat No.SH30256.01)，4℃，1500rpm，5min离心(Thermo)。弃去上清后，加入100μl/孔用样品稀释液(2％BSA-PBS)稀释的Alexa Fluor-647标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-605-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-605-088)，放于4℃冰箱孵育1小时。孵育结束后，4℃，1500rpm，5min离心(Thermo)洗涤3次。最后弃去上清，加入80μl/孔PBS重悬细胞，流式细胞仪(BD,Canto II)检测荧光信号强弱。
实验结果见图2A～图2D，图3A～图3D及表13-14。实验结果表明，本公开提供的抗人p95HER2抗体与CHO-K1-hup95HER2细胞有很好的结合作用，优于阳性对照或基本相当。
表13鼠源p95HER2抗体与CHO-K1-hup95HER2细胞结合实验结果
表14嵌合p95HER2抗体和人源化p95HER2抗体与CHO-K1-hu p95HER2细胞结合实验结果
2.p95HER2抗体与293T-cyno p95HER2细胞的结合实验
通过抗体与293T-cyno p95HER2细胞FACS实验检测抗体与猴p95HER2的结合力。具体实验方法如下：
收集293T-cyno p95HER2细胞，将细胞密度调整至5×105cells/ml，以100μl/孔的体积加入96孔培养板(Corning，Cat No.3799)中，4℃，1500rpm，5min离心。弃去上清，加入50μl/孔4％FBS-PBS buffer，放于4℃冰箱孵育1小时，孵育结束后，4℃，1500rpm，5min离心。弃去上清后，加入100μl/孔用样品稀释液(2％FBS-PBS)稀释的不同浓度待测抗体，放于4℃冰箱孵育1小时，孵育结束后，4℃，1500rpm，5min离心。弃去上清，加入 250μl/孔PBS(HyClone，Cat No.SH30256.01)，4℃，1500rpm，5min离心(Thermo)。弃去上清后，加入100μl/孔用样品稀释液(2％FBS-PBS)稀释的AlexaFluor-647标记的羊抗人二抗(Jackson Immuno Research，Cat No.109-605-088)，放于4℃冰箱孵育1小时。孵育结束后，4℃，1500rpm，5min离心(Thermo)洗涤3次。最后弃去上清，加入80μl/孔PBS重悬细胞，流式细胞仪(BD,Canto II)检测荧光信号强弱。具体结果详见表15。根据图4和表15所示结果可知，p95HER2抗体Hab02.11与293T-cyno p95HER2细胞的结合优于阳性对照611mab-hIgG1。
表15 p95HER2人源化抗体与293T-cynop95HER2细胞结合实验结果
3.p95HER2抗体的Biacore测定
该实验用Biacore 8K(GE)仪器，采用多循环动力学测定待测p95HER2抗体与人p95HER2(hup95HER2.ECD-Fc)、猴P95HER2(cyno p95HER2.ECD-Fc)的亲和力。
实验运行缓冲液为1×HBS-EP+缓冲溶液(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(Cat.#BR-1006-69，GE)，流经池温度设置为25℃，样品仓温度设置为16℃。两者都用运行缓冲液预处理。用ProteinA生物传感芯片(Cat.#29127556,GE)亲和捕获一定量的待测抗体，然后于芯片表面流经一定浓度的人、猴p95HER2抗原，利用Biacore 8K仪器(GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，pH1.5的甘氨酸-盐酸再生溶液(Cat.#BR-1003-54,GE)将抗原-抗体复合物洗净再生。具体地，通过注射溶液中不同浓度的人p95HER2和猴p95HER2抗原240s来检测其结合过程，流速为30μL/min，从50nM起始，以1:1稀释，设置一系列浓度梯度；解离时间长达900s，最后用10mM甘氨酸-盐酸溶液(pH 1.5)以30μL/min的流速洗涤30s完成芯片表面的再生。实验得到的数据用GE Biacore 8K Evaluation version 2.0软件以(1:1)Langmuir模型进行拟合，得出结合速率(Ka)、解离速率(Kd)和亲和力数值(KD)，具体见表16-17所示。实验结果表明，本公开提供的p95HER2抗体均能与人p95HER2以高亲和力结合，优于阳性对照。Hab02.11、ChAb02、Hab04.11和ChAb04均能与猴p95HER2以高亲和力结合，优于对照。
表16 p95HER2抗体与人p95HER2蛋白的反应亲和力
表17 p95HER2抗体与猴p95HER2蛋白的反应亲和力
4.p95HER2抗体的IHC染色
