WO2024088325A1 - 抗体及其应用 - Google Patents

抗体及其应用 Download PDF

Info

Publication number
WO2024088325A1
WO2024088325A1 PCT/CN2023/126593 CN2023126593W WO2024088325A1 WO 2024088325 A1 WO2024088325 A1 WO 2024088325A1 CN 2023126593 W CN2023126593 W CN 2023126593W WO 2024088325 A1 WO2024088325 A1 WO 2024088325A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
antigen
binding fragment
amino acid
Prior art date
Application number
PCT/CN2023/126593
Other languages
English (en)
French (fr)
Inventor
周亮
任鹏举
王鹏
李宗海
Original Assignee
科济生物医药（上海）有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 科济生物医药（上海）有限公司 filed Critical 科济生物医药（上海）有限公司
Publication of WO2024088325A1 publication Critical patent/WO2024088325A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of tumor immunotherapy or diagnosis, and more specifically, to antibodies specifically binding to CLDN6 and applications thereof.
  • CLDN tight junction protein
  • CLDN proteins contain four transmembrane (TM) helices (TM1, TM2, TM3, and TM4) and two extracellular loops (EL1 and EL2), with both the N-terminus and C-terminus located in the cytoplasm.
  • TM transmembrane
  • EL1 and EL2 extracellular loops
  • CLDN proteins play a role in various human diseases and pathologies.
  • CLDN6 is a member of the CLDN gene family. It is not expressed in normal adult tissues, but is highly expressed in a variety of solid tumor tissues such as ovarian cancer, testicular cancer, and endometrial cancer. Therefore, CLDN6 is a potential target for cancer treatment in the Claudin family.
  • the extracellular segment sequence homology of CLDN6 and its family members CLDN9 and CLDN4 is very high, so it is very challenging to develop antibodies that specifically bind to CLDN6.
  • the present invention aims to provide an antibody that specifically recognizes CLDN6.
  • the present invention also relates to a method for preparing an anti-CLDN6 specific antibody, including a humanized antibody preparation technology and a phage library screening technology.
  • the present invention also relates to the study of the characteristics and specificity of anti-CLDN6 antibodies (including but not limited to scFv forms).
  • the present invention provides a chimeric antigen receptor (CAR) targeting CLDN6 and a method for preparing the same.
  • the present invention also provides an isolated nucleic acid encoding an anti-CLDN6 antibody of the present invention and a chimeric antigen receptor targeting CLDN6.
  • the present invention also provides a host cell comprising the nucleic acid of the present invention.
  • the method further comprises culturing the host cell of the present invention to prepare the antibody or the CAR.
  • the antibody and/or CAR of the present invention are used to treat tumors or tumor diagnosis.
  • an antibody or antigen-binding fragment that recognizes CLDN6 is provided, selected from the group consisting of:
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the HCDR3 of the heavy chain variable region has at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR3 of SEQ ID NO: 41;
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 represented by GYYMN (SEQ ID NO: 35); and/or
  • HCDR3 represented by RDYYX1GSX2X3YAX4DY (SEQ ID NO: 52 ) , wherein X1 is Y or L or a conservatively substituted amino acid residue of Y or L, X2 is G or N or a conservatively substituted amino acid residue of G or N, X3 is F or S or a conservatively substituted amino acid residue of F or S, and X4 is M or L or a conservatively substituted amino acid residue of M or L;
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR1, HCDR2 and HCDR3 set forth in SEQ ID NOs: 35, 36 and 37;
  • the antibody or antigen-binding fragment comprises a light chain variable region, wherein LCDR3 of the light chain variable region is identical to SEQ ID LCDR3 as set forth in NO: 42, 43 or 44 has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity;
  • the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 represented by QASQSVSNNLN (SEQ ID NO: 38); and/or
  • LCDR3 represented by X5QHRX6X7WT (SEQ ID NO: 53 ), wherein X5 is L or Q or a conservatively substituted amino acid residue of L or Q, X6 is Y or F or a conservatively substituted amino acid residue of Y or F, and X7 is L or M or a conservatively substituted amino acid residue of L or M;
  • the antibody or antigen-binding fragment comprises a light chain variable region, wherein LCDR1, LCDR2 and LCDR3 of the light chain variable region have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 38, 39 and 40;
  • the antibody or antigen-binding fragment comprises the heavy chain variable region described in any one of (1) to (3) and the light chain variable region described in any one of (4) to (6);
  • the antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), wherein the variant comprises at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).
  • the HCDR2 of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:36.
  • the antibody or antigen-binding fragment is selected from the group consisting of:
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 35, and/or HCDR2 shown in SEQ ID NO: 36, and/or HCDR3 shown in SEQ ID NO: 37 or 41;
  • the antibody or antigen-binding fragment comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 shown in SEQ ID NO: 38, and/or LCDR2 shown in SEQ ID NO: 39, and/or LCDR3 shown in SEQ ID NO: 40, 42, 43 or 44;
  • the antibody or antigen-binding fragment comprises the heavy chain variable region described in (1) and the light chain variable region described in (2);
  • the antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (3), wherein the variant comprises at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (3).
  • the antibody or antigen-binding fragment is selected from the group consisting of:
  • the antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40; or
  • the antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 35, SEQ ID NO: 36, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, or SEQ ID NO: 40; or
  • the antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 42; or
  • the antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 43; or
  • the antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 41, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 42; or
  • the antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 41, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 43; or
  • the antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 44; or
  • the antibody or antigen-binding fragment described in any one of (1) to (7) is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), and the variant contains at least one and no more than 7, 6, 5, 4, 3 or 2 amino acid changes in 1, 2, 3, 4, 5 or 6 CDR regions, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).
  • an antibody or antigen-binding fragment that recognizes CLDN6 is provided, selected from the group consisting of:
  • the antibody or antigen-binding fragment comprises a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 or 5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the antibody or antigen-binding fragment comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:3, 7, 9 or 11, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the antibody or antigen-binding fragment comprises the heavy chain variable region described in (1) and the light chain variable region described in (2);
  • the antibody or antigen-binding fragment is a variant of the antibody or antigen-binding fragment described in any one of (1) to (3), wherein the variant comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in VH or VL, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (3).
  • the aforementioned antibody or antigen-binding fragment is selected from the following group:
  • the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:3 an amino acid sequence, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:3, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO:7, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the heavy chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the light chain variable region of the antibody or antigen-binding fragment has the amino acid sequence shown in SEQ ID NO:11, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or
  • the antibody or antigen-binding fragment described in any one of (1) to (7) is a variant of the antibody or antigen-binding fragment described in any one of (1) to (7), wherein the variant comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in VH or VL, and has the same or similar activity as the antibody or antigen-binding fragment described in any one of (1) to (7).
  • an antibody or antigen-binding fragment that recognizes CLDN6 comprising a heavy chain variable region and a light chain variable region,
  • the light chain variable region comprises the sequence shown in SEQ ID NO: 3, 7, 9, 11, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the above sequence;
  • the heavy chain variable region comprises HCDR1 shown in SEQ ID NO:35, HCDR2 shown in SEQ ID NO:36, and HCDR3 shown in SEQ ID NO:37 or 41.
  • the heavy chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:1, 5, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequence.
  • an antibody or antigen-binding fragment that recognizes CLDN6 comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO: 1, 5, or an amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the above sequence; and
  • the light chain variable region comprises LCDR1 shown in SEQ ID NO:38, LCDR2 shown in SEQ ID NO:39, and LCDR3 shown in SEQ ID NO:40, 42, 43 or 44.
  • the light chain variable region of the antibody or antigen-binding fragment comprises the sequence shown in SEQ ID NO:3, 7, 9 or 11, or an amino acid sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the above sequence.
  • the antibody or antigen-binding fragment described in any of the preceding items is selected from a whole antibody, scFv, a single domain antibody, a Fab fragment, a Fab' fragment, a Fv fragment, a F(ab') 2 fragment, a Fd fragment, a dAb fragment, a multifunctional antibody, an IgG4 antibody, a scFv-Fc antibody, a hybridoma antibody, a chimeric antibody, a humanized antibody, a fully human antibody or a monoclonal antibody.
  • the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 13, 14, 15, 16, 17, 18 or 19, or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the above sequence, or comprises at least one and no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid changes in the above sequence.
  • the antibody or antigen-binding fragment binds to CLDN6 and does not significantly bind to CLDN4 or CLDN9; and/or, the antibody or antigen-binding fragment binds to cells expressing CLDN6 and does not significantly bind to cells expressing CLDN4, CLDN9 or a combination thereof.
  • an immunoconjugate comprising: the antibody or antigen-binding fragment described in any one of aspects 1 to 4, and a functional molecule connected thereto.
  • the functional molecule is selected from: a molecule targeting a tumor surface marker, a molecule that inhibits tumors, a molecule targeting a surface marker of an immune cell, or a detectable marker.
  • the molecule targeting a tumor surface marker is an antibody or a ligand that binds to other tumor surface markers except CLDN6.
  • the tumor-suppressing molecule is an anti-tumor cytokine or an anti-tumor toxin.
  • the cytokine is selected from: IL-7, IL-12, IL-15, IL-18, IL-21, type I interferon or TNF- ⁇ .
  • the molecule targeting the surface marker of immune cells is an antibody that binds to the surface marker of T cells, which forms a T cell-involved antigen binding protein with the antibody or antigen-binding fragment described in any one of the first to fourth aspects.
  • Bifunctional antibodies are provided.
  • the surface marker of the targeted immune cell is selected from: CD3, CD16, and CD28.
  • the antibody targeting the surface marker of an immune cell is an anti-CD3 antibody.
  • the immunoconjugate further comprises a connecting peptide between the antibody or antigen-binding fragment described in any one of the first to fourth aspects and the functional molecule connected thereto.
  • a chimeric receptor comprising an extracellular region, wherein the extracellular region comprises the antibody or antigen-binding fragment according to any one of the first to fourth aspects;
  • the chimeric receptor includes: a chimeric antigen receptor (CAR), a chimeric T cell receptor, a T cell antigen coupler (TAC), a synthetic polypeptide receptor (synNotch) or a combination thereof.
  • CAR chimeric antigen receptor
  • TAC T cell antigen coupler
  • SynNotch synthetic polypeptide receptor
  • the chimeric receptor is a chimeric antigen receptor (CAR), which comprises the antibody or antigen-binding fragment described in any one of the first to fourth aspects, a transmembrane region and an intracellular signaling region.
  • CAR chimeric antigen receptor
  • the antibody or antigen-binding fragment is linked to the transmembrane region via a hinge domain.
  • the transmembrane region of the chimeric receptor comprises a transmembrane region selected from the ⁇ , ⁇ , ⁇ or ⁇ chain of TCR, CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD134, CD137, CD152, CD154 or PD1.
  • the transmembrane region is selected from the transmembrane domain of CD8 or CD28.
  • the transmembrane region is selected from the sequence shown in SEQ ID NO: 25 or 22, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.
  • the intracellular signaling region of the chimeric receptor comprises a primary signaling domain.
  • the primary signaling domain is selected from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”), CD66d or CD3 ⁇ .
  • the intracellular signaling region of the chimeric receptor further comprises one or more co-stimulatory signaling domains.
  • the co-stimulatory signal domain is selected from the intracellular signaling region of CARD11, CD2, CD5, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, Fc ⁇ RI ⁇ , MyD88, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB or 4-1BBL, or a combination thereof.
  • the co-stimulatory signaling domain is selected from the intracellular signaling domains of CD28 and/or CD137.
  • the intracellular signaling region of the chimeric receptor is selected from the sequence shown in SEQ ID NO: 24, or SEQ ID NO: 23 and 24, or the sequence shown in SEQ ID NO: 26 and 24, or the sequence of SEQ ID NO: 23, 26 and 24, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the above sequences.
  • the hinge region of the chimeric receptor is derived from CD8, IgG4 or IgG1.
  • the hinge region comprises the sequence shown in SEQ ID NO: 21, 27, 28, 29 or 30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the above sequence.
  • the chimeric receptor comprises:
  • the antibody or antigen-binding fragment described in any one of the first to fourth aspects the transmembrane region of CD8/CD28, the intracellular signaling region of CD28, CD137 and CD3 ⁇ .
  • the chimeric receptor comprises an amino acid sequence as shown in any of SEQ ID NO: 13, 14, 15, 16, 17, 18 or 19 connected to the sequence shown in any of SEQ ID NO: 45, 46 or 47, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the above sequences.
  • the chimeric receptor comprises a sequence as shown in SEQ ID NO: 48 or 49, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the above sequence.
  • nucleic acid encoding the antibody or antigen-binding fragment of any one of the first to fourth aspects, the immunoconjugate of the fifth aspect, or the chimeric receptor of the sixth aspect is provided.
  • a vector which comprises the nucleic acid described in the seventh aspect.
  • a cell which comprises the antibody or antigen-binding fragment described in any one of the first to fourth aspects, the immunoconjugate described in the fifth aspect, the chimeric receptor described in the sixth aspect, the nucleic acid described in the seventh aspect, and/or the vector described in the eighth aspect.
  • the cell comprises a T cell, a natural killer cell, a natural killer T cell, a NK92 cell, a cytotoxic T cell, a dendritic cell, a macrophage, a CIK cell, a pluripotent stem cell, a stem cell-derived immune cell, or a combination thereof.
  • the T cells include natural T cells and/or T cells induced from pluripotent stem cells.
  • the T cells include autologous T cells and/or allogeneic T cells.
  • the T cells are primary T cells.
  • the T cells are derived from human autologous T cells.
  • the cell binds to cells expressing CLDN6 and does not significantly bind to cells expressing CLDN4, CLDN9, or a combination thereof.
  • the cell further carries the coding sequence of an exogenous cytokine; and/or it further expresses a chimeric receptor that does not target CLDN6; and/or it further expresses a chemokine; and/or it further expresses a chemokine receptor; and/or it further expresses a safety switch; and/or it further expresses an inhibitory molecule.
  • the cells further carry exogenous cytokines including coding sequences of IL-7, IL-12, IL-15, IL-18, IL-21, type I interferon or TNF- ⁇ .
  • the cells further express a chemokine comprising CCL19 or CCL21.
  • the cells further express chemokine receptors, including CCR2, CCR4, CCR5, CXCR2, CXCR4 or CXCR5.
  • chemokine receptors including CCR2, CCR4, CCR5, CXCR2, CXCR4 or CXCR5.
  • the cell also expresses a safety switch, which includes iCaspase-9, Truancated EGFR or RQR8.
  • the cells also express inhibitory molecules, including siRNA that reduces PD-1 expression or a protein that blocks PD-L1.
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of the first to fourth aspects of claims, the immunoconjugate of the fifth aspect, the chimeric receptor of the sixth aspect, the nucleic acid of the seventh aspect, the vector of the eighth aspect and/or the cell of the ninth aspect, and a pharmaceutically acceptable adjuvant.
  • a combination drug is provided, wherein the antibody or antigen-binding fragment described in any one of the first to fourth aspects, the immunoconjugate described in the fifth aspect, the chimeric receptor described in the sixth aspect, the cell described in the ninth aspect, or the pharmaceutical composition described in the tenth aspect is administered in combination with an agent that enhances their function.
  • the drug is used in combination with a chemotherapeutic drug; and/or is administered in combination with an agent that improves one or more side effects associated therewith; and/or is administered in combination with a cell expressing a chimeric antigen receptor other than CLDN6; and/or is administered in combination with an agent that treats a disease associated with the expression of CLDN6.
  • the agent comprises an antibody or antigen binding fragment, a cell, RNA, a vaccine, an oncolytic virus, a checkpoint inhibitor, a BKT inhibitor, a chemotherapeutic, a radiotherapeutic, a hormonal therapeutic, a toxin, an immunotherapeutic, or a combination thereof.
  • a method for preparing the antibody or antigen-binding fragment described in any one of aspects 1 to 4, the immunoconjugate described in aspect 5, or the chimeric receptor described in aspect 6 is provided, the method comprising culturing the cells described in aspect 9, and isolating the antibody or antigen-binding fragment, immunoconjugate or chimeric receptor expressed by the cells.
  • kits comprising the antibody or antigen-binding fragment described in any one of the first to fourth aspects, the immunoconjugate described in the fifth aspect, the chimeric receptor described in the sixth aspect, the nucleic acid described in the seventh aspect, the vector described in the eighth aspect, the cell described in the ninth aspect and/or the pharmaceutical composition described in the tenth aspect.
  • the fourteenth aspect uses of the antibody or antigen-binding fragment described in any one of the first to fourth aspects, the immunoconjugate described in the fifth aspect, the cell described in the ninth aspect, the pharmaceutical composition described in the tenth aspect and/or the kit described in the thirteenth aspect, (1) killing cells expressing CLDN6; (2) inhibiting the proliferation of cells expressing CLDN6; (3) mediating the alleviation of diseases or tumors; (4) preventing tumor formation or re-formation; (5) inhibiting the metastasis of cells expressing CLDN6; (6) preparing drugs for treating/diagnosing diseases.
  • the disease expresses CLDN6.
  • the disease is selected from an inflammatory disorder, an infection, an autoimmune disease, and a tumor.
  • the tumor is a solid tumor.
  • the tumor is ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonal tumors, ovarian epithelial cancer, non-small cell lung cancer, non-squamous non-small cell lung cancer, endometrial cancer, or a combination thereof.
  • the present invention provides a method for the following uses and comprising the antibody or antigen described in the first aspect.
  • a method for treating/diagnosing a disease comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment as described in any one of the first to fourth aspects, or the immunoconjugate as described in the fifth aspect, or the cell as described in the ninth aspect, or the pharmaceutical composition as described in the tenth aspect, or the kit as described in the thirteenth aspect.
  • the disease is selected from an inflammatory disorder, an infection, an autoimmune disease, and a tumor.
  • the subject is a human.
  • the cells are autologous or allogeneic T cells to the subject.
  • the antibody or antigen-binding fragment as described in any one of the first to fourth aspects, the immunoconjugate as described in the fifth aspect, the cell as described in the ninth aspect, the pharmaceutical composition as described in the tenth aspect and/or the kit as described in the thirteenth aspect, which is used for treating/diagnosing a disease comprising the expression of CLDN6.
  • the disease is selected from an inflammatory disorder, an infection, an autoimmune disease, and a tumor.
  • the tumor is a solid tumor.
  • the tumor is ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonal tumors, ovarian epithelial cancer, non-small cell lung cancer, non-squamous non-small cell lung cancer, endometrial cancer, or a combination thereof.
  • Figure 1 shows the flow cytometry results of 293T-CLDN4, 293T-CLDN6, and 293T-CLDN9 stably transfected cell lines;
  • FIG2 shows that antibody H1 (scFv-huFc) specifically binds to 293T-CLDN6 cells
  • Figure 3 shows the EC50 of antibody H1 binding to 293T-CLDN6 cells
  • Figure 4 shows that antibodies P1, P2, P3, and P4 all specifically bind to 293T-CLDN6 cells
  • FIG5 shows the EC50 of antibodies P1, P2, P3 and P4 binding to 293T-CLDN6 cells
  • Figure 6 shows that antibodies H1, M1, and M2 all specifically bind to 293T-CLDN6 cells
  • FIG7 shows the EC50 of the binding of antibodies M1 and M2 to 293T-CLDN6 cells
  • Figure 8 shows the positive rates of H1-28Z CAR T cells and P4-28Z CAR T cells
  • Figure 9 shows the in vitro specific killing of target cells by H1-28Z CAR T cells and P4-28Z CAR T cells;
  • Figure 10 shows the in vitro killing of target cells by H1-28Z CAR T cells and P4-28Z CAR T cells;
  • Figure 11 shows the anti-tumor effects of H1-28Z CAR T cells and P4-28Z CAR T cells on subcutaneous transplanted tumors in NPG mice bearing human ovarian cancer cells (Figure 11A); changes in mouse body weight (Figure 11B); changes in tumor weight (Figure 11C); and survival of human T cells in the peripheral blood of mice ( Figure 11D).
  • the present invention provides a humanized antibody (including fragments thereof, such as a heavy chain variable region (VH), a light chain variable region (VL), and scFv) that specifically recognizes CLDN6, a chimeric receptor containing the antibody, a nucleic acid encoding the antibody, and a cell expressing the antibody.
  • the antibody of the present invention can be used to prepare targeted anti-tumor drugs and drugs for diagnosing tumors.
  • the present invention also provides methods for preparing and using the antibody and cells expressing the antibody.
  • the term "about” refers to the usual error range of each value that is easily known to those skilled in the art. A value or parameter, including embodiments that point to the value or parameter itself. For example, a description of "about X” includes “X”. “About” or “comprising” can mean a range of ⁇ 1%, ⁇ 2%, ⁇ 3%, ⁇ 4%, ⁇ 5%, ⁇ 6%, ⁇ 7%, ⁇ 8%, ⁇ 9%, ⁇ 10%, ⁇ 11%, ⁇ 12%, ⁇ 13%, ⁇ 14%, ⁇ 15%, ⁇ 16%, ⁇ 17%, ⁇ 18%, ⁇ 19%, ⁇ 20%, ⁇ 25%, ⁇ 30% of the value. Alternatively, particularly with respect to biological systems or methods, the term can refer to within an order of magnitude of a value, such as within about 5 times or within about 2 times of a value.
  • any concentration range, percentage range, ratio range or integer range described herein should be understood to include any integer within the range, and, where appropriate, fractional values thereof (e.g., tenths and hundredths of an integer).
  • CLDN6 Claudin 6
  • the gene encoding the human CLDN6 protein is located at 16p13.3 on the p-arm of human chromosome 16 and is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, rats, zebrafish and frogs.
  • the CLDN 6 polypeptide has an amino acid sequence or a fragment thereof having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% homology or identity with the amino acid sequence encoded by the transcript expressed by the gene of NCBI GenBank Gene ID: 9074, and/or may optionally include at most one or at most two or at most three conservative amino acid substitutions.
  • the amino acid sequence of the full length of human CLDN6 is shown in SEQ ID NO: 32.
  • CLDN9 refers to the most closely related family member of CLDN6.
  • the gene encoding the human CLDN9 protein is located at 16p13.3 on human chromosome 16 and consists of a single exon spanning approximately 2.1 kBp.
  • the full-length amino acid sequence of human CLDN9 is shown in SEQ ID NO:33.
  • CLDN4 Claudin 4
  • the gene encoding the human CLDN4 protein spans approximately 1.82 kBp at chromosome location 17q11.23.
  • Exemplarily, the full-length amino acid sequence of human CLDN4 is shown in SEQ ID NO:31.
  • polypeptide refers to polymers of any length composed of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids.
  • Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds.
  • the term refers to both short chains (commonly referred to as peptides, oligopeptides and oligomers in the art) and longer chains (commonly referred to as proteins in the art), and there are many types of proteins.
  • Polypeptides include, for example, biologically active fragments, basic homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.
  • the polymer can be linear, cyclic or branched, it can contain modified amino acids, particularly conservatively modified amino acids, and it can be interrupted by non-amino acids.
  • amino acid polymers such as those that have been modified by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer-RNA mediated amino addition such as arginylation, ubiquitination, or any other manipulation such as conjugation with a labeling component.
  • amino acid refers to natural and/or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptide mimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide.
  • the term also includes a polypeptide expressed by a specified nucleic acid sequence.
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen and is used in the broadest sense herein and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, multi-chain or single-chain, or complete immunoglobulins, and may be derived from natural sources or from recombinant sources, as long as it exhibits the desired antigen binding activity.
  • antigen-binding fragment refers to antibodies and immunologically active portions of antibodies, ie, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • the humanized antibodies H1, P1, P2, P3, P4, M1, and M2 of the present invention can specifically bind to CLDN6, but not to CLDN4 and CLDN9.
  • the antigen binding specificity of the antibodies H1, P1, P2, P3, P4, M1, and M2 of the present invention is better than that of the hybridoma antibody SC27.105; further, the antigen binding specificity of the antibodies H1, P1, P2, P3, P4, M1, and M2 of the present invention is better than that of the control antibody C46-S (from patent WO2015150327A1).
  • telomere binding refers to a measurable and reproducible interaction, such as binding of a target and an antibody, where the binding is determinative of the presence of the target in the presence of other molecules.
  • an antibody that specifically binds to a target is one that binds to that target with greater affinity, more readily and/or for a longer duration than it binds to other targets.
  • the antibody or ligand recognizes and binds to an associated binding partner protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.
  • specific binding may include but does not require exclusive binding.
  • Multispecificity refers to an antibody having binding specificity for at least two different sites on an antigen.
  • Antibody fragment refers to at least a portion of an intact antibody or a recombinant variant thereof, and may refer to the antigen binding domain of an intact antibody, such as the antigenicity determining variable region, which is sufficient to confer recognition and specific binding to a target, such as an antigen, on the antibody fragment.
  • antibody fragments include, but are not limited to (i) Fab fragments consisting of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region; (v) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments; (vi) single-chain Fv molecule antigen binding sites; (vii) bispecific single-chain Fv dimers; (viii) "dibodies” or “tribodies", multivalent or multispecific fragments constructed by gene fusion; (ix) scFv genetically fused to the same or different antibodies; (x) linear antibodies; (xi) camelid VHH domains; and (xii) single-domain antibodies such as sdAb (VH or VL). Antibody fragments
  • scFv refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are connected by a short flexible peptide linker, can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • scFv can have VL and VH in any order, for example, for the N-terminus and C-terminus of the polypeptide, scFv can include VL-linker-VH, and can also include VH-linker-VL.
  • the linker can be any amino acid of any length.
  • linker refers to a peptide linker composed of amino acid (e.g., glycine and/or serine) residues, which is used alone or in combination to connect the heavy chain variable region and the light chain variable region together.
  • the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4 or (Gly4Ser)3. In one embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser). In one embodiment, the linker further includes charged residues, such as lysine and/or glutamic acid, which can increase solubility. In one embodiment, the linker further includes one or more proline.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in the antibody molecule in its native conformation, and generally determines the antibody class.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their native conformation.
  • K and ⁇ light chains refer to the two major antibody light chain isotypes.
  • the "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain.
  • the heavy chain constant domains corresponding to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • variable region or variable domain refers to the domain of an antibody heavy chain or light chain that is involved in antibody antigen binding.
  • the heavy and light chain variable domains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain comprising four conserved FRs and three CDRs. .
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • antibodies that bind to a particular antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using a VH or VL domain from an antibody that binds to the antigen. .
  • hypervariable region or “complementarity determining region” or “CDR” refers to each region of an antibody variable domain whose sequence is hypervariable and/or forms structurally defined loops ("hypervariable loops") and/or contains residues that contact the antigen ("antigen contacts"), a non-contiguous amino acid sequence within an antibody variable region that confers specificity and/or binding affinity to the antibody.
  • the CDRs can be identified by a numbering system selected from the group consisting of Kabat, Chothia, IMGT, Gelfand, Aho, and AbM. For example, they can be identified by the Kabat numbering system.
  • an antibody can include six CDRs: three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3).
  • Fc region or “Fc” is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • FR Framework (FR) refers to variable domain residues other than hypervariable region (CDR) residues.
  • CDR hypervariable region
  • the FR of a variable domain is usually composed of four FR domains: FR1, FR2, FR3 and FR4. Therefore, in VH (or VL), CDR and FR sequences usually appear in the following order:
  • CDR residues and other residues in the variable domain are numbered herein according to Kabat et al., supra.
  • natural antibody refers to naturally occurring immunoglobulin molecules with a variety of structures.
  • a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From N-terminus to C-terminus, each heavy chain has a variable region (VH), which is also called a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), which is also called a variable light chain domain or a light chain variable domain, followed by a light chain constant (CL) structure.
  • VH variable heavy chain domain
  • VL variable region
  • the light chains of antibodies can be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of their constant domains.
  • whole antibody full length antibody
  • intact antibody refers to a complete full-length antibody having a structure substantially similar to a native antibody structure or having a heavy chain containing an Fc region as defined herein or including an antigen-binding region.
  • single domain antibody refers to a type of antibody that lacks the antibody light chain and only has the heavy chain variable region. Because of its small molecular weight, it is also called nanobody.
  • single domain antibody refers to an antibody comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain.
  • the single domain antibody is a human single domain antibody.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homologous antibodies, that is, the individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or generated during the preparation of the monoclonal antibody preparation, which variants are usually present in small amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • the term "monoclonal” indicates that the nature of the antibody is obtained from a substantially homologous antibody population, and is not considered to require that the antibody be prepared by any particular method.
  • it can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci.
  • chimeric antibody refers to a portion of an antibody heavy chain and/or light chain that is derived from a specific source or species, while the remainder of the heavy chain and/or light chain is derived from antibodies of different sources or species.
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region.
  • a chimeric antibody is a "type switching" antibody, in which the type or subclass has been changed by the type or subclass of the parent antibody.
  • a chimeric antibody includes an antigen binding fragment thereof.
  • a chimeric antibody is a "humanized antibody”.
  • humanization is used for non-human antibodies, such as rodents or primates, etc., and is a hybrid immunoglobulin containing a minimal sequence derived from a non-human immunoglobulin, an immunoglobulin chain or a fragment thereof.
  • Humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one (generally two) variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies.
  • Humanized antibodies may optionally comprise at least a portion of an antibody constant region derived from a human antibody.
  • a variety of techniques for humanizing antibodies or antibody fragments well known in the art are basically to replace corresponding human antibody sequences with rodent CDRs or CDR sequences, i.e., CDR grafting.
  • Humanized antibodies are often human antibodies, in which some CDR residues and possibly some FR residues are replaced by residues from similar sites in rodent antibodies.
  • humanized antibodies may include mutations, such as mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo.
  • the human domains to be used to prepare the humanized antibodies may be selected to reduce antigenicity.
  • the variable domain sequences of rodent antibodies are screened against the complete library of known human variable domain sequences. The human sequences resolved for rodents can then be accepted as human.
  • FR is used for humanized antibodies.
  • a specific FR derived from a consensus sequence is used, wherein the consensus sequence is a consensus sequence of all human antibodies with a light chain or heavy chain of a specific subgroup.
  • FR can be used for several different humanized antibodies.
  • some FR residues of a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., an antibody from which the CDR residues are derived
  • FR The region outside the CDR of VH and VL is called the framework region (hereinafter referred to as FR); in one example, a cDNA encoding the amino acid sequence of VH composed of the amino acid sequence of the CDR of VH of a non-human animal antibody and the amino acid sequence of the FR of VH of an arbitrary human antibody, and a cDNA encoding the amino acid sequence of VL composed of the amino acid sequence of the CDR of VL of a non-human animal antibody and the amino acid sequence of the FR of VL of an arbitrary human antibody are constructed, and a humanized antibody expression vector is constructed and introduced into animal or fungal or bacterial cells to express it.
  • Mouse-derived hybridoma antibodies produce immune side effects because the constant region can be recognized by the human immune system, inducing human anti-mouse antibody response (HAMA).
  • HAMA human anti-mouse antibody response
  • Humanized antibodies have lower immunogenicity, are closer to natural antibodies in the human body, and can improve drug efficacy and safety.
  • the humanized antibodies disclosed in the present invention have reduced immunogenicity in human subjects.
  • the present invention humanizes hybridoma antibody SC27.105 (from WO2016073649A1).
  • the antibodies H1, P1, P2, P3, P4, M1, and M2 of the present application have low immunogenicity in human subjects.
  • the antibodies H1, P1, P2, P3, P4, M1, and M2 of the present application are lower than the hybridoma antibody SC27.105.
  • Low or reduced immunogenicity is characterized by the ability to treat patients long-term with measurable symptom relief and low and/or acceptable toxicity.
  • Low or acceptable immunogenicity and/or high affinity, as well as other suitable characteristics, can help achieve therapeutic outcomes.
  • “Low or reduced immunogenicity” is defined herein as patients with a proportion of HAHA, HACA or HAMA responses below 90%, such as below 80%, below 70%, below 60%, below 50%, below 40%, below 30%, below 20%, below 10%.
  • recombinant antibody refers to an antibody produced using recombinant DNA technology, for example, an antibody or antibody fragment expressed by a phage or yeast expression system or a mammalian cell expression system.
  • the term should also be understood to refer to an antibody produced by: synthesizing a DNA molecule encoding the antibody and expressing the antibody protein from the DNA molecule; or synthesizing the amino acid sequence of the antibody, wherein the DNA or amino acid sequence is obtained by using recombinant DNA or amino acid sequence technology available and well known in the art.
  • the antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having the desired one or more activities. For example, a variety of methods are known in the art for generating phage display libraries and screening the libraries for antibodies having the desired binding properties. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., Human Press, Totowa, NJ, 2001) and further described in, for example, McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks, Meth. Mol.
  • VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which is then screened for phage binding to the antigen, as described in Winter et al., Ann. Rev. Immunol. 12:433-455 (1994).
  • Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas.
  • natural The library (e.g., by humans) thus provides a single source of antibodies to a variety of non-self antigens and self antigens without any immunization, as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
  • natural libraries can also be prepared synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequences to encode hypervariable CDR3 regions and achieve rearrangement in vitro, as described by Hoogenboom, J. Mol. Biol. 227: 381-388 (1992).
  • amino acid sequence variants of antibodies are provided herein.
  • parent antibody refers to an antibody provided herein or an antibody obtained after mutation, or affinity maturation, etc., according to the antibody provided herein.
  • the parent antibody may be a naturally occurring antibody, or a variant or modified version of a naturally occurring antibody.
  • the parent antibody may refer to the antibody itself, a composition comprising the parent antibody, or its encoded amino acid sequence.
  • affinity matured antibody refers to an antibody with one or more alterations in one or more hypervariable regions (CDRs) compared to a parent antibody, which alterations result in an improvement in the affinity of the antibody for antigen.
  • variant refers to a polypeptide having substantially the same amino acid sequence as the sequence of the antibody provided herein or one or more activities encoded by substantially the same nucleotide sequence.
  • the variant has the same or similar activity as the antibody provided in the Examples of the present application.
  • the variant can be a variant antibody or antibody variant based on the amino acid sequence of the antibody provided herein.
  • variant antibody or “antibody variant” includes an antibody sequence that is different from the parent antibody sequence due to at least one amino acid modification compared to the parent.
  • the variant antibody sequence herein preferably has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with the parent antibody sequence.
  • Antibody variants may refer to the antibody itself or to a composition comprising the antibody variant.
  • the amino acid sequence variants of an antibody may be prepared by introducing suitable modifications to the nucleotide sequence encoding the antibody or by peptide synthesis.
  • amino acid modification includes amino acid substitutions, additions and/or deletions
  • amino acid substitutions means replacing an amino acid at a specific position in a parent polypeptide sequence with another amino acid
  • amino acid insertions means adding an amino acid at a specific position in a parent polypeptide sequence
  • amino acid deletions means removing an amino acid at a specific position in a parent polypeptide sequence. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, eg, antigen binding.
  • modification refers to a change in the state or structure of a protein or polypeptide of the present invention.
  • the modification may be chemical, structural or functional.
  • conservative modification or “conservative sequence modification” means an amino acid modification that does not significantly affect or change the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions of an amino acid residue by another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, as shown in Table 1.
  • amino acid residues in the CDR region or in the framework region of an antibody of the present invention can be replaced with other amino acid residues of the same side chain family, and the altered antibody (variant antibody) can be tested for retained function.
  • Non-conservative substitutions entail exchanging a member of one of these groups for a member of another group.
  • a substitution variant includes one or more hypervariable regions or FR residues that replace a parent antibody (e.g., humanized or human antibody).