MAb04可作为p95HER2特异性的IHC染色抗体，具体检测方法如下所示：分别用SKBR3细胞切片,SKBR3-p95HER2细胞切片，SKBR3肿瘤切片，SKBR3-p95HER2肿瘤切片，PDX(patient-drivedxenotransplantation,PDX)切片进行IHC染色。染色步骤如下：石蜡切片在全自动组织脱水机经二甲苯、梯度酒精脱蜡水化。用EDTA修复液(PH9.0)(福州迈新生物，稀释比例1：50)用高压热修复的方法进行抗原修复。经修复后的切片自然冷却至室温，用PBS洗涤切片，5min 3次。用3％过氧化氢溶液(abcam，ab64218)阻断内源性过氧化物酶，孵育15min。PBS洗涤切片，5min 3次。10％正常羊血清封闭切片20min阻断非特异染色。甩去血清，直接滴加一抗(抗体稀释液Dako antibody diluent)，湿盒内4℃孵育过夜。取出复温至室温，PBST(0.05％吐温20)洗涤切片，10min 3次。滴加对应一抗的HRP标记的二抗，室温孵育60min，PBST洗涤切片，10min 3次。配置DAB显色液，显微镜下显色，每张切片统一显色时间。充分水洗后，在全自动组织染色机上进行复染苏木素，脱水后封片。
IHC染色结果见图5。实验结果表明，本公开的p95HER2抗体mAb04可特异性地针对p95HER2着色。
实施例8 p95HER2抗体的体外功能实验
1.p95HER2抗体的内吞实验
抗人p95HER2抗体的内吞能力的检测，通过使抗体与DT3C蛋白(M.Yamaguchi et al,Biochem.Biophys.Res.Commun.,2014 Nov 28；454(4):600-3.doi:10.1016/j.bbrc.2014.10.133)结合，模拟一个DAR2的ADC，检测抗体-DT3C复合物对细胞产生的杀伤来表征。将SKBR3-p95HER2过表达细胞系以胰酶消化后，用含10％胎牛血清的McCoy's 5A培养基重悬，以5000个细胞/孔的密度接种至96孔平底板，置于37℃、5％CO2培养箱中培养24小时。将抗体用含10％胎牛血清的培养基进行梯度稀释，然后与两倍质量浓度的DT3C溶液等体积混合，室温静置30分钟。将抗体与DT3C的偶联物加入细胞中，置于37℃、5％CO2培养箱中培养72小时。实验结束时，以CellTiter-Glo检测细胞活率，再转化为抑制率(抑制率＝1-细胞活率百分比)。
实验结果如图6A～图6B所示，p95HER2抗体Hab02.11、Hab04.11和DT3C的偶联物对SKBR3-p95HER2细胞具有较好的杀伤作用，杀伤效果与抗体和两种细胞的结合能力相关。说明Hab02.11和Hab04.11抗体具有较好的内吞能力。
2.p95HER2-MMAE ADC的偶联
使用超滤管(Millpore,货号：UFC903096)将Hab02.11超滤换液至BufferA(50mM磷酸盐,150mM NaCl,1mM EDTA,pH6.50)，抗体浓度控制在10mg/mL。向抗体中加入3.6倍当量10mM TCEP(Sigma，货号75259)25℃，300r/min恒温金属浴(Eppendorf,型号Thermo Mixer C)孵育3h,还原抗体内部二硫键。向部分还原蛋白溶液中加入10倍当量DMSO溶解VcMMAE(MCE,货号HY-15575)；25℃，300r/min恒温金属浴孵育3h。使用超滤管对ADC样品进行超滤换液至BufferA，换液体积1000倍以上，去除游离毒素。采用HIC和LC-MS方法对样品进行分析，获得DAR4ADC终样品。
3.p95HER2-MMAE ADC对SKBR3-p95HER2细胞的杀伤实验
将SKBR3-p95HER2过表达细胞系以胰酶消化后，用含10％胎牛血清的McCoy's 5A培养基重悬，以5000个细胞/孔的密度接种至96孔平底板，置于37℃、5％CO2培养箱中培养24小时。将抗体用含10％胎牛血清的培养基进行梯度稀释，然后将Hab02.11-MMAE ADC分子(DAR4)或其对照分子加入细胞中，置于37℃、5％CO2培养箱中培养72小时。实验结束时，以CellTiter-Glo检测细胞活率，再转化为抑制率(抑制率＝1-细胞活率百分比)。
实验结果见图7，结果表明，Hab02.11-MMAE ADC分子对SKBR3-p95HER2细胞具有较好的杀伤作用，说明其具有较好的对表达靶点抗原的细胞杀伤的能力。
4.p95HER2-MMAE ADC对OE19-p95HER2 tet on细胞或T47D-p95HER2 tet on细胞的杀伤实验
将稳定表达p95HER2tet-on系统的肿瘤细胞系OE19细胞系或稳定表达p95HER2 tet-on系统的肿瘤细胞系T47D细胞系用2μg/mL多西环素诱导3天，以胰酶消化收集后，用含2μg/mL多西环素的培养基重悬，以3500个细胞/孔的密度接种至96孔平底板中，置于37℃、5％CO2培养箱中孵育24小时。将抗体用含2μg/mL多西环素的培养基进行梯度稀释，然后将Hab02.11-MMAE ADC分子(DAR4)或其对照分子加入细胞中，置于37℃、5％CO2培养箱中继续孵育7天。每48h补充一次多西环素(终浓度2μg/mL)。7天后，以CellTiter-Glo检测细胞活率，再转化为抑制率(抑制率＝1-细胞活率百分比)。实验结果见图8-9，结果表明，Hab02.11-MMAE ADC对OE19-p95HER2细胞和T47D-P95HER2细胞具有较好的杀伤作用，说明其有较好的对表达靶点抗原的细胞杀伤的能力。
实施例9 p95HER2/HER2双特异性抗体的生产和结合活性检测
1.p95HER2/HER2双抗的构建和生产
设计多种形式的p95HER2/HER2双抗(图10A～图10C)：