  • the variant selected for further study will have changes (e.g., improve) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will substantially maintain certain biological properties of the parent antibody.
  • Exemplary substitution variants are affinity matured antibodies, which can, for example, be conventionally prepared using affinity maturation techniques (such as those described herein) based on phage display. In short, one or more CDR or FR residues are mutated and the variant antibody is displayed on a phage and a specific biological activity (e.g., binding affinity) is screened.
  • Changes can be made in the CDR regions, for example, to improve antibody affinity.
  • Such changes can be made in CDR "hot spots", i.e., codon-encoded residues that are mutated at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008), and/or residues that are antigen-depleted, and the resulting variant VH or VL is tested for binding affinity.
  • Affinity maturation by constructing secondary libraries and reselecting therefrom has been described, e.g., in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human press, Totowa, NJ, (2001)).
  • affinity maturation diversity is introduced into the variable gene species selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then generated.
  • the library is then screened to identify any antibody variants with the desired affinity.
  • Another method of introducing diversity includes a CDR-directed approach in which several CDR residues (e.g., 4-6 residues at a time) are randomized.
  • substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not significantly reduce the ability of the antibody to bind to antigen.
  • conservative changes e.g., conservative modifications described herein
  • Such changes may, for example, be outside of the residues that contact the antigen in the CDRs.
  • each CDR is unchanged, or contains no more than one, two or three amino acid substitutions.
  • amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with an N-terminal methionyl residue.
  • Other insertion variants of the antibody molecule include fusing the N-terminus or C-terminus of the antibody to an enzyme or polypeptide, which increases the serum half-life of the antibody.
  • anti-CLDN6 antibody refers to an antibody that can bind to CLDN6 with sufficient affinity, which can be used as a diagnostic agent and/or therapeutic agent targeting CLDN6.
  • the degree of binding of the anti-CLDN6 antibody to an unrelated, non-CLDN6 protein is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10% of the antibody to CLDN6, as determined by an enzyme-linked immunosorbent assay (ELISA).
  • the degree of binding of the anti-CLDN6 antibody to CLDN4, or to CLDN9 is comparable to the binding level detected in a negative control group.
  • the anti-CLDN6 antibody binds to an epitope of CLDN6 that is conserved between CLDN6 derived from different species.
  • chimeric T cell receptor includes recombinant polypeptides derived from various polypeptides constituting TCR, which are capable of binding to surface antigens on target cells and interacting with other polypeptides of the complete TCR complex, usually co-localized on the surface of T cells.
  • the chimeric T cell receptor is composed of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least a portion of the TCR extracellular domain, a transmembrane domain, and a stimulatory domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit and the antibody domain are operatively connected, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3 ⁇ or CD3 ⁇ , and the chimeric T cell receptor is integrated into the TCR expressed on the T cell.
  • T cell antigen coupler includes three functional domains: (1) an antigen-binding domain, which can be a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into close proximity with the TCR receptor; and (3) a transmembrane domain and an intracellular domain of the CD4 co-receptor, which is linked to the protein kinase LCK and catalyzes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as the initial step in T cell activation.
  • an antigen-binding domain which can be a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties
  • DARPin an extracellular domain
  • TAC receptor binds to CD3
  • TAC receptor a transmembrane domain and an intracellular domain of
  • chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • transmembrane domain is connected to the extracellular antigen binding domain.
  • the transmembrane domain can be derived from natural or synthetic. When the transmembrane domain is of natural origin, it can be derived from any membrane-bound protein or transmembrane protein. On the one hand, whenever CAR binds to the target, the transmembrane domain can conduct the signal to the intracellular domain.
  • the transmembrane domain includes: ⁇ , ⁇ , ⁇ , ⁇ or ⁇ chains of T cell receptors; CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; IL2 receptor p55 ( ⁇ chain), p75 ( ⁇ chain) or ⁇ chain; Fc ⁇ RIII.
  • hydrophobic residues such as leucine and valine may be included.
  • phenylalanine, tryptophan and valine triplets may be present at both ends of the synthetic transmembrane domain.
  • the transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane domain is derived, and/or one or more amino acids associated with the intracellular region of the protein from which the transmembrane domain is derived.
  • the transmembrane domain may be connected to the extracellular region of the CAR via a "hinge region". The use of a "hinge region" provides more flexibility and accessibility to the extracellular antigen binding domain.
  • the hinge region may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25 to 50 amino acids.
  • the hinge region can be derived from all or part of the extracellular region of naturally occurring molecules such as CD8, CD4, CD28, Fc ⁇ RIII or IgG1, IgG4, or from all or part of the antibody constant region.
  • the hinge region can also be a synthetic sequence.
  • intracellular signaling region or “intracellular signaling domain” refers to the intracellular portion of the CAR molecule, which is responsible for activating at least one normal effector function of the immune cell into which the CAR has been introduced.
  • the intracellular signaling domain produces cell lytic activity and auxiliary activity that can promote CAR-containing cells (eg, CAR-T cells), including secreted cytokines.
  • Signaling domain refers to a protein portion that transduces effector signaling function signals and guides cells to perform specific functions.
  • the signaling domain for CAR can be a cytoplasmic sequence of a T cell receptor, and a cytoplasmic sequence of a co-receptor that synergistically triggers signal transduction after antigen receptor engagement, and any derivatives or variants of these sequences, as well as any synthetic sequence with the same function.
  • the intracellular signaling domain may include a stimulatory molecule (also referred to as a stimulatory molecule, a primary signal molecule) and/or a functional signaling domain of a co-stimulatory molecule.
  • the intracellular signaling domain may include a primary signal molecule, exemplarily, the primary signal molecule is derived from those domains of a molecule responsible for the first stimulation or antigen-dependent stimulation.
  • the signal generated by TCR alone is not sufficient to fully activate T cells, and a second and/or co-stimulatory signal is also required.
  • the intracellular signaling domain may include a co-stimulatory intracellular signaling domain, exemplarily, the co-stimulatory intracellular signaling domain includes those domains of a molecule responsible for co-stimulatory signals or antigen-independent stimulation.
  • the intracellular signaling domain may include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain of the molecule, or a functional fragment thereof.
  • a stimulatory molecule may be a ⁇ chain bound to a T cell receptor complex; for example, a cytoplasmic signaling domain further includes a functional signaling domain of one or more co-stimulatory molecules, such as 4-1BB (i.e., CD137), intracellular sequences of CD27 and/or CD28.
  • 4-1BB i.e., CD137
  • CAR comprises a chimeric fusion protein
  • the protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain
  • the intracellular signaling domain contains a functional signaling domain derived from a stimulatory molecule.
  • CAR comprises a chimeric fusion protein
  • the protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain
  • the intracellular signaling domain contains a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule.
  • CAR comprises a chimeric fusion protein
  • the protein comprises an extracellular antigen recognition domain, a transmembrane domain, and an intracellular transduction domain
  • the intracellular signaling domain comprises at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule.
  • CAR comprises an optional "leader sequence" at the amino acid (ND end) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.
  • the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.
  • leader sequence (also called a signal peptide) is operably linked to the CAR nucleic acid sequence and positioned so that the newly synthesized polypeptide is directed into the secretory pathway of the cell.
  • the leader sequence is usually positioned at the 5' end of the nucleic acid sequence encoding the polypeptide.
  • the signal peptide includes the signal sequence of a naturally occurring protein of the CAR or a synthetic non-naturally occurring signal sequence.
  • the signal peptide is selected from CD8 ⁇ , GM-CSF receptor ⁇ and IgG1 heavy chain.
  • the term "primary signal molecule” or “stimulatory molecule” regulates the initial activation of the TCR complex in a stimulatory manner.
  • the primary signal is triggered by the binding of, for example, a TCR/CD3 complex to an MHC molecule loaded with a peptide, thereby mediating a T cell response (including but not limited to, proliferation, activation, differentiation, etc.).
  • the primary signal molecule that acts in a stimulatory manner may include a signal transduction motif of an immunoreceptor tyrosine activation motif or an ITAM.
  • Examples of functional signal transduction domains (primary signal domains) of primary signal molecules containing ITAMs that are particularly useful in the present invention include but are not limited to, derived from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also referred to as "ICOS"), CD66d, DAP10 and DAP12 sequences, in the CAR of the present invention, in any one or more of the CARs of the present invention, the intracellular signal transduction domain includes an intracellular signal transduction sequence, such as the primary signal domain of CD3 ⁇ .
  • costimulatory signal domain or "costimulatory molecule” generally refers to an intracellular domain of a costimulatory molecule that can bind to a cell stimulatory signal molecule, such as TCR/CD3, and the combination leads to T cell proliferation and/or upregulation or downregulation of key molecules.
  • Costimulatory molecules are generally associated binding partners on T cells, which specifically bind to costimulatory ligands, thereby mediating the co-stimulatory response of T cells, for example, including but not limited to proliferation.
  • Costimulatory molecules are cell surface molecules or their ligands that are non-antigen receptors required for an effective immune response.
  • the costimulatory intracellular signaling domain can be derived from the intracellular portion of a costimulatory molecule.
  • Costimulatory molecules can appear in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins) and activating NK cell receptors.
  • Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), GITR, CD30, CD40, ICOS, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83, etc.
  • CD3 ⁇ also known as CD3Zeta
  • TCR ⁇ or " ⁇ chain”
  • CD3 ⁇ domain is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to 164 of GenBan Accession No. BAG36664.1, and its functional orthologs - equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • the amino acid sequence of CD3 ⁇ is shown in SEQ ID NO: 24.
  • 4-1BB also known as CD137
  • CD137 refers to a member of the TNFR superfamily having the amino acid sequence of GenBank Acc.No.AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • the "4-1BB co-stimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc.No.AAA62478.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • the amino acid sequence of the 4-1BB co-stimulatory domain is shown in SEQ ID NO: 26.
  • chemokine is a polypeptide with a molecular weight of 8 to 10 kDa. It is the largest cytokine family. Its main function is to recruit monocytes, neutrophils, lymphocytes, etc. in the blood into specific lymphoid organs and tissues as well as the site of infection.
  • chemokine receptor refers to a class of seven-transmembrane G protein-coupled receptors that mediate the function of chemokines. (GPCR), usually expressed on the cell membranes of immune cells, neutrophils, endothelial cells, etc. Chemokines and chemokine receptors play an important role in mediating cell migration, proliferation and resisting pathogen invasion, and are closely related to the occurrence and development of inflammation and cancer in the immune environment.
  • safety switch is intended to improve the safety of CAR-T therapy by designing a rapid and reversible "off” or “on” safety switch for CAR-T cells to minimize treatment-related toxicity.
  • CAR-T cell therapy has excellent clinical characteristics, when tumor burden is unpredictable and the activity of T cells is uncontrolled, it can trigger potentially fatal side effects such as severe CRS.
  • severe CRS requires appropriate monitoring using the CRS grading system as a guide and precise regulation using small molecule-based safety switches.
  • a humanized anti-CLDN6 specific antibody is obtained by using conventional humanized antibody preparation technology in the art, and a mutant of a humanized anti-CLDN6 specific antibody is obtained by CDR region randomization mutation technology and phage screening technology. These molecules show fine specificity.
  • the antibody only recognizes CLDN6, and 293T cells, OVCAR3 cells and OV90 cells expressing CLDN6, and does not recognize cells expressing CLDN4, CLDN9 or a combination thereof. If not otherwise specified in the present invention, CLDN6 herein refers to human CLDN6.
  • the present invention includes antibodies having scFv sequences fused to one or more heavy chain constant regions to form antibodies having human immunoglobulin Fc regions to produce bivalent proteins, thereby increasing the overall affinity and stability of the antibody.
  • the Fc portion allows other molecules (including but not limited to fluorescent dyes, cytotoxins, radioisotopes, etc.) to be directly conjugated to antibodies, such as for use in antigen quantification studies, so as to immobilize antibodies for affinity measurements, for directed delivery of therapeutics, for testing Fc-mediated cytotoxicity using immune effector cells, and many other applications.
  • the antibodies or antibody fragments of the present invention are based on the use of humanized antibody preparation technology and phage screening technology to obtain single-chain antibody fragments (scFv), the amino acid sequence of which confers specificity to the antibody or antibody fragment against CLDN6 and forms the basis of all antibodies disclosed herein. Therefore, the scFv can be used to design a series of different "antibodies or antibody fragments", including, for example, full-length antibodies, fragments thereof such as F(ab')2, fusion proteins, multivalent antibodies, i.e., antibodies with more than one specificity for the same antigen or different antigens, for example, bispecific T cell binding antibodies (BiTEs), tribodies, etc. (see Cuesta et al., Multivalent antibodies: when design surpasses evolution, Trends in Biotechnology 28: 355-362, 2010).
  • the present invention provides full-length antibodies, whose heavy and light chains can be full-length (e.g., the antibody can include at least one, preferably two, complete heavy chains, and at least one, preferably two, complete light chains) or can include antigen binding portions (Fab, F(ab')2, Fv or scFv).
  • the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE.
  • the choice of antibody type will depend on the immune effector function that the designed antibody is intended to elicit.
  • suitable amino acid sequences of the constant regions of various immunoglobulin isotypes and methods for producing a wide range of antibodies are known to those skilled in the art.
  • the present invention provides an antibody or antigen-binding fragment that recognizes CLDN6, comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1 shown in GYYMN (SEQ ID NO: 35); and/or EINPATGSTTYNQKFKA (SEQ ID NO: NO: 36 ); and/or HCDR2 shown in RDYYX1GSX2X3YAX4DY (SEQ ID NO: 52 ) , wherein X1 is Y or L, X2 is G or N, X3 is F or S, and X4 is M or L; and/or the antibody comprises a light chain variable region, wherein the light chain variable region comprises LCDR1 shown in QASQSVSNNLN (SEQ ID NO: 38); and/or LCDR2 shown in GASKLED (SEQ ID NO: 39 ); and/or LCDR3 shown in X5QHRX6X7WT (SEQ ID NO: 53), wherein X5 is L or Q, X6 is Y
  • the antibody or antigen binding fragment may include at least one CDR in the heavy chain variable region; and/or the antibody or antigen binding fragment may include at least one CDR in the light chain variable region. In certain embodiments, the antibody or antigen binding fragment may include one CDR, two CDRs, or three CDRs in the heavy chain variable region. In certain embodiments, the antibody or antigen binding fragment may include one CDR, two CDRs, or three CDRs in the light chain variable region.
  • the antibody may comprise one CDR, two CDRs or three CDRs in the heavy chain variable region, and one CDR, two CDRs or three CDRs in the light chain variable region.
  • one CDR in the heavy chain variable region may be included, and one CDR, two CDRs or three CDRs in the light chain variable region may be included; for example, two CDRs in the heavy chain variable region may be included, and one CDR, two CDRs or three CDRs in the light chain variable region may be included; for example, three CDRs in the heavy chain variable region may be included, and one CDR, two CDRs or three CDRs in the light chain variable region may be included; for example, three CDRs in the heavy chain variable region may be included, and three CDRs in the light chain variable region may be included.
  • the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region, wherein the HCDR3 of the heavy chain variable region has at least 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the HCDR3 shown in SEQ ID NO: 41; and/or
  • the antibodies or antigen-binding fragments of the present invention comprise a light chain variable region, wherein the LCDR3 of the light chain variable region has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the LCDR3 shown in SEQ ID NO: 42, 43 or 44.
  • the antibody may comprise the heavy chain variable region and/or the light chain variable region.
  • the heavy chain variable region may comprise a heavy chain CDR1 (HCDR1), and the HCDR1 may comprise an amino acid sequence as shown in SEQ ID NO:35.
  • the heavy chain variable region may comprise a heavy chain CDR2 (HCDR2), and the HCDR2 may comprise an amino acid sequence as shown in SEQ ID NO:36.
  • the heavy chain variable region may comprise a heavy chain CDR3 (HCDR3), and the HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NO:37 or 41.
  • the heavy chain variable region may comprise HCDR1 and HCDR3, and the HCDR1 may comprise an amino acid sequence as shown in SEQ ID NO:35, and the HCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NO:37 or 41.
  • the heavy chain variable region may comprise HCDR1 and HCDR2, and the HCDR1 may comprise an amino acid sequence as shown in SEQ ID NO:35, and the HCDR2 may comprise an amino acid sequence as shown in SEQ ID NO:36.
  • the heavy chain variable region may comprise HCDR2 and HCDR3, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 37 or 41.
  • the heavy chain variable region may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 35, the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 36, and the HCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 37 or 41.
  • the light chain variable region may comprise
  • the light chain variable region may comprise a light chain CDR1 (LCDR1), wherein the LCDR1 may comprise an amino acid sequence as shown in SEQ ID NO: 38.
  • the light chain variable region may comprise a light chain CDR2 (LCDR2), wherein the LCDR2 may comprise an amino acid sequence as shown in SEQ ID NO: 39.
  • the light chain variable region may comprise a light chain CDR3 (LCDR3), wherein the LCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 40, 42, 43 or 44.
  • the light chain variable region may comprise LCDR1 and LCDR3, wherein the LCDR1 may comprise an amino acid sequence as shown in SEQ ID NO: 38, wherein the LCDR3 may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 40, 42, 43 or 44.
  • the light chain variable region may comprise LCDR1 and LCDR2, wherein the LCDR1 may comprise an amino acid sequence as shown in SEQ ID NO: 38, wherein the LCDR2 may comprise an amino acid sequence as shown in SEQ ID NO: 39.
  • the light chain variable region may comprise LCDR2 and LCDR3, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 39, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 40, 42, 43 or 44.
  • the light chain variable region may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 38, the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 39, and the LCDR3 may comprise the amino acid sequence shown in any one of SEQ ID NO: 40, 42, 43 or 44.
  • the antibody may comprise the heavy chain variable region and the light chain variable region.
  • the antibody comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 37, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40; or the antibody comprises HCDR1 shown in SEQ ID NO: 35, HCDR2 shown in SEQ ID NO: 36, HCDR3 shown in SEQ ID NO: 41, LCDR1 shown in SEQ ID NO: 38, LCDR2 shown in SEQ ID NO: 39, and LCDR3 shown in SEQ ID NO: 40.
  • the CDR can be determined by a numbering system selected from the following: Kabat, Chothia, IMGT, Gelfand, Aho and AbM.
  • the antibody recognizing CLDN6 provided by the present invention may comprise a CDR sequence or a combination thereof determined by any of the above numbering systems, and the CDRs in the heavy chain and/or light chain constituting the antibody do not have to be determined by the same numbering system.
  • one or some CDRs in an antibody may be determined by the Kabat system, and other CDRs may be determined by any one or a combination of the above numbering systems.
  • the CDRs of the antibody may be determined by a numbering system, for example, by the Kabat numbering system; for example, by the Chothia numbering system; for example, by the IMGT numbering system; for example, by the Gelfand numbering system; for example, by the Aho numbering system; for example, by the AbM numbering system.
  • a numbering system for example, by the Kabat numbering system; for example, by the Chothia numbering system; for example, by the IMGT numbering system; for example, by the Gelfand numbering system; for example, by the Aho numbering system; for example, by the AbM numbering system.
  • the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO:1 or 5 or a variant thereof.
  • the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 3, 7, 9 or 11 or a variant thereof.
  • the heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 1 or 5, or a variant thereof, and the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 3, 7, 9 or 11, or a variant thereof.
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 1, or a variant thereof, and the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 3, 7, 9 or 11, or a variant thereof;
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 5, or a variant thereof, and the light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NO: 3, 7, 9 or 11, or a variant thereof.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:3.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:1
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:3.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:5
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:9.
  • the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 1
  • the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the present invention provides an antibody that recognizes CLDN6, comprising a heavy chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NO: 1 or 5, or a variant of the above sequence.
  • the present invention provides an antibody that recognizes CLDN6, comprising a light chain variable region, which light chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO: 3, 7, 9 or 11, or a variant of the above sequence.
  • the present invention provides an antibody that recognizes CLDN6, comprising the above-mentioned heavy chain variable region and light chain variable region.
  • the heavy chain and light chain variable region sequences can bind to CLDN6, the heavy chain and light chain variable region sequences can be “mixed and matched” to generate the anti-CLDN6 binding molecules of the present invention.
  • the present invention provides variants of antibodies that bind to CLDN6 or variants of fragments thereof.
  • Such variants comprise a heavy chain variable region, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%,
  • the variant of the antibody of the present invention or the variant of its fragment comprises a heavy chain and/or light chain variable region having at least 80% the same sequence as the heavy chain or light chain variable region of the antibody of the present invention.
  • the amino acid sequence identity of the heavy chain and/or light chain variable region is at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%, particularly 96%, more particularly 97%, even more particularly 98%, most particularly 99%, including, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.
  • the variant can be obtained by methods such as yeast library screening, phage library screening, and point mutation with the antibody described in the present application as the parent antibody.
  • the present invention provides an antibody that specifically binds to CLDN6, wherein the antibody is a whole antibody, scFv, single domain antibody, Fab fragment, Fab' fragment, Fv fragment, F(ab') 2 fragment, Fd fragment, dAb fragment or multifunctional antibody.
  • the antibody is a hybridoma antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the antibody is a monoclonal antibody.
  • anti-CLDN6 antibodies provided herein can be identified, screened, or characterized for physical/chemical properties and/or biological activity by a variety of assays known in the art, including, for example, ELISA, biacore, Western blot, and flow cytometric analysis. Suitable assays are described in detail in the Examples.
  • affinity refers to the sum of the forces of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including the use of Biacore to determine the affinity of an antibody.
  • KD dissociation constant
  • the KD of an antibody refers to the equilibrium dissociation constant of an antibody-antigen interaction.
  • affinity for a CLDN antigen herein is expressed as the EC50 of the antibody.
  • EC50 concentration for 50% of maximal effect, refers to the concentration that can cause 50% of the maximal effect.
  • the method for determining binding affinity uses surface plasmon resonance.
  • Surface plasmon resonance is an optical phenomenon that can be used to analyze real-time biospecific interactions by, for example, detecting changes in protein concentrations in a biochip matrix using a Biacore system.
  • no significant binding is characterized by reduced affinity of the antibody to the antigen, a faster dissociation rate and/or a lower binding signal, particularly a faster dissociation rate and/or a lower binding signal.
  • no significant binding refers to the level of binding of the antibody to the antigen protein or polypeptide that is not statistically significantly above the background; the so-called background is the binding level detected in the absence of the antibody, or the level of binding detected in the presence of a negative control protein (e.g., an isotype control antibody).
  • a biosensor analysis e.g., Biacore
  • Biacore a biosensor analysis
  • PBS is used instead of the antibody to detect the antigen binding level.
  • the antibody of the present invention binds to CLDN6. In one example, the antibody of the present invention does not significantly bind to CLDN4 or CLDN9.
  • antigen refers to a substance that is recognized and specifically bound by an antigen binding unit.
  • Antigens may include peptides, proteins, glycoproteins, polysaccharides and lipids, parts thereof and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen may also refer to a molecule that triggers an immune response. Such an immune response may involve antibody production or activation of specific immunologically-competent cells, or both. It will be understood by those skilled in the art that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.
  • antigens may be derived from recombinant DNA or genomic DNA.
  • any DNA containing a nucleotide sequence or a partial nucleotide sequence encoding a protein that can cause immune translation can encode an antigen. It is clear to those skilled in the art that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. Including but not limited to, using partial nucleotide sequences of more than one gene, these nucleotide sequences can be arranged in various combinations to encode polypeptides that can cause a desired immune response. In addition, antigens may not need to be encoded by genes. Antigens may be produced synthetically, or may be derived from biological samples, or may be macromolecules other than polypeptides. The biological sample may include but is not limited to tissue samples, tumor samples, cells or fluids, and other biological components.
  • epitope refers to an antigen or part of an antigen that can be recognized by an antibody, B cell, T cell, or engineered cell.
  • an epitope can be a tumor epitope or a pathogen epitope recognized by an antibody; an antibody recognizes multiple epitopes within an antigen. Epitopes can also mutate.
  • antigenic determinant site is also called “antigenic epitope” or “epitope” or “antigenic determinant”, and includes any determinant or region that can be bound by an antibody.
  • An antigenic epitope is a region of an antigen that is bound by an antibody targeting the antigen, including specific amino acids that are in direct contact with the antibody.
  • an antigenic epitope may consist of a continuous sequence of a CLDN6 protein sequence, or may consist of a discontinuous three-dimensional structure of a CLDN6 protein sequence.
  • the antigen used herein is human CLDN6.
  • the present invention also provides an immunoconjugate, which includes the antibody described herein and one or more functional molecules connected thereto.
  • the antibody provided by the present invention has been described above, and the conjugate provided by the present invention includes all of its technical solutions.
  • the antibody and the functional molecule can form an immunoconjugate by covalent bonding, coupling, attachment, cross-linking, etc.
  • Connection or “fusion” are used interchangeably herein. It generally refers to connecting two or more chemical elements or components together by any means including chemical conjugation or recombinant methods.
  • “In-frame fusion” refers to connecting two or more ORFs in a manner that maintains the correct reading frame of the original open reading frame (ORF) to form a longer ORF that is continuous. Therefore, the resulting recombinant fusion protein is a single protein containing two or more fragments, which correspond to polypeptides encoded by the original ORF (these fragments are usually not so connected in the natural state). Although the reading frame is therefore continuous throughout the fusion fragment, these fragments can be physically or spatially separated by, for example, in-frame connecting sequences (e.g., "flexons").
  • the functional molecules are selected from: molecules targeting tumor surface markers, molecules that inhibit tumors, molecules that target immune
  • the molecule targeting the tumor surface marker can be an antibody or ligand that binds to the tumor surface marker, which can synergize with the antibody of the present invention to more accurately target tumor cells.
  • the tumor-suppressing molecule comprises a cytotoxic agent, including but not limited to: radioisotopes, chemotherapeutic agents, growth inhibitors, enzymes and fragments thereof, antibiotics, toxins.
  • the tumor-suppressing molecule is an anti-tumor cytokine, including but not limited to: IL-2, IL-7, IL-12, IL-15, type I IFN, TNF- ⁇ .
  • the molecule of the surface marker of the targeted immune cell is an antibody or ligand that binds to the surface marker of the immune cell, which can recognize the immune cell, and it carries the antibody of the present invention to the immune cell, and the antibody of the present invention can target the immune cell to the tumor cell, thereby inducing the immune cell to specifically kill the tumor cell.
  • the surface marker of the immune cell is selected from CD3, CD6, CD28, NKG2A, NKG2C, NKG2D, CD94, CD159a, CD159c, CD158, CD56, LIR/ILT2, CD244, CD226, CD2, CD16, CD161, and the immune cell is selected from T cells, NK cells, and NKT cells.
  • the molecule of the surface marker of the targeted immune cell is an antibody that binds to the surface marker of the T cell, which forms a bifunctional antibody involving T cells with the antibody of the present invention.
  • the detectable marker includes, but is not limited to, fluorescent markers, chromogenic markers; for example, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and non-radioactive paramagnetic metal ions.
  • the marker used to label the antibody for detection and/or analysis and/or diagnostic purposes depends on the specific detection/analysis/diagnosis technology and/or method used, such as immunohistochemical staining (tissue) samples, flow cytometry, etc.
  • the immunoconjugates include antibody-drug conjugates (ADCs) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids, auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF), dolastatin, calicheamicin or derivatives thereof, anthracyclines (e.g., daunomycin or doxorubicin), methotrexate, vindesine, taxanes (e.g., docetaxel, paclitaxel, leronate, tesetaxel and oxtataxel), trichothecenes, CC1065.
  • ADCs antibody-drug conjugates
  • drugs including but not limited to maytansinoids, auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF), dolastatin, calicheamicin or derivatives thereof, anthracyclines (e.g.
  • Another aspect of the present invention provides nucleic acid molecules encoding the immunoconjugates of the present invention.
  • the relevant sequence can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • the present invention also provides a chimeric receptor, which generally refers to an expression product of a fusion molecule formed by connecting DNA fragments or cDNAs corresponding to proteins from different sources using genetic recombination technology, and may include an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes an antigen binding domain.
  • the extracellular domain includes an antibody provided by the present invention, which has been described in detail above, and the chimeric receptor provided by the present invention includes all of its technical solutions.
  • the chimeric receptor includes Including but not limited to: chimeric antigen receptor (CAR), chimeric T cell receptor, T cell antigen coupler (TAC), synthetic polypeptide receptor (synNotch).
  • the cells expressing CAR can be targeted to bind to the antigen of interest by engineering an antigen binding domain that specifically binds to the antigen of interest into the CAR.
  • the chimeric receptor of the present invention is a chimeric antigen receptor (CAR).
  • the chimeric antigen receptor generally comprises an extracellular antigen binding region or an antibody.
  • the extracellular antigen binding region may be completely human.
  • the extracellular antigen binding region may be humanized.
  • the extracellular antigen binding region may be mouse-derived, or the chimera in the extracellular antigen binding region may be composed of amino acid sequences from at least two different animals.
  • the extracellular antigen binding region may be non-human.
  • the chimeric antigen receptor can also be designed to include a variety of antigen binding regions, including single-chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fab) selected from libraries, single domain fragments, or natural ligands that bind to their cognate receptors.
  • scFv single-chain variable fragments
  • Fab fragment antigen binding regions
  • the extracellular antigen binding region may include scFv, Fab, or natural ligands, and any derivatives thereof.
  • the extracellular antigen binding region may refer to a molecule other than a complete antibody, which may include a portion of a complete antibody and may bind to an antigen bound by the complete antibody.
  • antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; bifunctional antibodies, linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed by antibody fragments.
  • the extracellular antigen binding region such as scFv, Fab, or natural ligand, may be part of a CAR that determines antigen specificity.
  • the extracellular antigen binding region may bind to any complementary target.
  • the extracellular antigen binding region may be derived from an antibody of a known variable region sequence.
  • the extracellular antigen binding region may be obtained from an antibody sequence obtained from an available mouse hybridoma. Alternatively, the extracellular antigen binding region can be obtained from whole exosome cleavage sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TILs).
  • TILs tumor infiltrating lymphocytes
  • the binding specificity of the extracellular antigen binding region of CAR can be determined by complementary determining region or CDR, such as light chain CDR and/or heavy chain CDR. In some embodiments, the binding specificity of the extracellular antigen binding region of CAR can be determined by light chain variable region and/or heavy chain variable region.
  • the extracellular region of the CAR includes a hinge or a spacer, and the hinge and the spacer can be used interchangeably.
  • the hinge can be considered as a part of the CAR used to provide flexibility to the extracellular antigen binding region.
  • the hinge can be used to detect CAR on the cell surface of the cell, especially when the antibody detecting the extracellular antigen binding region does not work or is not available.
  • the hinge may not belong to an immunoglobulin, but to another molecule, such as the natural hinge of the CD8 ⁇ molecule.
  • the CD8 ⁇ hinge may contain cysteine and proline residues known to play a role in the interaction between CD8 auxiliary receptors and MHC molecules.
  • the hinge can be adjusted according to the extracellular antigen binding region used.
  • the hinge can be of any length.
  • the hinge can be a natural hinge region of IgG1, IgG4, or a mutated hinge region thereof, or other structural parts outside the hinge region.
  • the hinge may comprise an amino acid sequence as shown in SEQ ID NO: 21, 27, 28, 29 or 30.
  • the transmembrane domain (or structural region) of CAR can anchor CAR to the plasma membrane of the cell.
  • the natural transmembrane portion of CD28 can be used for CAR.
  • the natural transmembrane portion of CD8 ⁇ can also be used in CAR.
  • CD8 can be a protein with at least 85, 90, 95, 96, 97, 98, 99 or 100% identity with NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity.
  • CD8 nucleic acid molecule can be a protein encoding CD8
  • a polynucleotide of a polypeptide in some cases, the transmembrane region can be a natural transmembrane portion of CD28, and “CD28” can refer to a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity with NCBI Reference No.: NP_006130 or a fragment thereof having stimulatory activity.
  • a "CD28 nucleic acid molecule” can be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion can include a CD8 alpha region.
  • the transmembrane domain can include an amino acid sequence as shown in SEQ ID NO: 22 or 25.
  • the (thin) intracellular signaling region of CAR may be responsible for activating at least one of the effector functions of the immune response cells comprising the CAR.
  • CAR can induce the effector function of T cells, for example, the effector function is cytolytic activity or auxiliary activity, including the secretion of cytokines, such as IL-2, TNF- ⁇ , ⁇ -IFN, etc.
  • the term intracellular signaling region refers to a protein portion that transduces effector function signals and guides cells to perform specific functions. Although the entire intracellular signaling region can usually be used, in many cases, it is not necessary to use the entire chain of the signaling domain. In some embodiments, a truncated portion of the intracellular signaling region is used. In some embodiments, the term intracellular signaling region is therefore intended to include any truncated portion of the intracellular signaling region sufficient to transduce effector function signals.
  • Preferred examples of the signaling domain (or structural region) used in CAR may include the cytoplasmic sequence of the T cell receptor (TCR) and a co-receptor that cooperates to initiate signal transduction after target-receptor binding, as well as any derivative or variant sequence thereof and any synthetic sequence of these sequences having the same functionality.
  • TCR T cell receptor
  • CD3 ⁇ domain also known as the T cell receptor CD3 ⁇ chain or CD247.
  • This domain is part of the T cell receptor-CD3 complex and plays an important role in coupling antigen recognition of several intracellular signal transduction pathways with the primary effector activation of T cells.
  • CD3 ⁇ refers primarily to human CD3 ⁇ and its isoforms, as known from Swissprot entry P20963, including proteins with essentially the same sequence.
  • the full T cell receptor CD3 ⁇ chain is not required, and any derivative thereof comprising the signaling domain of the T cell receptor CD3 ⁇ chain is suitable, including any functional equivalent thereof.
  • the signaling domain of the CD3 ⁇ chain may comprise the amino acid sequence shown in SEQ ID NO:24.
  • the intracellular signaling domain (or structural region) of CAR can be selected from any one of the costimulatory domains in Table 2.
  • the domain can be modified so that the identity with the reference domain can be about 50% to about 100%.
  • Any one of the domains in Table 1 can be modified so that the modified form can contain about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or up to about 100% identity.
  • the intracellular signaling region of the CAR may further comprise one or more co-stimulatory domains.
  • the intracellular signaling region may comprise a single co-stimulatory domain, such as a ⁇ chain (first generation CAR) or a co-stimulatory domain thereof with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling region may comprise two co-stimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • the co-stimulatory domain of the CD28 may comprise an amino acid sequence as shown in SEQ ID NO:23.
  • these co-stimulatory domains can produce downstream activation of kinase pathways, thereby supporting gene transcription and functional cellular responses.
  • proximal signaling proteins associated with the CD28 phosphatidylinositol-4,5-bisphosphate 3-kinase
  • 4-1BB/OX40 TNF-receptor-associated factor adaptor protein
  • the signal generated by CAR may be combined with an auxiliary or costimulatory signal.
  • the costimulatory signal domain the chimeric antigen receptor-like complex can be designed to include several possible costimulatory signal domains.
  • the binding of T cell receptors alone is not enough to induce the complete activation of T cells as cytotoxic T cells.
  • Complete productive T cell activation requires a second costimulatory signal. It has been reported that several receptors that provide costimulation for T cell activation include but are not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88 and 4-1BB.
  • the signal transduction pathways used by these costimulatory molecules can all synergize with the main T cell receptor activation signal.