(1)图10A所示抗体为由VH1-linker-VH2-CH1-Fc和VL1-linker-VL2-CL组成的DVD-Ig形式抗体，其中，VH1和VL1靶向HER2，VH2和VL2靶向p95HER2，linker可省略。
(2)图10B所示抗体为由VH1-linker-VH2-CH1-Fc和VL1-linker-VL2-CL组成的DVD-Ig形式抗体，其中，VH1和VL1靶向p95HER2，VH2和VL2靶向HER2，linker可省略。
(3)图10C所示抗体为由VH1-CH1-Fc和VL1-CL-VH2-linker-VL2组成的四链抗体，其中其中，VH1和VL1靶向p95HER2，VH2和VL2靶向HER2，linker可省略。
双抗的具体序列参见表18，其中，HER2抗体pertuzumab和trastuzumab的CDR序列 参见表19。
按所设计结构，分别在pTT5载体上构建双特异性抗体轻重链表达质粒，并于HEK293细胞中表达，然后采用实施例1所述ProteinA亲和层析的方法进行纯化。纯化后符合纯度要求的蛋白，透析换液并进行后续检测，对不同形式p95HER2/HER2双抗形式进行评价。
表18 p95HER2/HER2双抗序列信息
表19 HER2抗体CDR分析
2.p95HER2/HER2双抗与CHO-K1 human HER2细胞的结合实验
p95HER2/HER2双抗与HER2的结合力通过抗体与CHO-K1-hu HER2细胞系的FACS实验来检测。具体实验方法如下：
收集CHO-K1-hu HER2细胞，将细胞密度调整至5×105cells/ml，以100μl/孔的体积加入96孔培养板(Corning，Cat No.3799)中，4℃，1500rpm，5min离心。弃去上清，加入250μl/孔PBS(HyClone，Cat No.SH30256.01)，4℃，1500rpm，5min离心。弃去上清后，加入100μl/孔用样品稀释液(2％BSA-PBS)稀释的不同浓度待测抗体，放于4℃冰箱孵育1小时，孵育结束后，4℃，1500rpm，5min离心。弃去上清，加入250μl/孔PBS(HyClone，Cat No.SH30256.01)，4℃，1500rpm，5min离心(Thermo)。弃去上清后，加入100μl/孔用样品稀释液(2％BSA-PBS)稀释的Alexa Fluor-647标记的羊抗人二抗(Jackson Immuno Research，CatNo.109-605-088)，放于4℃冰箱孵育1小时。孵育结束后，4℃，1500rpm，5min离心(Thermo)洗涤3次。最后弃去上清，加入80μl/孔PBS重悬细胞，流式细胞仪(BD,Canto II)检测荧光信号强弱。
实验结果见图11A～图11B，BsAb02-P抗体(图10A双抗形式)结合HER2的能力与HER2单抗(trastuzumab-hIgG1,pertuzumab-hIgG1)相当，而BsAb01-T(图10C所示双抗结构)结合HER2的能力，与Trastuzumab-hIgG1单抗相比，显著下降，表明图10A所示结构优于图10C，图10A所示双抗结构不影响双抗与HER2的结合。
3.p95HER2/HER2双抗的内吞实验
参照实施例8检测p95HER2/HER2双抗的内吞能力，实验结果详见图12A～图12B：BsAb02-P(10A所示双抗结构，HER2抗体部分靶向HER2胞外结构域的结构域II)的内吞能力优于BsAb02-P-B(图10B所示双抗结构域，HER2抗体部分靶向HER2胞外结构域的结构域II)，同时还优于BsAb02-T(图10A所示双抗结构域，HER2抗体部分靶向HER2胞外结构域的结构域IV)。
4.p95HER2/HER2双抗BsAb02-P的Biacore测定
该实验用Biacore 8K(GE)仪器，采用多循环动力学测定待测抗体BsAb02-P与人p95HER2(hup95HER2.ECD-Fc)和HER2的亲和力。
实验运行缓冲液为1×HBS-EP+缓冲溶液(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)(Cat.#BR-1006-69，GE)，流经池温度设置为25℃，样品仓温度设置为16℃。两者都用运行缓冲液预处理。用ProteinA生物传感芯片(Cat.#29127556,GE)亲和捕获一定量的待测抗体，然后于芯片表面流经一定浓度的人p95HER2抗原或人HER2 抗原，利用Biacore 8K仪器(GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，pH1.5的甘氨酸-盐酸再生溶液(Cat.#BR-1003-54,GE)将抗原-抗体复合物洗净再生。具体地，通过注射溶液中不同浓度的人p95HER2和人HER2抗原240s来检测其结合过程，流速为30μL/min，从50nM起始，以1:1稀释，设置一系列浓度梯度；解离时间长达900s，最后用10mM甘氨酸-盐酸溶液(pH 1.5)以30μL/min的流速洗涤30s完成芯片表面的再生。
实验得到的数据用GE Biacore 8K Evaluation version 2.0软件以(1:1)Langmuir模型进行拟合，得出结合速率(Ka)、解离速率(Kd)和亲和力数值(KD)，具体见表20-21所示。实验结果表明，本公开的p95HER2/HER2双抗能与人p95HER2和HER2以高亲和力结合。