  • the signals provided by these costimulatory signal transduction regions can synergize with the main effector activation signals derived from one or more ITAM motifs (such as CD3zeta signal transduction domains), and the requirements for T cell activation can be completed.
  • the addition of a co-stimulatory domain to the chimeric antigen receptor-like complex can enhance the efficacy and durability of the engineered cells.
  • the T cell signaling domain and the co-stimulatory domain are fused to each other to form a signaling region.
  • the present invention provides cells (e.g., T cells) that are engineered to express CAR, wherein the cells expressing CAR (e.g., CAR-T cells) exhibit anti-tumor properties.
  • CAR e.g., CAR-T cells
  • cells are transduced with CAR, and CAR is expressed on the cell surface.
  • cells e.g., T cells
  • the viral vector is a reverse transcription vector.
  • the viral vector is a lentiviral vector.
  • cells can stably express CAR.
  • cells e.g., T cells
  • the CAR comprises an antigen binding domain that targets CLDN6.
  • the CLDN6 binding portion of the CAR is a scFv, which is a functional antibody fragment that retains comparable binding affinity to the IgG antibody from which it is derived, e.g., it binds to the antigen with comparable efficacy; it thereby provides a biochemical response, e.g., activating an immune response, inhibiting the initiation of signal transduction from its target antigen, inhibiting kinase activity, etc.
  • the anti-CLDN6 antigen binding domain of the CAR comprises a scFv sequence as shown in any of SEQ ID NOs: 13, 14, 15, 16, 17, 18, or 19.
  • the anti-CLDN6 antigen binding domain of CAR is a humanized antibody or a fragment thereof. In some embodiments, the anti-CLDN6 antigen binding domain of CAR is a fully human antibody or a fragment thereof. In some embodiments, the anti-CLDN6 antigen binding domain of CAR is a murine antibody or a fragment thereof.
  • the CAR of the present invention combines the antigen binding domain of a specific antibody with an intracellular signaling molecule.
  • intracellular signaling molecules include, but are not limited to, CD3 ⁇ chain, 4-1BB and CD28 signaling modules and combinations thereof.
  • CLDN6-CAR comprises at least one intracellular signaling domain selected from CD137 (4-1BB) signaling domain, CD28 signaling domain, CD3 ⁇ signaling domain, and any combination thereof.
  • CLDN6-CAR comprises at least one intracellular signaling domain from one or more costimulatory molecules other than CD137 (4-1BB) or CD28.
  • the sequence of CLDN6-CAR includes: an extracellular domain shown in SEQ ID NO: 13, a hinge domain shown in SEQ ID NO: 21, a transmembrane domain shown in SEQ ID NO: 22, a co-stimulatory signal domain shown in SEQ ID NO: 23, and a primary signal domain (H1-28Z) shown in SEQ ID NO: 24; or an extracellular domain shown in SEQ ID NO: 17, a hinge domain shown in SEQ ID NO: 21, a transmembrane domain shown in SEQ ID NO: 22, a co-stimulatory signal domain shown in SEQ ID NO: 23, and a primary signal domain (P4-28Z) shown in SEQ ID NO: 24.
  • amino acid sequence of the chimeric antigen receptor comprises a sequence as shown in any one of SEQ ID NOs: 13, 14, 15, 16, 17, 18 or 19 linked to a sequence as shown in any one of SEQ ID NOs: 45, 46 or 47, respectively.
  • the amino acid sequence of the chimeric antigen receptor comprises: the sequence set forth in SEQ ID NO: 13 linked to the sequence set forth in SEQ ID NO: 45; or the sequence set forth in SEQ ID NO: 13 linked to the sequence set forth in SEQ ID NO: 46; or the sequence set forth in SEQ ID NO: 13 linked to the sequence set forth in SEQ ID NO: 47; or the sequence set forth in SEQ ID NO: 14 linked to the sequence set forth in SEQ ID NO: 45; or the sequence set forth in SEQ ID NO: 14 linked to the sequence set forth in SEQ ID NO: 46; or the sequence set forth in SEQ ID NO: 14 linked to the sequence set forth in SEQ ID NO: 47; or the sequence set forth in SEQ ID NO: 15 linked to the sequence set forth in SEQ ID NO: 45; or the sequence set forth in SEQ ID NO: 15 linked to the sequence set forth in SEQ ID NO: 46; or the sequence set forth in SEQ ID NO: 15 linked to the sequence set forth in SEQ ID NO: 47 or the sequence shown in S
  • transmembrane domain and intracellular domain of the chimeric antigen receptor mentioned above can be replaced by conventional transmembrane domains and intracellular domains selected by those skilled in the art, and all of them fall within the protection scope of the present application.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, oligonucleotides, fragments generated by PCR, fragments generated by any of connection, chain scission, endonuclease action and exonuclease action.
  • Nucleic acid sequences include natural nucleotide sequences, and also implicitly include conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences.
  • Modified nucleotides may have changes in sugar moieties and/or pyrimidine or purine base moieties. For example, sugar modifications include replacement of one or more hydroxyls with halogens, alkyl groups, amines and azido groups, or sugars may be functionalized with ethers or esters.
  • codon refers to the three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by the ribosome into an amino acid residue.
  • codon optimization refers to the frequency of occurrence of synonymous codons (codons encoding the same amino acid) in coding DNA, which has a preference in different species, and the codons that are usually rare in the highly expressed genes of a given species are replaced by the common codons of the highly expressed genes in this species. Codon degeneracy allows multiple nucleotide sequences to encode the same polypeptide. Degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by mixed bases and/or deoxyinosine residues.
  • coding refers to the inherent properties of a specific nucleotide sequence in a polynucleotide such as a gene, DNA or mRNA, i.e., the sequence can serve as a template to synthesize other polymers and macromolecules with a specified nucleotide sequence (e.g., rRNA, tRNA and mRNA) or a specified amino acid sequence in a biological process and the biological properties resulting therefrom. Therefore, when transcription and translation corresponding to the mRNA of a gene in a cell or other biological system cause protein production, the gene, cDNA or RNA encodes the protein.
  • a specified nucleotide sequence e.g., rRNA, tRNA and mRNA
  • Coding strands (its nucleotide sequence is identical to the mRNA sequence, usually provided in a sequence table) and non-coding strands (used as templates for transcription of a gene or cDNA) can both be referred to as coded proteins or other products of the gene or cDNA.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the sequence of proteins along the polypeptide (protein) chain. The order of amino acids in a nucleic acid sequence. Therefore, a nucleic acid sequence encodes an amino acid sequence.
  • a "nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences encoding the same amino acid sequence that are degenerate forms of each other. When used to refer to a nucleotide sequence, the term “sequence” as used herein includes DNA or RNA, and may be single-stranded or double-stranded.
  • target sequence refers to a sequence that is complementary to a guide sequence, and the complementary pairing between the target sequence and the guide sequence promotes the formation of a CRISPR complex.
  • a target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide.
  • the target sequence is located in the nucleus or cytoplasm of a cell.
  • identity refers to the subunit sequence identity between two nucleic acid molecules, or between two polypeptide molecules.
  • a subunit position is occupied by the same monomer subunit in both molecules, such as when two DNA molecules are both occupied by adenosine at one position, then they are homologous or identical at that position.
  • the homology between two sequences is the number of matching or homologous positions divided by the total number of positions in the sequences, and the result is multiplied by 100 to produce a percentage of sequence identity. For example, when 80% of the positions in two sequences (e.g., 8 bp in a nucleic acid molecule 10 bp long) are homologous, then the two sequences have 80% homology.
  • transfection or “transduction” or “transformation” refers to the introduction of exogenous nucleic acid into a host cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran mediated transfection, polybrene mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.
  • Transfected” or “transformed” or “transduced” cells are cells that have been transfected, transformed or transduced with exogenous nucleic acid. The cell includes primary subject cells and their progeny.
  • expression vector refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • the expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system.
  • Expression vectors include all those known in the art, such as plasmids, viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • the plasmid vector may also comprise a selection marker that provides identification and/or screening of cells receiving the vector.
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. Non-plasmid and non-viral compounds that promote nucleic acid transfer into cells may also be included, such as polylysine compounds, liposomes, etc.
  • the vector may be associated or combined with any cell penetration technology (such as sonoporation or electroporation or its derivative technology).
  • the selection of the vector depends mainly on the size of the nucleic acid to be inserted into the vector and the specific host cell to be forwarded by the vector.
  • each vector contains different components.
  • Vector components generally include but are not limited to: origin of replication, selectable marker gene, promoter, ribosome binding site (RBS), signal sequence, heterologous nucleic acid insert and transcription termination sequence.
  • Physical methods for introducing vectors into cells include calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc.
  • Chemical methods for introducing vectors into cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Retrovirus refers to the genus of the Retroviridae family. Retroviruses are retroviruses that are capable of infecting non-dividing cells. Unique among transcriptional viruses; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most effective methods for gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means of achieving significant levels of gene transfer in vivo. "Integrating lentiviral vectors (LV)” refer to these vectors as non-limiting examples, which can integrate the genome of target cells. “Non-integrating lentiviral vectors (NILV)” relatively refer to effective gene delivery vectors, which do not integrate the genome of target cells through the action of viral integrase.
  • endogenous refers to any material that originates from or is produced in an organism, cell, tissue or system itself, such as a nucleic acid molecule or a polypeptide.
  • exogenous refers to any substance, such as a nucleic acid molecule, polypeptide, cell, introduced into an organism, cell, tissue, or system, or produced outside of an organism, cell, tissue, or system.
  • foreign protein may be a protein that is exogenously introduced into cells and recognizes a target antigen, such as an exogenous receptor (ie, the aforementioned “chimeric receptor” herein).
  • the term "host” refers to a recipient of a transplant, and in some embodiments, may be an individual, such as a human, into whom foreign cells are implanted.
  • isolated means changed or removed from the natural state.
  • a nucleic acid or peptide naturally present in a living animal is not “isolated”, but the nucleic acid or peptide partially or completely separated from its coexistent in the natural state is “isolated”.
  • An isolated nucleic acid or protein may exist in a substantially pure form, or may exist in a non-natural environment such as a host cell.
  • An "isolated” material may also be provided by artificial assembly methods, such as by chemical synthesis or recombinant expression.
  • expression refers to the transcription and/or translation of a specific nucleotide sequence driven by a promoter.
  • promoter is a DNA sequence that RNA polymerase recognizes, binds to, and starts to transcribe. It is an important component of a gene, and its main function is to regulate the start time and expression level of gene transcription.
  • the nucleic acid encoding CAR is operably connected to a promoter.
  • the present invention provides isolated nucleic acids, vectors and host cells comprising the nucleic acids or vectors encoding antibodies or fragments thereof that recognize CLDN6.
  • the nucleic acids may be in intact cells, in cell lysates or in partially purified or substantially purified form.
  • the nucleic acids of the present invention can be obtained using standard molecular biology techniques, for example, cDNA encoding the light and heavy chains of antibodies or encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • cDNA encoding the light and heavy chains of antibodies or encoding VH and VL segments can be obtained by standard PCR amplification or cDNA cloning techniques.
  • antibodies obtained from immunoglobulin gene libraries e.g., using phage display technology
  • one or more nucleic acids encoding antibodies can be recovered from the library.
  • Methods for introducing exogenous nucleic acids into host cells are generally known in the art and can vary with the host cells used.
  • the nucleic acid molecule of the present invention is selected from SEQ ID NO: 2 or 6 encoding the heavy chain variable region, and/or selected from SEQ ID NO: 4, 8, 10 or 12 encoding the light chain variable region.
  • it is a nucleic acid molecule comprising a heavy chain variable region sequence of SEQ ID NO: 2 and a light chain variable region sequence of SEQ ID NO: 4; or a heavy chain variable region sequence of SEQ ID NO: 2 and a light chain variable region sequence of SEQ ID NO: 8; or a heavy chain variable region sequence of SEQ ID NO: 2 and a light chain variable region sequence of SEQ ID NO: 10; or a heavy chain variable region sequence of SEQ ID NO: 6
  • one or more vectors comprising the above-described nucleic acids are provided.
  • cell refers to a cell of human or non-human animal origin.
  • host cell refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell.
  • Host cells include “transformants” and “transformed cells”, which include the transformed primary cells and the offspring derived therefrom (regardless of the number of passages).
  • the nucleic acid content of the offspring may not be exactly the same as that of the parent cell, and may contain mutations. Mutant offspring having the same function or biological activity as that screened or selected for in the original transformed cell are included herein.
  • the term "positive” for a particular marker refers to the detectable presence of a particular marker (usually a surface marker) on or in a cell.
  • a surface marker refers to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is significantly higher than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, and/or is substantially similar to the level of cells known to be positive for the marker, and/or is significantly higher than the level of cells known to be negative for the marker.
  • negative for a particular marker refers to a substantial detectable absence of a particular marker (typically a surface marker) on or in the cell.
  • a surface marker refers to the presence of surface expression detected by flow cytometry, e.g., by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry at a level that is significantly higher than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, and/or significantly lower than the level of cells known to be positive for the marker, and/or substantially similar to the level of cells known to be negative for the marker.
  • CLDN6-positive host cells refers to host cells that express CLDN6 on the cell surface, which cells can be detected by, for example, flow cytometry using antibodies that specifically recognize epitopes on CLDN6.
  • the host cell is an immune cell.
  • immune cell refers to cells that participate in immune response and produce immune effectors, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages, mast cells, neutrophils, eosinophils, and/or basophils, etc.
  • the immune cells are T cells, NK cells, and NKT cells.
  • the T cells can be autologous T cells, heterologous T cells, and allogeneic T cells.
  • the NK cells can be allogeneic NK cells.
  • immune effector function or immune effector response refers to immune cells, such as functions or reactions that enhance or promote immune attacks on target cells.
  • immune function or response refers to the properties of T cells or NK cells that promote the killing of target cells or inhibit growth or proliferation.
  • artificially modified cells with immune cell function refers to cells or cell lines that do not have immune effects. After artificial modification or stimulation by a stimulant, the cell acquires immune cell function.
  • 293T cells are artificially modified to have immune cell function; for example, stem cells are induced in vitro to differentiate into immune cells.
  • Stem cells can be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells.
  • cells are generally primary cells, such as those isolated directly from an individual and/or isolated and frozen from an individual.
  • cells include one or more subsets of T cells or other cell types, such as full T cell populations, CD4+ cells, CD8+ cells and subsets thereof, such as those defined by function, activation state, maturation, differentiation potential, amplification, recycling, location, and/or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion, and/or degree of differentiation.
  • cells can be allogeneic and/or autologous.
  • T cells may be pluripotent stem cells from bone marrow that differentiate and mature into mature T cells with immune activity in the thymus.
  • T cells may be a cell population with specific phenotypic characteristics, or a mixed cell population with different phenotypic characteristics, such as “T cells” may be cells containing at least one T cell subpopulation: memory stem cell-like T cells (stem cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs), effector memory T cells (Tem), naive T (TN) cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells (e.g., TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells), cytotoxic T cells, and/or mucosa
  • T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from infection sites, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of techniques known to those skilled in the art, such as FicollTM separation can be used to obtain T cells from blood collected from an individual.
  • cells from circulating blood of an individual are obtained by single blood sampling.
  • Single collection products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • cells collected by single collection can be washed to remove plasma molecules and the cells are placed in a suitable buffer or culture medium for subsequent processing steps.
  • T cells can be obtained from healthy donors, or from cells derived from patients diagnosed with tumors. The conventional method for preparing CAR-T in the art is adopted, and T cells obtained by continuing to culture after activation of PBMC cells with magnetic beads of anti-CD3 and CD28 antibodies are used, and CAR-T cells are obtained after lentiviral infection.
  • peripheral blood mononuclear cell refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc.
  • activation and “activation” are used interchangeably and may refer to the process by which a cell changes from a quiescent state to an active state.
  • the process may include a response to an antigen, migration, and/or a phenotypic or genetic change in a functionally active state.
  • activation may refer to the process by which NK cells, T cells are gradually activated.
  • T cell activation or “T cell activation” refers to a state of T cells that are sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • Genetic engineering generally involves introducing a nucleic acid encoding the recombinant or engineered part into a cell, for example, by retroviral or lentiviral transduction, transfection or transformation, or by transposon, electroporation. In some embodiments, this is done by first stimulating the cells, for example, by combining them with a stimulant that induces a response, such as proliferation, survival and/or activation, for example, detected by the expression of cytokines or activation markers, and then transducing the activated cells and expanding them in culture to a number sufficient for clinical use.
  • a stimulant that induces a response, such as proliferation, survival and/or activation, for example, detected by the expression of cytokines or activation markers
  • a host cell comprising the aforementioned nucleic acid herein.
  • the host cell comprises (e.g., is transduced with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody.
  • the host cell is eukaryotic, e.g., a 293T cell.
  • the host cell expresses the chimeric receptor described herein.
  • the host cell comprises a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, a regulatory T cell, a NK92 cell, and/or a stem cell-derived immune cell.
  • the T cells are derived from natural T cells and/or T cells induced by pluripotent stem cells; preferably, the T cells are autologous/allogeneic T cells; preferably, the T cells are primary T cells; preferably, the T cells are derived from human autologous T cells.
  • the T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells (Tregs), effector memory T cells (Tem), ⁇ T cells, or a combination thereof.
  • the host cell binds to cells expressing CLDN6 and does not significantly bind to cells expressing CLDN4, CLDN9, or a combination thereof.
  • the host cell further carries an exogenous cytokine coding sequence.
  • the host cell may also express another chimeric receptor in addition to the above-mentioned antigen-binding receptor.
  • the host cell may also express a chemokine receptor.
  • the host cell may also express a safety switch.
  • a method of preparing an anti-CLDN6 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for expressing the antibody as described above, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • the nucleic acid encoding the antibody of the present invention can be integrated into an expression vector.
  • a variety of expression vectors can be used for protein expression.
  • the expression vector may include a self-replicating extrachromosomal vector, or a vector integrated into the host genome.
  • the expression vector used in the present invention includes but is not limited to those that enable protein to be expressed in mammalian cells, bacteria, insect cells, yeast and in vitro systems. As known in the art, a variety of expression vectors are commercially available or otherwise obtainable. Can be used in the present invention to express antibodies.
  • the host cell is administered in combination with an agent that enhances its function, preferably, in combination with a chemotherapeutic drug. and/or the host cell is administered in combination with an agent that improves one or more side effects associated therewith; and/or the host cell is administered in combination with a host cell expressing a chimeric antigen receptor targeting a protein other than CLDN6.
  • compositions comprising
  • the antibodies, immunoconjugates, chimeric receptors, and host cells of the present invention can be used to prepare pharmaceutical compositions or diagnostic reagents.
  • the composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when appropriately administered to animals or humans.
  • specific examples of some substances that can serve as pharmaceutically acceptable carriers or their components are sugars, such as lactose, glucose, mannose, sucrose, dextran; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl
  • compositions described herein may contain one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the parent compound and do not produce any adverse toxicological effects. Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc., and salts derived from non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
  • non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc.
  • non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.
  • Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.
  • antioxidants include, but are not limited to, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium pyrosulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium pyrosulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecit
  • the composition of the present invention can be prepared into various dosage forms as needed, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient type, age, weight and general disease condition, and the mode of administration.
  • the mode of administration can be, for example, parenteral administration (such as injection) or other treatment methods.
  • the "parenteral" administration of the immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques. It can also be administered to the patient via an artery, intradermally, intratumorally, intranodally, intramedullary, or intraperitoneally.
  • the compositions may be isotonic, i.e., they may have the same osmotic pressure as blood and tears.
  • the desired isotonicity of the compositions of the present invention may be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. If desired, the viscosity of the composition may be increased.
  • Pharmaceutically acceptable thickeners can be used to maintain selected levels. Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer etc. The preferred concentration of thickener will depend on selected reagent. Obviously, the selection of suitable carriers and other additives will depend on the property of definite route of administration and specific dosage form, for example liquid dosage form.
  • the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention is administered in combination with other known active agents or treatments.
  • Combined administration refers to the administration of two or more different treatments to an individual. In one embodiment, one treatment is still continuing when the second treatment begins, so there is overlap in administration. In one embodiment, after the administration of one treatment ends, the second treatment begins. In one embodiment, combined administration makes the treatment more effective. In some embodiments, the effects of the two treatments can be partially additive, completely additive, or greater than additive.
  • the treatments described in the "combination administration” include, but are not limited to, one or more of the following: surgery, chemotherapy, radiation, immunosuppressants (e.g., cyclosporin, azathioprine, methotrexate, mycophenolate mofetil and FK506), antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cyclophosphamide, fludarabine, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation.
  • the treatments described in the "combination administration” also include immunomodulators such as interferon ⁇ , interferon ⁇ , TGF- ⁇ 2 peptide inhibitors, or poly-ICLC.
  • the composition comprises another therapeutic agent.
  • the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention is administered in combination with a pharmaceutical agent that enhances its function.
  • the other therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and/or a pharmaceutically acceptable salt or analog thereof.
  • chemotherapeutic agents include alkylating agents, platinum-based agents, angiogenesis inhibitors (e.g., VEGF pathway inhibitors, tyrosine kinase inhibitors EGF pathway inhibitors), and mTOR inhibitors.
  • the therapeutic agent includes, but is not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibin (TM) (vinorelbine, 5'-dehydrogensulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL), and other compounds derived from camptothecin and its analogs; podophyllotoxin derivatives, such as etoposide, teniposide, and midoxazolidinone; alkylating agents cisplatin, cyclophosphamide, and dapoxetine; Phosphoramide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, brequizine, uracil mustard, chlorprofen and dacarbazine; antimetabolites, including cytarabine,
  • the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin and 5-fluorouracil.
  • the additional therapeutic agents include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides) and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including ⁇ and ⁇ ) interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2, etc.
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides) and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including ⁇ and ⁇ ) interleukin 12, retinoic acid, and tissue inhibitors of metall
  • the anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells of the present invention are Cells, pharmaceutical compositions or kits are administered in combination with inhibitors of inhibitory molecules.
  • Inhibitory molecules include PD1, PD-L1, CTLA-4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR ⁇ .
  • Inhibitory nucleic acids such as dsRNA, such as siRNA or shRNA, can be used to inhibit the expression of inhibitory molecules in CAR-expressing cells.
  • the inhibitor is shRNA.
  • inhibitory molecules are inhibited in CAR-expressing cells.
  • dsRNA molecules that inhibit the expression of inhibitory molecules can be connected to nucleic acids encoding components (e.g., all components) of CAR.
  • the inhibitor of inhibitory signals can be, for example, antibodies or antibody fragments that bind to inhibitory molecules.
  • the active agent can be an antibody or antibody fragment (e.g., ipilimumab, Tremelimumab) that binds to PD1, PD-L1, PD-L2 or CTLA4.
  • the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention is co-administered with an agent that improves one or more side effects associated therewith.
  • the side effects include, but are not limited to, CRS. Symptoms of CRS include high fever, nausea, transient hypotension, hypoxia, and the like.
  • the co-administered agents provided by the present invention can manage the elevated levels of soluble factors caused by anti-CLDN6 antibodies, immunoconjugates, chimeric receptor-modified host cells, pharmaceutical compositions or kits. Soluble factors elevated in individuals include IFN- ⁇ , TNF ⁇ , IL-2 and/or IL-6.
  • the agent administered to improve side effects may be an active agent that neutralizes one or more of these soluble factors.
  • active agents include, but are not limited to, steroids, TNF ⁇ inhibitors and/or IL-6 inhibitors.
  • TNF ⁇ inhibitors include, but are not limited to, Etanercept.
  • IL-6 inhibitors include, but are not limited to, Tocilizumab (toc).
  • the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention is administered in combination with a cell expressing a chimeric antigen receptor other than targeting CLDN6. In one embodiment, it is administered in combination with an agent for treating a disease associated with expression of CLDN6.
  • the agent comprises an antibody, a cell, an RNA, a vaccine, an oncolytic virus, a checkpoint inhibitor, a BKT inhibitor, a chemical drug, a radiotherapeutic agent, a hormonal therapeutic agent, a toxin, an immunotherapeutic agent or a combination thereof.
  • the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention and another therapeutic agent can be administered simultaneously, in the same composition or in separate compositions, or sequentially.
  • the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention can be administered first, and then another therapeutic agent can be administered.
  • another therapeutic agent can be administered first, and then the anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention can be administered.
  • kits comprising the antibodies, immunoconjugates, chimeric receptors, nucleic acids, or host cells described herein.
  • the kits may include therapeutic or preventive compositions containing an effective amount of the antibodies, chimeric receptors, nucleic acids, or host cells described herein in one or more unit dosage forms.
  • the kits contain a sterile container that may contain the therapeutic or preventive composition; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, blister packs, or other suitable containers known in the art.
  • Such containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding drugs.
  • kits contain antibodies, Immunoconjugates, chimeric receptors, nucleic acids or host cells, and instructions for administering the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein to an individual.
  • the instructions generally include methods of using the antibodies, immunoconjugates, chimeric receptors, nucleic acids or host cells described herein to treat or prevent cancer or tumors.
  • the kit comprises a host cell described herein and may include about 1 ⁇ 10 4 cells to about 1 ⁇ 10 6 cells.
  • the kit can include at least about 1 ⁇ 10 5 cells, at least about 1 ⁇ 10 6 cells, at least about 1 ⁇ 10 7 cells, at least about 4 ⁇ 10 7 cells, at least about 5 ⁇ 10 7 cells, at least about 6 ⁇ 10 7 cells, at least about 6 ⁇ 10 7 cells, 8 ⁇ 10 7 cells, at least about 9 ⁇ 10 7 cells, at least about 1 ⁇ 10 8 cells, at least about 2 ⁇ 10 8 cells, at least about 3 ⁇ 10 8 cells, at least about 4 ⁇ 10 8 cells, at least about 5 ⁇ 10 8 cells, at least about 6 ⁇ 10 8 cells, at least about 6 ⁇ 10 8 cells, at least about 8 ⁇ 10 8 cells, at least about 9 ⁇ 10 8 cells, at least about 1 ⁇ 10 9 cells, at least about 2 ⁇ 10 9 cells, at least about 3 ⁇ 10 9 cells, at least about 4 ⁇ 10 9 cells, at least about 5 ⁇ 10 9 cells 10 9 cells, at least about 6 ⁇ 10 9 cells, at least about 8 ⁇ 10 9 cells, at least about 9 ⁇ 10 9 cells, at least about 1 ⁇ 10 10 cells, at least about
  • the kit may include allogeneic cells. In some embodiments, the kit may include cells that may include genomic modifications. In some embodiments, the kit may include "off-the-shelf" cells. In some embodiments, the kit may include cells that may be expanded for clinical use. In some cases, the kit may include contents for research purposes.
  • the instructions include at least one of the following: a description of the therapeutic agent; dosage regimens and administration for treating or preventing a tumor or its symptoms; precautions, warnings, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and/or references.
  • the instructions can be printed directly on the container (if any), or as a label on the container, or as a separate paper, brochure, card, or folder provided in or in the container.
  • the instructions provide methods for administering the antibodies described herein for treating or preventing tumors.
  • the instructions provide methods for administering the antibodies of the present invention before, after, or simultaneously with the administration of a chemotherapeutic agent.
  • modulate refers to a positive or negative change. Examples of modulation include 1%, 2%, 10%, 25%, 50%, 75%, or 100% change. In a specific embodiment, it refers to a negative change.
  • treatment refers to intervention measures that attempt to change the course of a disease, which can be either preventive or intervention in the clinical pathological process.
  • the therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, relieving or improving the prognosis, etc.
  • the term does not mean that the disease is completely cured, or any symptoms are completely eliminated, or that there is an effect on all symptoms or consequences.
  • anti-tumor effect refers to a biological effect that can manifest in many forms, including but not limited to, The reduction of tumor volume, the reduction of tumor cell number, the reduction of metastatic tumor number, the increase of life span, the reduction of tumor cell proliferation, the reduction of tumor cell survival, or the improvement of various physiological symptoms related to cancer.
  • Anti-tumor effect can also be manifested as the ability of the peptides, polynucleotides, cells and antibodies of the present invention to prevent tumor occurrence from the beginning.
  • prevention refers to the prevention or prophylactic treatment of a disease or disease state by providing a method to prevent the occurrence or recurrence of a disease in a subject who is predisposed to developing the disease but has not yet been diagnosed with the disease, or by attempting to intervene before the disease develops (such as rejection of a cell transplant).
  • autologous refers to any substance obtained from an individual and subsequently reintroduced into the same individual.
  • allogeneic refers to any material introduced into an individual that comes from a different individual of the same species as the individual. When two or more individuals differ in genes at one or more loci, the individuals are referred to as being allogeneic. In some aspects, allogeneic material from individuals of the same species can be genetically different enough to cause antigenic interactions.
  • xenogeneic refers to a transplant that originates from an individual of a different species.
  • tumor or cancer refers to a disease characterized by rapid and uncontrolled abnormal cell growth. Tumor cells or cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system.
  • disease associated with CLDN6 expression or “disease associated with expression of CLDN6” includes, but is not limited to, diseases associated with CLDN6 expression, or disorders associated with cells expressing CLDN6, such as ovarian cancer, breast cancer, cervical cancer, gastric cancer, lung cancer, testicular cancer, germ cell and embryonic tumors, ovarian epithelial cancer, non-small cell lung cancer, non-squamous non-small cell lung cancer, endometrial cancer, etc.
  • detection includes quantitative or qualitative detection.
  • the antibodies of the present invention can be used to detect the presence of CLDN6 in a biological sample, including blood, serum, cells or tissues.
  • tumor antigen refers to an antigen that appears or is overexpressed during the development and progression of a hyperproliferative disease.
  • the hyperproliferative disorder of the present invention refers to a tumor.
  • the tumor antigen described in the present invention may be a solid tumor antigen or a hematological tumor antigen.
  • the tumor antigens of the present invention include, but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit ⁇ (IL-13R ⁇ ); interleukin 11 receptor ⁇ (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor
  • the pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.
  • subject is intended to include living organisms that can elicit an immune response, and refers to any animal, such as a mammal or marsupial.
  • the subject of the present invention includes, but is not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
  • an effective amount refers to an amount that provides therapeutic or preventive benefits, and is effective in achieving the desired therapeutic or preventive results at a certain dosage and for the necessary length of time. It can be determined by a physician based on individual differences in age, weight, tumor size, degree of infection, degree of metastasis, etc. of the patient (individual).
  • Any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit provided herein can be used in a therapeutic method.
  • Any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit provided by the present invention provides one or more of the following by causing an antigen-specific response to CLDN6: targeting and destroying CLDN6-expressing tumor cells, reducing or eliminating tumors, promoting the infiltration of immune cells into tumor sites, and enhancing/prolonging anti-tumor responses. Since CLDN6 is expressed at undetectable levels in normal (i.e., non-cancerous) tissues, any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit provided by the present invention is believed to avoid targeting/destroying normal tissues and cells.
  • any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit for use as a medicament is provided.
  • any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit for treating a disease is provided.
  • any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit for use in a method of treatment is provided.
  • the present invention provides any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit for use in a method of treating an individual suffering from a disease, the method comprising administering to the individual an effective amount of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit. In one embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.
  • the "individual" is preferably a human.
  • the present invention provides any anti-CLDN6 antibody, immunoconjugate, chimeric receptor modified host cell, pharmaceutical composition or kit for use in the preparation or formulation of a drug.
  • the drug is used to treat a disease.
  • the drug is used in a method for treating a disease, the method comprising administering an effective amount of the drug to a sick individual.
  • the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.
  • the "individual" is preferably a human.
  • the present invention provides a method for treating a disease.
  • the method comprises administering an effective amount of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit to an individual suffering from a disease expressing CLDN6.
  • the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.
  • the "individual" is preferably a human.
  • the present invention provides a pharmaceutical preparation comprising any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit provided herein, for example for use in any of the above-mentioned treatment methods.
  • the pharmaceutical preparation comprises any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical preparation comprises any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit provided herein and at least one additional therapeutic agent.
  • the pharmaceutical preparation is for use in treating a disease.
  • the pharmaceutical preparation is administered to a diseased individual.
  • the "individual" according to any of the above embodiments is preferably a human.
  • the present invention provides a method for preparing a medicament or pharmaceutical preparation, the method comprising mixing any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit provided herein with a pharmaceutical carrier, for example, for use in any of the above-mentioned treatment methods.
  • the method for preparing a medicament or pharmaceutical preparation further comprises adding at least one additional therapeutic agent to the medicament or pharmaceutical preparation.
  • Any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention can be used alone for treatment or in combination with other agents for treatment.
  • any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention can be co-administered with at least one additional therapeutic agent.
  • Such combination therapy described above includes combined administration (wherein two or more therapeutic agents are contained in the same or separate preparations) and separate administration, in which case, administration of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention may occur before, simultaneously with, and/or after administration of the additional therapeutic agent or reagent.
  • administration of any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention and administration of the additional therapeutic agent occur within about one month, or within about one week, two weeks or three weeks, or within about one day, two days, three days, four days, five days or six days of each other.
  • Any anti-CLDN6 antibody, immunoconjugate, chimeric receptor-modified host cell, pharmaceutical composition or kit of the present invention can be administered by any suitable means, including parenteral administration, intrapulmonary administration or intranasal administration, and, if necessary for treatment, intralesional administration.
  • Parenteral infusion includes intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, which depends in part on whether the administration is short-term or long-term.
  • a variety of dosing regimens are contemplated herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse injection.
  • the preparation containing the immunoreactive cell population administered to an individual contains multiple immunoreactive cells that effectively treat and/or prevent a specific indication or disease. Therefore, a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • a preparation containing about 1 ⁇ 10 4 to about 1 ⁇ 10 10 immunoreactive cells is administered.
  • the preparation will contain about 1 ⁇ 10 5 to about 1 ⁇ 10 9 immunoreactive cells, about 5 ⁇ 10 5 to about 5 ⁇ 10 8 immunoreactive cells, or about 1 ⁇ 10 6 to about 1 ⁇ 10 7 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to the individual will vary between a wide range. The doctor will ultimately determine the appropriate dose to be used.
  • chimeric receptors are used to stimulate host cell-mediated immune responses.
  • a T cell-mediated immune response is an immune response involving T cell activation.
  • Activated antigen-specific cytotoxic T cells can induce apoptosis in target cells that display exogenous antigen epitopes on their surfaces, such as cancer cells that display tumor antigens.
  • chimeric antigen receptors are used to provide anti-tumor immunity in mammals. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.
  • the method for treating a subject with a tumor may involve administering one or more host cells of the present invention to a subject in need of treatment.
  • the host cells can bind to tumor target molecules and induce cancer cell death.
  • the present invention also provides a method for treating a pathogen infection in an individual, comprising administering a therapeutically effective amount of the host of the present invention to the individual.
  • the frequency of administration of the immunoreactive cells of the present invention will depend on factors including the disease being treated, the components of the specific immunoreactive cells and the mode of administration.
  • the administration may be 4 times, 3 times, 2 times or once a day, every other day, Every three days, every four days, every five days, every six days, once a week, once every eight days, once every nine days, every ten days, once a week, or twice a month.
  • the immune response cells of the present application have improved viability, they can be administered not only in a therapeutically effective amount lower than similar immune response cells that do not express exogenous type I interferon, but also in a lower frequency of administration to obtain at least similar, and preferably more significant, therapeutic effects.
  • the present invention provides a humanized antibody that specifically recognizes CLDN6, and the CAR T cells prepared from the antibody show good killing effect on target cells in vivo and in vitro.