表20 p95HER2/HER2双抗与人p95HER2蛋白的反应亲和力
表21 p95HER2/HER2双抗与人HER2蛋白的反应亲和力
实施例10.p95HER2/HER2双抗ADC的偶联和功能实验
1.BsAb02-P双抗ADC的偶联
使用超滤管(Millpore,货号：UFC903096)将BsAb02-P超滤换液至BufferA(50mM磷酸盐,150mM NaCl,1mM EDTA,pH6.50)，抗体浓度控制在10mg/mL。向抗体中加入1.8倍当量和3.6倍当量10mM TCEP(Sigma，货号75259)；25℃，300r/min恒温金属浴(Eppendorf,型号Thermo Mixer C)孵育3h,还原抗体内部二硫键。向部分还原蛋白溶液中加入10倍当量DMSO溶解VcMMAE(MCE,货号HY-15575)；25℃，300r/min恒温金属浴孵育3h。使用超滤管对ADC样品进行超滤换液至Buffer A，换液体积1000倍以上，去除游离毒素。采用HIC和LC-MS方法对样品进行分析，获得DAR2和DAR4终样品。
2.p95HER2/HER2双抗和双抗ADC与CHO-K1 hu p95HER2或CHO-K1-hu HER2细胞的结合实验
p95HER2/HER2双抗BsAb02-P和对应双抗ADC与p95HER2和HER2的结合力通过FACS实验来检测。将细胞收集到96孔细胞板中，抗体加入后信号的强弱被用于判断抗体和CHO-K1-hup95HER2和CHO-K1-hu HER2的结合活性。具体实验方法如下：
收集CHO-K1-hu p95HER2或CHO-K1-hu HER2细胞，将细胞密度调整至5×105cells/ml，以100μl/孔的体积加入96孔培养板(Corning，Cat No.3799)中，4℃，1500rpm，5min离心。弃去上清，加入250μl/孔PBS(HyClone，CatNo.SH30256.01)，4℃，1500rpm，5min离心。弃去上清后，加入100μl/孔用样品稀释液(2％BSA-PBS)稀释的不同浓度待测抗体(BsAb02-P或对应ADC)，放于4℃冰箱孵育1小时，孵育结束后，4℃，1500rpm，5min离心。弃去上清，加入250μl/孔PBS(HyClone，CatNo.SH30256.01)，4℃，1500rpm，5min离心(Thermo)。弃去上清后，加入100μl/孔用样品稀释液(2％BSA-PBS)稀释的Alexa  Fluor-647标记的羊抗人二抗(Jackson Immuno Research，Cat No.109-605-088)，放于4℃冰箱孵育1小时。孵育结束后，4℃，1500rpm，5min离心(Thermo)洗涤3次。最后弃去上清，加入80μl/孔PBS重悬细胞，流式细胞仪(BD,Canto II)检测荧光信号强弱。
实验结果见图13A～图13B。实验结果表明，BsAb02-P及其ADC与CHO-K1-hu p95HER2细胞和CHO-K1-hu HER2有很好的结合作用，MMAE毒素的偶联不影响抗体与靶蛋白的结合。
3.p95HER2/HER2双抗ADC对SKBR3-p95HER2细胞或SKBR3细胞的杀伤实验
将SKBR3细胞系和SKBR3-p95HER2过表达细胞系以胰酶消化后，用含10％胎牛血清的McCoy's 5A培养基重悬，以5000个细胞/孔的密度接种至96孔平底板，置于37℃、5％CO2培养箱中培养24小时。将抗体用含10％胎牛血清的培养基进行梯度稀释，然后将BsAb02-P-MMAE或其对照分子加入细胞中，置于37℃、5％CO2培养箱中培养72小时。实验结束时，以CellTiter-Glo检测细胞活率，再转化为抑制率(抑制率＝1-细胞活率百分比)。
实验结果见图14A～图14B，结果表明，BsAb02-P-MMAE ADC(DAR2)和BsAb02-P-MMAE ADC(DAR4)对HER2阳性的SKBR3细胞系和HER2阳性p95HER2阳性的SKBR3-P95HER2过表达细胞系具有较好的杀伤作用。
4.p95HER2/HER2双抗ADC对OE19-p95HER2细胞或OE19细胞的杀伤实验
将稳定表达p95tet-on系统的肿瘤细胞系OE19细胞系用2μg/mL多西环素诱导3天，以胰酶消化收集后，用含2μg/mL多西环素的培养基重悬，以3500个细胞/孔的密度接种至96孔平底板中，置于37℃、5％CO2培养箱中孵育24小时。将抗体用含2μg/mL多西环素的培养基进行梯度稀释，然后将BsAb02-P-MMAE或其对照分子加入细胞中，置于37℃、5％CO2培养箱中继续孵育7天。每48h补充一次多西环素(终浓度2μg/mL)。7天后，以CellTiter-Glo检测细胞活率，再转化为抑制率(抑制率＝1-细胞活率百分比)。按照相同方法检测p95HER2/HER2双抗ADC对OE19细胞的杀伤作用，区别在于，不使用多西环素诱导p95HER2的表达。
实验结果见图15A～图15B，结果表明，BsAb02-P-MMAE ADC(DAR2)和BsAb02-P-MMAE ADC(DAR4)对HER2阳性的OE19细胞系和HER2阳性p95HER2阳性的OE19-P95HER2过表达细胞系具有较好的杀伤作用。
5.p95HER2/HER2双抗ADC对T47D-p95HER2细胞的杀伤实验
将稳定表达p95tet-on系统的肿瘤细胞系T47D细胞系用2μg/mL多西环素诱导3天，以胰酶消化收集后，用含2μg/mL多西环素的培养基重悬，以3500个细胞/孔的密度接种至96孔平底板中，置于37℃、5％CO2培养箱中孵育24小时。将抗体用含2μg/mL多西环素的培养基进行梯度稀释，然后将p95HER2/HER2双抗和MMAE偶联的ADC分子(BsAb02-P-MMAE)或其对照分子加入细胞中，置于37℃、5％CO2培养箱中继续孵育7天。每48h补充一次多西环素(终浓度2μg/mL)。7天后，以CellTiter-Glo检测细胞活率。再转化为抑制率(抑制率＝1-细胞活率百分比)。按照相同方法检测p95HER2/HER2双抗ADC对T47D细胞的杀伤作用，区别在于，不使用多西环素诱导p95HER2的表达。
实验结果见图16A～图16B，结果表明，BsAb02-P-MMAE ADC(DAR2)和BsAb02-P-MMAE ADC(DAR4)对HER2极弱阳性p95HER2阳性的T47D-p95HER2过表达细胞系具有较好的杀伤作用。
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，不用于限定本发明的保护范围。

Claims (29)

  1. 特异性结合人p95HER2的抗体或抗原结合片段，其包括重链可变区(VH)和轻链可变区(VL)，其中：
    (1)所述重链可变区包括SEQ ID NO:18，78或86所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:19，79或87所示VL中所含的LCDR1、LCDR2和LCDR3；
    (2)所述重链可变区包括SEQ ID NO:16，76或84所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:17，77或85所示VL中所含的LCDR1、LCDR2和LCDR3；
    (3)所述重链可变区包括SEQ ID NO:20，80或88所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:21，81或89所示VL中所含的LCDR1、LCDR2和LCDR3；
    (4)所述重链可变区包括SEQ ID NO:22，82或90所示VH中所含的HCDR1、HCDR2和HCDR3，所述轻链可变区包括SEQ ID NO:23，83或91所示VL中所含的LCDR1、LCDR2和LCDR3；或
    (5)所述重链可变区和所述轻链可变区包括与第(1)-(4)组的任一组中所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性的序列，或至多发生3个插入、缺失或替换突变的序列；优选地，所述替换突变发生在“DDD”中的第二个“D”，优选突变为“DND”，或所述替换突变发生在“NG”中的“N”，优选突变为“SG”；
    优选地，所述HCDR1-3和所述LCDR1-3根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定，更优选地，根据Kabat编号系统确定，最优选地，所述HCDR1-3和所述LCDR1-3选自表7或表12。
  2. 特异性结合人p95HER2的抗体或抗原结合片段，其包括重链可变区(VH)和轻链可变区(VL)，所述重链可变区包括HCDR1、HCDR2和HCDR3，所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中，所述HCDR1-3和所述LCDR1-3选自表7或表12；
    优选地，
    (1)所述HCDR1选自SEQ ID NO:37，40和42，所述HCDR2选自SEQ ID NO:38，41和43，所述HCDR3选自SEQ ID NO:39和44；所述LCDR1选自SEQ ID NO:45和48，所述LCDR2选自SEQ ID NO:46和49，所述LCDR3选自SEQ ID NO:47；
    (2)所述HCDR1选自SEQ ID NO:24，27和29，所述HCDR2选自SEQ ID NO:25，28，30和92，所述HCDR3选自SEQ ID NO:26和31；所述LCDR1选自SEQ ID NO:32和35，所述LCDR2选自SEQ ID NO:33和36，所述LCDR3选自SEQ ID NO:34；
    (3)所述HCDR1选自SEQ ID NO:50，53，55和93，所述HCDR2选自SEQ ID NO:51，54和56，所述HCDR3选自SEQ ID NO:52和57；所述LCDR1选自SEQ ID NO:58和61，所述LCDR2选自SEQ ID NO:59和62，所述LCDR3选自SEQ ID NO:60；
    (4)所述HCDR1选自SEQ ID NO:63，66和68，所述HCDR2选自SEQ ID NO:64， 67和69，所述HCDR3选自SEQ ID NO:65和70；所述LCDR1选自SEQ ID NO:71和74，所述LCDR2选自SEQ ID NO:72和75，所述LCDR3选自SEQ ID NO:73；或，