  • mouse anti-SC27.105 (from WO2016073649A1) is used as the parent antibody, and the parent antibody sequence is compared with the germline sequence of the IMGT database to screen the heavy chain CDR transplantation template 1 and the light chain CDR transplantation template 2 of the SC27.105 antibody.
  • the LCDR region of the SC27.105 antibody replaces the CDR region of the antibody template 2 to form the light chain variable region VL of the humanized antibody H1 (amino acid sequence see SEQ ID NO: 3).
  • the 5th serine (Serine, S) in the heavy chain CDR2 of the SC27.105 antibody is mutated to alanine (Alanine, A), and then the parent antibody HCDR1, mutated HCDR2, and HCDR3 replace the CDR region of the transplantation template 2 to form the heavy chain variable region VH of the humanized antibody H1 (amino acid sequence see SEQ ID NO: 1).
  • the framework region in the VH amino acid sequence of the humanized antibody H1 is amino acids 1-30, 36-49, 67-98, and 113-123 in SEQ ID NO:1; the framework region in the VL amino acid sequence of the humanized antibody H1 is amino acids 1-23, 35-49, 57-88, and 97-106 in SEQ ID NO:3.
  • H1 heavy chain variable region (SEQ ID NO: 1), the CDR region is underlined, and the framework region is the sequence other than the CDR.
  • the CDR region of the H1 light chain variable region (SEQ ID NO: 3) is underlined, and the framework region is the sequence other than the CDR.
  • 293T-CLDN6, 293T-CLDN4, 293T-CLDN9 cells 2 ⁇ 10 5 cells/well in 96-well round bottom
  • the culture plate was washed with PBS; antibody H1 was added, incubated at 4°C, centrifuged, supernatant was discarded, and then washed with PBS; Goat-anti-Mouse FITC was added, incubated at 4°C; centrifuged, supernatant was discarded, and then washed, and detected by flow cytometry.
  • the results were statistically analyzed using FlowJo vX0.7 and graphed using GraphPad Prism8.0.
  • phage libraries H1-VH and H1-VL, were constructed, with a library capacity of 1E+9.
  • the phage library was incubated with 293T cells for 1-2 hours, and the supernatant was centrifuged and incubated with 293T-CLDN6 cells for 1-2 hours and washed; the phages on the cells were eluted, the eluate was neutralized, and then Escherichia coli TG1 was infected. After expansion culture, the phages were purified and used for the next round of screening.
  • amino acid sequence of VH of antibody P1 is shown in SEQ ID NO: 5
  • amino acid sequence of VL is shown in SEQ ID NO: 3
  • amino acid sequence of HCDR3 is shown in SEQ ID NO: 41
  • amino acid sequence of scFv is shown in SEQ ID NO: 14.
  • amino acid sequence of VH of antibody P2 is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 7; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 42; and the amino acid sequence of scFv is shown in SEQ ID NO: 15.
  • amino acid sequence of VH of antibody P3 is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 9; the amino acid sequence of LCDR3 is shown in SEQ ID NO: 43; and the amino acid sequence of scFv is shown in SEQ ID NO: 16.
  • amino acid sequence of VH of antibody P4 is shown in SEQ ID NO: 1, and the amino acid sequence of VL is shown in SEQ ID NO: 11; the sequence of LCDR3 is shown in SEQ ID NO: 44; and the amino acid sequence of scFv is shown in SEQ ID NO: 17.
  • the heavy chain variable region of antibody P1 in Example 3 was combined with the light chain variable regions of antibodies P2 and P3, respectively, to obtain antibodies M1 and M2.
  • a purified single-chain antibody (scFv) was obtained.
  • M1 and M2 were co-incubated with 293T-CLDN6, 293T-CLDN4 and 293T-CLDN9 cell lines, respectively.
  • the scFv bound to the cells was fluorescently labeled and detected by flow cytometry.
  • the experimental data were analyzed using FlowJo software to calculate the mean fluorescence intensity (MFI).
  • M1 and M2 specifically bind to 293T-CLDN6 cells, but not to 293T-CLDN4 and 293T-CLDN9 cells.
  • FIG7 shows that both M1 and M2 significantly bound to 293T-CLDN6 cells, with EC50 values of 23.04 nM and 24.71 nM, respectively, which increased their affinity by nearly 20 times compared to antibody H1.
  • PRRLSIN-cPPT.EF-1 ⁇ lentiviral plasmids PRRLSIN-cPPT.EF-1 ⁇ -H1-28Z and PRRLSIN-cPPT.EF-1 ⁇ -P4-28Z expressing the second-generation chimeric antigen receptors of antibodies H1 and P4 were constructed.
  • the amino acid sequence of H1-28Z is shown in SEQ ID NO: 48; the amino acid sequence of P4-28Z is shown in SEQ ID NO: 49.
  • the amino acid sequence of the control group C46-S-28Z is shown in SEQ ID NO: 51.
  • the conventional method for preparing CAR-T in the art is adopted, and T cells obtained by further culturing PBMC cells after activation with magnetic beads of anti-CD3 and CD28 antibodies are used, and CAR-T cells are obtained after lentivirus infection.
  • the lentivirus was packaged using the calcium phosphate method, and the viral supernatant was purified using PEG8000/NaCl. After purification, the virus was infected with CD3/CD28 magnetic beads at an MOI of 20, and T cells activated for 48 hours were obtained to express H1-28Z, P4-28Z, and C46-S-28Z. T cells not transfected with the virus were considered UTD. On the fifth day after infection, the CAR positive rate was detected by FACS. The primary antibody was Biotin-anti-F(ab')2-488 (Jackson ImmunoResearch), and the secondary antibody was SA-PE (eBioscience). The results are shown in Figure 8.
  • the target cells 293T-CLDN4 cells, 293T-CLDN6 cells, 293T-CLDN9 cells, OVCAR3 cells (human ovarian cancer cells, ATCC) and OV90 cells (human ovarian cancer cells, ATCC) were adjusted to a density of 0.2x10 ⁇ 6/mL using 1640 culture medium, and 50 ⁇ l was added to each well of a 96-well cell culture plate, i.e., 10,000 target cells per well. Then, 50 ⁇ l of effector cells (CAR-T cells, UTD as a control) were added according to a 1:1 effector-target ratio. After incubation at 37°C for 16 hours, the supernatant was collected for detection using an LDH kit, and finally the OD490 reading was performed using a microplate reader.
  • H1CAR-T cells and P4CAR-T cells had no killing effect on 293T-CLDN4 cells and 293T-CLDN9 cells; for 293T-CLDN6, OVCAR3 and OV90 cells expressing CLDN6, they all showed obvious killing effects, and the killing rate was about 60%-90% when the effector-target ratio was 3:1.
  • the results showed that H1CAR-T and P4CAR-T cells had specific in vitro killing effects on cells expressing CLDN6.
  • C46-S CAR-T cells had nonspecific cell killing effects.
  • Target OV90 ovarian cancer cells were inoculated into RTCA test well plates at 1E+04 cells/pore. After 20 hours, UTD, H1-28Z-CAR T, and P4-28Z-CAR T cells were added at an effector-target ratio of 1:1. After co-culture for 8 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, and 80 hours, the target cell lysis rate was detected. As shown in Figure 10, H1-28Z-CAR T and P4-28Z-CAR T cells showed a significant difference in the lysis rate of target cells. P4-28Z-CAR T showed significant killing effect on OV90 cells, with the killing rate reaching over 90% after 48 hours and approaching 100% after 72 hours.
  • Example 8 Antitumor effect of CLDN6-CAR T cells on subcutaneous transplanted tumors in NPG mice bearing human ovarian cancer cells
  • mice 5 ⁇ 10 6 OV90 cells were subcutaneously inoculated in NPG mice. The average tumor volume was about 234 mm 3 13 days after inoculation. The mice were divided into 4 groups and injected with UTD (3x10 6 ), H1-28Z (1x10 6 ), H1-28Z (3x10 6 ), and P4-28Z (3x10 6 ) through the tail vein, respectively. The results are shown in Figure 11A. 22 days after CAR-T injection, compared with UTD, the tumor inhibition rate of the H1-28Z (1x10 6 ) group was 74.11%, the tumor inhibition rate of the H1-28Z (3x10 6 ) group was 88.26%, and the tumor inhibition rate of the P4-28Z group was 70.81%.
  • FIG. 11B there was no significant change in the weight of mice in each CAR-T group.
  • Figure 11C shows that according to the change of tumor weight, the tumor inhibition rate of H1-28Z (1x10 6 ) group was 73.46%, the tumor inhibition rate of H1-28Z (3x10 6 ) group was 92.41%, and the tumor inhibition rate of P4-28Z group was 71.39%.
  • Figure 11D shows the survival of human T cells in the peripheral blood of mice in each group 11 days after CAR-T cell infusion.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明涉及特异性结合CLDN6的抗体及其应用。

Description

抗体及其应用 技术领域
本发明涉及肿瘤免疫治疗或诊断领域，更具体地，涉及特异性结合CLDN6抗体及其应用。
背景技术
紧密连接蛋白(CLDN)基因家族编码膜蛋白，该蛋白为紧密连接的重要组成部分。CLDN蛋白包含四个跨膜(TM)螺旋(TM1、TM2、TM3和TM4)和两个胞外环(EL1和EL2)，其N端和C端均位于胞质内。相邻细胞的CLDN蛋白的胞外环彼此相互作用以封闭细胞片层并调节管腔与基底外侧空间之间的细胞旁转运。CLDN蛋白在各种人类疾病和病理中起作用。CLDN6是CLDN基因家族的成员，在正常成人组织中不表达，而在多种实体肿瘤组织如卵巢癌、睾丸癌、子宫内膜癌上高表达，因此，CLDN6是Claudin家族中一个癌症治疗的潜力靶点。CLDN6与同家族成员CLDN9和CLDN4的胞外段序列同源性非常高，因此，开发特异性结合CLDN6的抗体具有非常高的挑战性。
发明内容
本发明目的在于提供特异性识别CLDN6的抗体。本发明还涉及制备抗CLDN6特异性抗体的方法，包括人源化抗体制备技术和噬菌体文库筛选技术。本发明还涉及抗CLDN6抗体(包括但不限于scFv形式)的特性及特异性的研究。本发明提供了靶向CLDN6嵌合抗原受体(CAR)及其制备方法。本发明还提供分离的编码本发明的抗CLDN6抗体和靶向CLDN6的嵌合抗原受体的核酸。本发明还提供宿主细胞，所述宿主细胞包含本发明的核酸。所述方法还包括培养本发明的宿主细胞以制备所述抗体或所述CAR。本发明的抗体和/或CAR用于治疗肿瘤或肿瘤诊断。
在第一方面，提供了识别CLDN6的抗体或抗原结合片段，选自下组：
(1)所述抗体或抗原结合片段包含重链可变区，所述重链可变区的HCDR3与SEQ ID NO：41所示的HCDR3具有至少95％、96％、97％、98％或99％的氨基酸序列同一性；
(2)所述抗体或抗原结合片段包含重链可变区，所述重链可变区包含GYYMN(SEQ ID NO：35)所示的HCDR1；和/或
EINPATGSTTYNQKFKA(SEQ ID NO：36)所示的HCDR2；和/或
RDYYX1GSX2X3YAX4DY(SEQ ID NO:52)所示的HCDR3，其中X1是Y或L或是Y或L的保守性取代氨基酸残基，X2是G或N或是G或N的保守性取代氨基酸残基，X3是F或S或是F或S的保守性取代氨基酸残基，X4是M或L或是M或L的保守性取代氨基酸残基；
(3)所述抗体或抗原结合片段包含重链可变区，所述重链可变区的HCDR1、HCDR2和HCDR3与SEQ ID NO：35、36和37所示的HCDR1、HCDR2和HCDR3具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性；
(4)所述抗体或抗原结合片段包含轻链可变区，所述轻链可变区的LCDR3与SEQ ID  NO：42、43或44所示的LCDR3具有至少60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性；
(5)所述抗体或抗原结合片段包含轻链可变区，所述轻链可变区包含QASQSVSNNLN(SEQ ID NO：38)所示的LCDR1；和/或
GASKLED(SEQ ID NO：39)所示的LCDR2；和/或
X5QHRX6X7WT(SEQ ID NO:53)所示的LCDR3，其中X5是L或Q或是L或Q的保守性取代氨基酸残基，X6是Y或F或是Y或F的保守性取代氨基酸残基，X7是L或M或是L或M的保守性取代氨基酸残基；
(6)所述抗体或抗原结合片段包含轻链可变区，所述轻链可变区的LCDR1、LCDR2和LCDR3与SEQ ID NO：38、39和40所示的LCDR1、LCDR2和LCDR3具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性；
(7)所述抗体或抗原结合片段包含(1)～(3)中任一项所述的重链可变区及(4)～(6)中任一项所述的轻链可变区；
(8)所述抗体或抗原结合片段是(1)～(7)中任一项所述的抗体或抗原结合片段的变体，所述变体在1、2、3、4、5或6个CDR区上共包含至少一个且不超过7、6、5、4、3或2个氨基酸改变，且具备与(1)～(7)中任一项所述的抗体或抗原结合片段相同或相似的活性。
在一优选例中，所述的抗体或抗原结合片段的HCDR2具有SEQ ID NO:36所示的氨基酸序列。
在一实施方式中，所述的抗体或抗原结合片段选自下组：
(1)所述的抗体或抗原结合片段包含重链可变区，所述重链可变区包含SEQ ID NO：35所示的HCDR1，和/或SEQ ID NO：36所示的HCDR2，和/或SEQ ID NO：37或41所示的HCDR3；
(2)所述的抗体或抗原结合片段包含轻链可变区，所述轻链可变区包含SEQ ID NO：38所示的LCDR1，和/或SEQ ID NO：39所示的LCDR2，和/或SEQ ID NO：40、42、43或44所示的LCDR3；
(3)所述的抗体或抗原结合片段包含(1)所述的重链可变区及(2)所述的轻链可变区；
(4)所述的抗体或抗原结合片段是(1)～(3)中任一项所述的抗体或抗原结合片段的变体，所述变体在1、2、3、4、5或6个CDR区上共包含至少一个且不超过7、6、5、4、3或2个氨基酸改变，且具备与(1)～(3)中任一项所述的抗体或抗原结合片段相同或相似的活性。
在一实施方式中，所述的抗体或抗原结合片段选自下组：
(1)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：40所示的LCDR3；或者
(2)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO： 36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：40所示的LCDR3；或者
(3)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：42所示的LCDR3；或者
(4)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：43所示的LCDR3；或者
(5)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：42所示的LCDR3；或者
(6)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：43所示的LCDR3；或者
(7)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：44所示的LCDR3；或者