    (5)所述HCDR1-3和所述LCDR1-3具有与第(1)-(4)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性，或至多发生3个插入、缺失或替换突变；优选地，所述替换突变发生在“DDD”中的第二个“D”，优选突变为“DND”，或所述替换突变发生在“NG”中的“N”，优选突变为“SG”。
  3. 根据权利要求1-2任一项所述抗体或抗原结合片段，其中，所述抗体或抗原结合片段为鼠源抗体，嵌合抗体，人源化抗体或全人抗体；
    优选地，所述抗体或抗原结合片段为人源化抗体，所述重链可变区和轻链可变区包括人源框架区和植入其中的所述HCDR1-3和LCDR1-3；
    更优选地，
    (1)所述重链可变区的框架区包括IGHV2-70*04的框架区1-3(HFR1-3)和IGHJ6*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV1-39*01的框架区1-3(LFR1-3)和IGKJ4*01的框架区4(HFR4)；
    (2)所述重链可变区的框架区包括IGHV2-70*01的框架区1-3(HFR1-3)和IGHJ6*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV3-11*01的框架区1-3(LFR1-3)和IGKJ2*01的框架区4(LFR4)；
    (3)所述重链可变区的框架区包括IGHV2-70*10的框架区1-3(HFR1-3)和IGHJ6*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV4-1*01的框架区1-3(LFR1-3)和IGKJ2*01的框架区4(LFR4)；
    (4)所述重链可变区的框架区包括IGHV33-7*01的框架区1-3(HFR1-3)和IGHJ1*01的框架区4(HFR4)，所述轻链可变区的框架区包括IGKV2-29*02的框架区1-3(LFR1-3)和IGKJ2*01的框架区4(LFR4)；或，
    (5)所述重链可变区的框架区和所述轻链可变区的框架区包括与所述HFR1-4和LFR1-4中的每个FR相比，具有至少80％同一性的序列，或至多发生10个插入、缺失或替换突变的序列；优选地，按自然顺序编号，(i)所述重链可变区包括下述突变中的一个或多个：I71V和A98V突变，所述轻链可变区包括D1L突变；(ii)所述重链可变区包括S30Y和/或K77G突变；(iii)所述重链可变区包括下述突变中的一个或多个：L4F、G27F、I29L、S30I、V71R、N76S、F78V、S79F和A96T，所述轻链可变区的框架区包括P43S突变；(iv)所述重链可变区包括下述突变中的一个或多个：S30N、K76Q和A97T，所述轻链可变区包括下述突变中的一个或多个：Y41L、A48S和G73E。
  4. 根据权利要求1-3任一项所述抗体或抗原结合片段，其中：
    (1)所述重链可变区包括SEQ ID NO:18，78或86所示序列，所述轻链可变区包括SEQ ID NO:19，79或87所示序列；
    (2)所述重链可变区包括SEQ ID NO:16，76或84所示序列，所述轻链可变区包括SEQ ID NO:17，77或85所示序列；
    (3)所述重链可变区包括SEQ ID NO:20，80或88所示序列，所述轻链可变区包括 SEQ ID NO:21，81或89所示序列；
    (4)所述重链可变区包括SEQ ID NO:22，82或90所示序列，所述轻链可变区包括SEQ ID NO:23，83或91所示序列；或
    (5)所述重链可变区和所述轻链可变区包括与第(1)-(4)组的任一组中所述重链可变区和轻链可变区相比，具有至少80％同一性的序列，或至多发生15个插入、缺失或替换突变的序列。
  5. 根据权利要求1-4任一项所述的抗体或抗原结合片段，其结合人p95HER2的KD小于10nM，2nM，1nM，0.5nM，0.2nM，0.01nM或0.06nM；
    任选地，所述抗体或抗原结合片段还结合猴p95HER2，优选KD小于10nM，3nM，1nM，0.5nM或0.1nM。
  6. 根据权利要求1-5任一项所述的抗体或抗原结合片段，其包含重链恒定区序列和/或轻链恒定区序列，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区片段包括CH1，铰链区，CH2，CH3或Fc；
    优选地，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；
    优选地，所述抗体或抗原结合片段包括完整的重链和轻链，所述重链由所述VH和重链恒定区组成，所述重链恒定区优选具有如SEQ ID NO:9所示序列，所述轻链由所述VL和轻链恒定区组成，所述轻链恒定区优选具有如SEQ ID NO:10所示序列。
  7. 根据权利要求1～6任一项所述的抗体或抗原结合片段，其中，所述抗原结合片段选自Fab，Fab’，Fab’-SH，(Fab’)2、scFv或双抗体(diabody)。
  8. 特异性结合人p95HER2的抗体或抗原结合片段，其与权利要求1～6任一项所述抗体或抗原结合片段竞争结合p95HER2，或与权利要求1～6任一项所述抗体或抗原结合片段结合相同或重叠的表位。
  9. 多特性抗体或抗原结合片段，其包括权利要求1～8任一项所述特异性结合人p95HER2的抗体或抗原结合片段，还包括结合其他抗原的结合单元，优选还包括结合HER2的抗体或抗原结合片段。
  10. 多特异性抗体或抗原结合片段，其包括第一肽链和第二肽链，以及第三肽链和第四肽链，其中：
    所述第一肽链和第三肽链包括VH1-(X1)n-VH2-CH1-Fc，所述X1为接头1，n是0或1；
    所述第二肽链和第四肽链包括VL1-(X2)n-VL2-CL，所述X2为接头2，n是0或1；
    所述VH1和VL1形成HER2结合结构域，所述VH2和VL2形成p95HER2结合结构域，或所述VH1和VL1形成p95HER2结合结构域，所述VH2和VL2形成HER2结合结构域。
  11. 根据权利要求10所述的多特异性抗体或抗原结合片段，其中，所述p95Her2结合结构域包括权利要求1～7任一项所述抗体或抗原结合片段。
  12. 根据权利要求10～11任一项所述的多特异性抗体或抗原结合片段，其中，所述HER2结合结构域结合HER2胞外结构域的结构域II或结构域IV；优选地，所述HER2结合结构域具有以下特征之一：
    (3)所述HER2结合结构域包括SEQ ID NO:5所示VH中的HCDR1-3和SEQ ID NO:6所示VL中的LCDR1-3；优选地，所述HCDR1-3具有如SEQ ID NO:108-110所示的序列，所述LCDR1-3具有如SEQ IDNO:111-113所示序列；更优选地，所述HER2结合结构域包括SEQ ID NO:5和SEQ ID NO:6所示序列；
    (4)所述HER2结合结构域包括SEQ ID NO:7所示VH中的HCDR1-3和SEQ ID NO:8所示VL中的LCDR1-3；优选地，所述HCDR1-3具有如SEQ ID NO:102-104所示的序列，所述LCDR1-3具有如SEQ IDNO:105-107所示序列；更优选地，所述HER2结合结构域包括SEQ ID NO:7和SEQ ID NO:8所示序列；
    (3)与第(1)或(2)组所述HER2抗原结合结构域竞争性结合HER2，或结合表位相同或重叠。
  13. 根据权利要求10～12任一项所述的多特异性抗体或抗原结合片段，其中，所述接头1和接头2为(G4S)n，n选自1，2，3，4，5或6；
    任选地，所述CH1-Fc具有具有与SEQ ID NO:9相同或至少具有80％同一性或至多存在20个缺失、插入或替换突变的序列；
    任选地，所述CL具有与SEQ ID NO:10相同或至少具有80％同一性或至多存在20个缺失、插入或替换突变的序列。
  14. 根据权利要求10～13任一项所述的多特异性抗体或抗原结合片段，其中，所述第一肽链和所述第三肽链具有与SEQ ID NO:94、96、98相同或至少具有80％同一性或至多存在30个缺失、插入或替换突变的序列，所述第二肽链和所述第四肽链具有与SEQ ID NO:95、97、99相同或至少具有80％同一性或至多存在30个缺失、插入或替换突变的序列。
  15. 如Ab-(L-D)n所示的抗体-药物偶联物或其药学上可接受的盐或溶剂合物，其中：
    Ab表示权利要求1～14任一项所示的抗体或抗原结合片段；
    L表示用于连接A与D的接头，优选为mc-vc-pAB或mc；
    D表示药物，优选为海兔毒素肽或其衍生物，更优选为MMAE或MMAF；
    n表示药物抗体比(drug-to-antibody ratio,DAR)，任选为1～10，优选为2或4。
  16. 根据权利要求15所示的抗体-药物偶联物或其药学上可接受的盐或溶剂合物，其中，所述抗体-药物偶联物具有如下式所示结构：
  17. 分离的核酸，其中，所述核酸编码权利要求1～14任一项所述抗体或抗原结合片段。
  18. 编码嵌合抗原受体(chimeric antigen receptor,CAR)的核酸，其中，所述CAR包括信号肽、抗原结合结构域、铰链区、跨膜区和胞内细胞传导区，所述抗原结合结构域包含权利要求1～14所述抗体或抗原结合结构域。
  19. 载体(vector)，其包含权利要求18或19所述的核酸。
  20. 宿主细胞，其包含权利要求17所述核酸或权利要求19所述载体或表达权利要求1～14任一项所述抗体或抗原结合片段；
    优选地，所述宿主细胞是原核细胞或真核细胞；
    更优选地，所述宿主细胞选自大肠杆菌、酵母、哺乳动物细胞或适合制备抗体或抗原结合片段地其他细胞，例如HEK293细胞或CHO细胞。
  21. 免疫效应细胞，其包含权利要求18所述核酸或权利要求19所述载体，或表达权利要求18所述核酸编码的嵌合抗原受体；优选地，所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞。
  22. 药物组合物，其包含权利要求1～14任一项所述的抗体或抗原结合片段或权利要求15～16任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂合物或权利要求21所述的免疫效应细胞，以及任选的药学上接受的运载体(carrier)，以及任选的其他治疗剂。
  23. 制备权利要求1～14任一项所述抗体或抗原结合片段或权利要求15～16任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂合物或权利要求21所述免疫效应细胞的方法，所述方法包括：
    (1)培养权利要求20所述宿主细胞表达权利要求1～14任一项所述抗体或抗原结合片段，任选地，所述方法还包括分离所述抗体或抗原结合片段；或，
    (2)在权利要求1～14任一项所述抗体或抗原结合片段上偶联药物，获得权利要求15～16任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂合物；或，
    (3)在免疫效应细胞中转入权利要求19所述核酸或权利要求19所述载体，获得包含所述核酸或载体或获得表达权利要求18所述核酸编码的嵌合抗原受体的免疫细胞。
  24. 权利要求1～14任一项所述抗体或抗原结合片段或权利要求15～16任一项所述抗体-药物偶联物或其药学上可接受的盐或溶剂合物或权利要求17或18所述核酸或权利要求19所述载体或权利要求20所述宿主细胞或权利要求21所述免疫效应细胞或权利要求22所述药物组合物或根据权利要求23所述方法制备的产物，在制备用于治疗癌症或肿瘤中药物中的用途；
    优选地，所述癌症或肿瘤选自实体瘤，例如，乳腺癌，胃癌，膀胱癌，卵巢癌，肺癌，前列腺癌，胰腺癌，结直肠癌，头颈癌，肺癌，胆道癌，尿路上皮癌，肝癌，食道癌或骨肉瘤；
    优选地，所述癌症或肿瘤表现为HER2+和/或p95HER2+；
    优选地，所述癌症或肿瘤表现出HER2靶向疗法耐药性，例如曲妥珠、帕妥珠、T-DM1、 DS-8201或RC48耐药性；
    优选地，所述药物还包括其他治疗剂，例如化疗药(紫杉烷，选自多西他赛或紫杉醇；蒽环类，选自阿霉素，表柔比星，柔红霉素或戊柔比星)，或免疫检查点抑制剂(PD-1抗体或PD-L1抗体)。
  25. 权利要求1～14任一项所述抗体或抗原结合片段或权利要求15～16任一项所述抗体-药物偶联物或其药学上可接受的盐或溶剂合物或权利要求17或18所述核酸或权利要求19所述载体或权利要求20所述宿主细胞或权利要求21所述免疫效应细胞或权利要求22所述药物组合物或根据权利要求23所述方法制备的产物，用于治疗癌症或肿瘤；
    优选地，所述癌症或肿瘤选自实体瘤，例如，乳腺癌，胃癌，膀胱癌，卵巢癌，肺癌，前列腺癌，胰腺癌，结直肠癌，头颈癌，肺癌，胆道癌，尿路上皮癌，肝癌，食道癌或骨肉瘤；
    优选地，所述癌症或肿瘤表现为HER2+和/或p95HER2+；
    优选地，所述癌症或肿瘤表现出HER2靶向疗法耐药性，例如曲妥珠、帕妥珠、T-DM1、DS-8201或RC48耐药性；
    优选地，所述治疗还包括其他治疗剂，例如化疗药(紫杉烷，选自多西他赛或紫杉醇；蒽环类，选自阿霉素，表柔比星，柔红霉素或戊柔比星)，或免疫检查点抑制剂(PD-1抗体或PD-L1抗体)。
  26. 治疗癌症或肿瘤的方法，其包括向受试者给予有效量的权利要求1～14任一项所述抗体或抗原结合片段或权利要求15～16任一项所述抗体-药物偶联物或其药学上可接受的盐或溶剂合物或权利要求17或18所述核酸或权利要求19所述载体或权利要求20所述宿主细胞或权利要求21所述免疫效应细胞或权利要求22所述药物组合物或根据权利要求23所述方法制备的产物；
    优选地，所述癌症或肿瘤选自实体瘤，例如，乳腺癌，胃癌，膀胱癌，卵巢癌，肺癌，前列腺癌，胰腺癌，结直肠癌，头颈癌，肺癌，胆道癌，尿路上皮癌，肝癌，食道癌或骨肉瘤；
    优选地，所述癌症或肿瘤表现为HER2+和/或p95HER2+；
    优选地，所述癌症或肿瘤表现出HER2靶向疗法耐药性，例如曲妥珠、帕妥珠、T-DM1、DS-8201或RC48耐药性；
    优选地，所述药物还包括其他治疗剂，例如化疗药(紫杉烷，选自多西他赛或紫杉醇；蒽环类，选自阿霉素，表柔比星，柔红霉素或戊柔比星)，或免疫检查点抑制剂(PD-1抗体或PD-L1抗体)。
  27. 检测p95HER2的方法，其包括在权利要求1～8任一项所述抗体或抗原结合片段能与p95HER2形成复合物的条件下，使待测样品与所述抗体或抗原结合片段接触；
    任选地，检测所述复合物的形成，指示样品中p95HER2的存在或表达水平；
    任选地，所述样品为癌症或肿瘤患者的样品，根据样品中p95HER2的存在或表达水平，鉴定所述患者是否能从p95HER特异性治疗中受益；
    任选地，所述方法还包括在HER2抗体或其抗原结合片段能与HER2形成复合物的条件下，使待测样品与所述HER2抗体或抗原结合片段接触，检测所述复合物的形成，指示样品中HER2的存在或表达水平，和任选地，所述样品为癌症或肿瘤患者的样品，根据所述HER2的存在或表达水平，鉴定所述患者是否能从HER2特异性治疗中受益。
    任选地，所述治疗包括权利要求26所述治疗癌症或肿瘤的方法；
    任选地，所述检测通过免疫组织化学(ICH)进行。
  28. 权利要求1～8任一项所述的抗体或抗原结合片段在制备用于检测p95HER2的试剂盒中的应用。
  29. 根据权利要求28所述应用制备得到的试剂盒。
PCT/CN2023/072966 2022-01-26 2023-01-18 p95HER2抗体及其应用 WO2023143322A1 (zh)

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