(8)所述的抗体或抗原结合片段是(1)～(7)中任一项所述的抗体或抗原结合片段的变体，所述变体在1、2、3、4、5或6个CDR区上共包含至少一个且不超过7、6、5、4、3或2个氨基酸改变，且具备与(1)～(7)中任一项所述的抗体或抗原结合片段相同或相似的活性。
第二方面，提供了识别CLDN6的抗体或抗原结合片段，选自下组：
(1)所述的抗体或抗原结合片段包含重链可变区，所述重链可变区包含SEQ ID NO:1或5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(2)所述的抗体或抗原结合片段包含轻链可变区，该轻链可变区包含SEQ ID NO:3、7、9或11所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
(3)所述的抗体或抗原结合片段包含(1)所述的重链可变区及(2)所述的轻链可变区；
(4)所述的抗体或抗原结合片段是(1)～(3)中任一项所述的抗体或抗原结合片段的变体，所述变体在VH或VL上包含至少一个且不超过20、15、10、9、8、7、6、5、4、3或2个氨基酸改变，且具备与(1)～(3)中任一项所述的抗体或抗原结合片段相同或相似的活性。
在一实施方式中，前述所述的抗体或抗原结合片段选自下组：
(1)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:3所示的氨基 酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(2)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:3所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(3)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:7所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(4)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:9所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(5)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:7所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(6)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:9所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(7)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:11所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
(8)所述的抗体或抗原结合片段是(1)～(7)中任一项所述的抗体或抗原结合片段的变体，所述变体在VH或VL上包含至少一个且不超过20、15、10、9、8、7、6、5、4、3或2个氨基酸改变，且具备与(1)～(7)中任一项所述的抗体或抗原结合片段相同或相似的活性。
第三方面，提供了识别CLDN6的抗体或抗原结合片段，包含重链可变区和轻链可变区， 所述轻链可变区包含SEQ ID NO:3、7、9、11所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；和
所述重链可变区包含SEQ ID NO:35所示的HCDR1，SEQ ID NO:36所示的HCDR2，和SEQ ID NO:37或41所示的HCDR3。
在一实施方式中，所述的抗体或抗原结合片段的重链可变区包含SEQ ID NO:1、5所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列。
第四方面，提供了识别CLDN6的抗体或抗原结合片段，包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:1、5所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；和
所述轻链可变区包含SEQ ID NO:38所示的LCDR1，SEQ ID NO:39所示的LCDR2，和SEQ ID NO:40、42、43或44所示的LCDR3。
在一实施方式中，所述的抗体或抗原结合片段的轻链可变区包含SEQ ID NO:3、7、9或11所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列。
在一实施方式中，前述任一项所述的抗体或抗原结合片段，选自全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体、IgG4抗体、scFv-Fc抗体、杂交瘤抗体、嵌合抗体、人源化抗体、全人源抗体或单克隆抗体。
在一实施方式中，所述的抗体或抗原结合片段，包含SEQ ID NO：13、14、15、16、17、18或19所示的氨基酸序列，或与上述序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或包含在上述序列中具有至少一个且不超过20、15、10、9、8、7、6、5、4、3或2个氨基酸改变。
在一实施方式中，所述抗体或抗原结合片段结合CLDN6，不显著结合CLDN4或CLDN9；和/或，所述抗体或抗原结合片段结合表达CLDN6的细胞，不显著结合表达CLDN4、CLDN9或其组合的细胞。
第五方面，提供了一种免疫缀合物，包括：第一方面至第四方面任一所述的抗体或抗原结合片段，以及与之连接的功能性分子。
在一实施方式中，所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。
在一实施方式中，所述的靶向肿瘤表面标志物的分子是结合除CLDN6外的其它肿瘤表面标志物的抗体或配体。
在一实施方式中，所述的抑制肿瘤的分子是抗肿瘤的细胞因子或抗肿瘤的毒素。
在一实施方式中，所述的细胞因子选自：IL-7、IL-12、IL-15、IL-18、IL-21、、I型干扰素或TNF-α。
在一实施方式中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与第一方面至第四方面任一所述的抗体或抗原结合片段形成T细胞参与的 双功能抗体。
在一实施方式中，所述的靶向免疫细胞的表面标志物选自：CD3、CD16、CD28。
在一实施方式中，所述的靶向免疫细胞的表面标志物的抗体是抗CD3抗体。
在一实施方式中，所述免疫缀合物中所述第一方面至第四方面任一所述的抗体或抗原结合片段，以及与之连接的功能性分子之间，还包括连接肽。
第六方面，提供了一种嵌合受体，包含胞外区，所述胞外区包含第一方面至第四方面任一所述的抗体或抗原结合片段；
所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)、合成多肽受体(synNotch)或其组合。
在一实施方式中，所述嵌合受体为嵌合抗原受体(CAR)，其包含第一方面至第四方面任一所述的抗体或抗原结合片段、跨膜区和胞内信号区。
在一实施方式中，所述抗体或抗原结合片段通过铰链域连接到所述跨膜区。
在一实施方式中，所述的嵌合受体的跨膜区包含选自TCR的α、β、γ或ζ链、CD3ε、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD27、CD28、CD33、CD37、CD45、CD64、CD80、CD134、CD137、CD152、CD154或PD1的跨膜区。
在一实施方式中，所述跨膜区选自CD8或CD28的跨膜结构域。
在一实施方式中，所述跨膜区选自SEQ ID NO:25或22所示的序列，或与其具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
在一实施方式中，所述嵌合受体的胞内信号区包含初级信号域。
在一实施方式中，所述初级信号域选自TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(也称作“ICOS”)、CD66d或CD3ζ。
在一实施方式中，所述嵌合受体的胞内信号区还包含一个或多个共刺激信号域。
在一实施方式中，所述共刺激信号域选自CARD11、CD2、CD5、CD7、CD27、CD28、CD30、CD40、CD54、CD83、OX40、CD137、CD134、CD150、CD152、CD223、CD270、PD-L2、PD-L1、CD278、DAP10、LAT、NKD2C、SLP76、TRIM、FcεRIγ、MyD88、ICAM-1、LFA-1(CD11a/CD18)、4-1BB或4-1BBL的胞内信号区或其组合。
在一实施方式中，所述共刺激信号域选自CD28和/或CD137的胞内信号域。
在一实施方式中，所述的嵌合受体的胞内信号区选自SEQ ID NO：24、或SEQ ID NO：23和24所示的序列，或SEQ ID NO：26和24所示的序列，或SEQ ID NO：23、26和24的序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
在一实施方式中，所述嵌合受体的铰链区来自CD8、lgG4或lgG1。
在一实施方式中，所述铰链区包含SEQ ID NO：21、27、28、29或30所示的序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
在一实施方式中，所述的嵌合受体包括：
第一至第四方面任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区和CD3ζ；或
第一至第四方面任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区、CD137的胞内信号区和CD3ζ；或
第一至第四方面任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区、CD28的胞内信号区和CD3ζ；或
第一至第四方面任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3ζ。
在一实施方式中，所述嵌合受体包含如SEQ ID NO：13、14、15、16、17、18或19任一所示的序列分别与SEQ ID NO：45、46或47任一所示的序列连接的氨基酸序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
在一实施方式中，所述嵌合受体包含如SEQ ID NO：48或49所示的序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
第七方面，提供了编码第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第六方面所述的嵌合受体的核酸。
第八方面，提供了一种载体，其包含第七方面所述的核酸。
第九方面，提供了一种细胞，其包含第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第六方面所述的嵌合受体、第七方面所述核酸、和/或第八方面所述的载体。
在一实施方式中，所述细胞包括T细胞、自然杀伤细胞、自然杀伤T细胞、NK92细胞、细胞毒性T细胞、树突细胞、巨噬细胞、CIK细胞、多能干细胞、干细胞衍生的免疫细胞或其组合。
在一实施方式中，所述T细胞包括天然的T细胞和/或经多能干细胞诱导产生的T细胞。
在一实施方式中，所述T细胞包括自体T细胞和/或同种异体T细胞。
在一实施方式中，所述T细胞为原代T细胞。
在一实施方式中，所述T细胞来源于人的自体T细胞。
在一实施方式中，所述细胞结合表达CLDN6的细胞，不显著结合表达CLDN4、CLDN9或其组合的细胞。
在一实施方式中，所述细胞还携带外源的细胞因子的编码序列；和/或其还表达非靶向CLDN6的嵌合受体；和/或其还表达趋化因子；和/或其还表达趋化因子受体；和/或其还表达安全开关；和/或其还表达抑制性分子。
在一实施方式中，所述细胞还携带外源的细胞因子包括IL-7、IL-12、IL-15、IL-18、IL-21、I型干扰素或TNF-α的编码序列。
在一实施方式中，所述细胞还表达趋化因子，所述趋化因子包括CCL19或CCL21。
在一实施方式中，所述细胞还表达趋化因子受体，所述趋化因子受体包括CCR2、CCR4、 CCR5、CXCR2、CXCR4或CXCR5。
在一实施方式中，所述细胞还表达安全开关，所述安全开关包括iCaspase-9、Truancated EGFR或RQR8。
在一实施方式中，所述细胞还表达抑制性分子，所述抑制性分子包括能降低PD-1表达的siRNA或阻断PD-L1的蛋白。
第十方面，提供了一种药物组合物，其包含权利要求第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第六方面所述的嵌合受体、第七方面所述核酸、第八方面所述的载体和/或第九方面所述的细胞，以及药学上可接受的佐剂。
第十一方面，提供了联合用药，第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第六方面所述的嵌合受体、第九方面所述的细胞、第十方面所述的药物组合物与增强其功能的药剂组合施用。
在一实施方式中，与化疗药物联用；和/或与改善其相关的一种或多种副作用的药剂联合施用；和/或与表达靶向CLDN6之外的嵌合抗原受体的细胞联合施用；和/或与治疗表达CLDN6相关疾病的药剂联合施用。
在一实施方式中，所述药剂包括抗体或抗原结合片段、细胞、RNA、疫苗、溶瘤病毒、检查点抑制剂、BKT抑制剂、化学药、放射治疗剂、激素治疗剂、毒素、免疫治疗剂或其组合。
第十二方面，提供了一种制备第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第六方面所述的嵌合受体的方法，所述方法包含培养第九方面所述的细胞，并分离出由所述细胞表达的所述抗体或抗原结合片段、免疫缀合物或嵌合受体。
第十三方面，提供了一种试剂盒，其特征在于，其包括第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第六方面所述的嵌合受体、第七方面所述核酸、第八方面所述的载体、第九方面所述的细胞和/或第十方面所述药物组合物。
第十四方面，提供了第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第九方面所述的细胞、第十方面所述的药物组合物和/或第十三方面所述的试剂盒的用途，(1)杀伤表达CLDN6的细胞；(2)抑制表达CLDN6的细胞的增殖；(3)介导疾病或肿瘤的缓解；(4)预防肿瘤形成或再形成；(5)抑制表达CLDN6的细胞的转移；(6)制备用于治疗/诊断疾病的药物。
在一实施方式中，所述疾病表达CLDN6。
在一实施方式中，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤。
在一实施方式中，所述肿瘤是实体瘤。
在一实施方式中，所述肿瘤是卵巢癌、乳腺癌、宫颈癌、胃癌、肺癌、睾丸癌、生殖细胞和胚胎肿瘤、卵巢上皮癌、非小细胞肺癌，非鳞状非小细胞肺癌、子宫内膜癌、或其组合。
在第十五方面，本发明提高一种用于以下用途的并且包含第一方面所述的抗体或抗原 结合片段、第二方面所述的免疫缀合物、第九方面所述的细胞、第十方面所述的药物组合物的药剂，
(1)杀伤表达CLDN6的细胞；
(2)抑制表达CLDN6的细胞的增殖；
(3)介导疾病或肿瘤的缓解；
(4)预防肿瘤形成或再形成；
(5)抑制表达CLDN6的细胞的转移；
(6)用于治疗/诊断疾病。
第十六方面，提供了一种治疗/诊断疾病的方法，其包括向有需要的受试者给予有效量的如第一方面至第四方面任一所述的抗体或抗原结合片段、或如第五方面所述的免疫缀合物、或如第九方面所述的细胞、或如第十方面所述的药物组合物、或如第十三方面所述的试剂盒。
在一实施方式中，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤。
在一实施方式中，所述受试者是人。
在一实施方式中，其中所述细胞对于所述受试者是自体的或同种异体的T细胞。
第十七方面，提供了如第一方面至第四方面任一所述的抗体或抗原结合片段、第五方面所述的免疫缀合物、第九方面所述的细胞、第十方面所述的药物组合物和/或第十三方面所述的试剂盒，其用于治疗/诊断疾病，所述疾病包含表达CLDN6。
在一实施方式中，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤。
在一实施方式中，所述肿瘤是实体瘤。
在一实施方式中，所述肿瘤是卵巢癌、乳腺癌、宫颈癌、胃癌、肺癌、睾丸癌、生殖细胞和胚胎肿瘤、卵巢上皮癌、非小细胞肺癌，非鳞状非小细胞肺癌、子宫内膜癌、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了293T-CLDN4、293T-CLDN6、293T-CLDN9稳转细胞系流式检测结果；
图2显示了抗体H1(scFv-huFc)特异性结合293T-CLDN6细胞；
图3显示了抗体H1与293T-CLDN6细胞结合的EC50；
图4显示了抗体P1、P2、P3和P4均特异性结合293T-CLDN6细胞；
图5显示了抗体P1、P2、P3和P4与293T-CLDN6细胞结合的EC50；
图6显示了抗体H1、M1和M2均特异性结合293T-CLDN6细胞；
图7显示了抗体M1和M2与293T-CLDN6细胞结合的EC50；
图8显示了H1-28Z CAR T细胞、P4-28Z CAR T细胞阳性率；
图9显示了H1-28Z CAR T细胞、P4-28Z CAR T细胞对靶细胞的体外特异性杀伤；
图10显示了H1-28Z CAR T细胞、P4-28Z CAR T细胞对靶细胞的体外杀伤；
图11显示了H1-28Z CAR T细胞、P4-28Z CAR T细胞对荷人卵巢癌细胞的NPG小鼠皮下移植瘤的抗肿瘤作用(图11A)；小鼠体重变化(图11B)；瘤重变化(图11C)；小鼠体内的外周血中人T细胞的存活情况(图11D)。
具体实施方式
本发明提供了特异性识别CLDN6的人源化抗体(包括其片段，例如重链可变区(VH)、轻链可变区(VL)、scFv)，含有此抗体的嵌合受体，编码此抗体的核酸，以及表达此抗体的细胞。本发明的抗体可以被应用于制备靶向抗肿瘤药物以及诊断肿瘤的药物。本发明还提供了制备和使用此抗体及表达此抗体的细胞的方法。
术语
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的相同含义。类似或等效于本文中描述的所有方法和材料都可以在本发明的实践或测试中使用，其中，本文描述的是合适的方法和材料。本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书，包括定义为准。此外，除非另有规定，材料、方法和实施例仅是说明性的，而并非旨在进行限制。根据本发明内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本发明的精神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本发明的各方面的举例说明)，并且功能等价的方法和组分在本发明的范围内。本发明包括对本发明的主题进行变型和修改来用于各种用途和条件。
除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围。这些技术充分解释于文献中。
公开内容中，请求保护的主题的各个方面均以范围形式呈现。应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述的或中间的值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。所述较小范围内可独立地包含这些较小范围的上下限，它们也属于请求保护的主题的范围，除非明确地排除所述范围的上下限。设定范围包含一个或两个限值时，请求保护的主题也包括排除所述限值之一个或两个的范围。这适用而无关范围的宽度。
术语“约”是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约” 值或参数，包括指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”。“约”或“包含”可意指该值的±1％、±2％、±3％、±4％、±5％、±6％、±7％、±8％、±9％、±10％、±11％、±12％、±13％、±14％、±15％、±16％、±17％、±18％、±19％、±20％、±25％、±30％的范围。可替代的，特别是关于生物系统或方法，该术语可指在数值的一个数量级内，例如在一个值的约5倍之内或在约2倍之内。
除非另外指出，本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值。
为了更易于理解本发明，首先定义一些术语。
术语“Claudin 6(CLDN6)”是CLDN家族的成员。编码人CLDN6蛋白的基因位于人16号染色体的p臂16p13.3处，并且在黑猩猩、恒河猴、狗、牛、小鼠、大鼠、斑马鱼和蛙中为保守的。CLDN 6多肽具有与由NCBI GenBank Gene ID：9074的基因表达的转录物编码的氨基酸序列具有至少约80％、至少约85％、至少约90％、至少约95％、至少约96％、至少约97％、至少约98％、至少约99％或100％同源性或同一性的氨基酸序列或其片段，和/或可任选地包括至多一个或至多两个或至多三个保守氨基酸取代。示例性，人CLDN6全长的氨基酸序列如SEQ ID NO:32所示。
术语“Claudin 9(CLDN9)”是CLDN6最密切相关的家族成员。编码人CLDN9蛋白的基因位于人16号染色体16p13.3处跨越约2.1kBp的单一外显子组成。示例性，人CLDN9全长的氨基酸序列如SEQ ID NO:33所示。
术语“Claudin 4(CLDN4)”也是CLDN家族的成员。编码人CLDN4蛋白的基因在染色体位置17q11.23处跨越约1.82kBp。示例性，人CLDN4全长的氨基酸序列如SEQ ID NO:31所示。
术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换使用，指任何长度的通过肽键共价连接的氨基酸残基组成的聚合物。蛋白质或肽必须含有至少两个氨基酸，对于氨基酸的最大数目没有限制。多肽包括含有彼此通过肽键连接的两个或两个以上氨基酸的任何肽或蛋白质。该术语既指短链(在本领域中通常称为肽、寡肽和寡聚物)，也指较长链(通常在本领域中成为蛋白质)，存在许多类型的蛋白质。多肽包括例如生物活性片段、基本同源多肽、寡肽、同二聚体、异二聚体、多肽变体、修饰的多肽、衍生物、类似物、融合蛋白等。多肽包括天然肽、重组肽或其组合。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。
术语“抗体”指特异性结合抗原的、源自免疫球蛋白分子的蛋白质或多肽序列，在本文中以最广义使用并且包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)、抗体片段、多链或单链的、或完整免疫球蛋白，并可以来源于天然来源或来自重组来源，只要其显示所需的抗原结合活性即可。
术语“抗原结合片段”指抗体和抗体的免疫学活性部分，即，含有与抗原特异性结合(与抗原有免疫反应)的抗原结合位点的分子。
本发明人源化抗体H1、P1、P2、P3、P4、M1、M2可以特异性结合CLDN6，不结合CLDN4和CLDN9。在具体的实施方式中，本发明抗体H1、P1、P2、P3、P4、M1、M2抗原结合特异性优于杂交瘤抗体SC27.105；进一步地，本发明抗体H1、P1、P2、P3、P4、M1、M2抗原结合特异性优于对照抗体C46-S(来自专利WO2015150327A1)。
术语“特异性结合”、“特异性识别”或“对……具有特异性”是指可测定的和可再现的相互作用，例如靶标和抗体的结合，在存在其它分子的情况下，所述结合是靶标存在的决定性因素。例如，特异性结合靶标的抗体，是与其它靶标结合相比，与该靶标的结合具有更大的亲和力，更容易和/或具有更长的持续时间的抗体。在一些实施方式中，抗体或配体识别和结合样品中存在的关联结合配偶体蛋白，但基本上不识别或结合样品中的其它分子。在一些实施方式中，特异性结合可包括但不需要排他性结合。“多特异性”是指抗体具有对抗原至少两个不同位点具有结合特异性。
“抗体片段”是指完整抗体的至少一部分或其重组变体，并可以指完整抗体的抗原结合结构域，例如抗原性决定可变区，其足以赋予抗体片段对靶标例如抗原的识别和特异性结合。抗体片段的实例包括但不限于(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点；(vii)双特异性单链Fv二聚体；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段；(ix)与相同或不同抗体遗传融合的scFv；(x)线性抗体；(xi)驼类VHH结构域；和(xii)单结构域抗体如sdAb(VH或VL)。可通过各种技术制备抗体片段，包括但不限于对完整抗体的蛋白酶水解消化以及通过重组宿主细胞产生。
术语“scFv”指，包含至少一个含有轻链可变区的抗体片段和至少一个含有重链可变区的抗体片段的融合蛋白，其中轻链可变区和重链可变区通过短的柔性肽接头连接，能够表达为单链多肽，并且其中scFv保持其所源自的完整抗体的特异性。本文中scFv可以以任何一种顺序具有VL和VH，例如项对于多肽的N端和C端，scFv可以包含VL-接头-VH，还可以包含VH-接头-VL。所述接头可以是任何长度的任何氨基酸。
术语“接头”或“柔性多肽接头”指由氨基酸(例如甘氨酸和/或丝氨酸)残基组成的肽接头，其单独或组合使用，以将重链可变区和轻链可变区连接在一起。在一实施方案中，柔性多肽接头是Gly/Ser接头，包括氨基酸序列(Gly-Gly-Gly-Gly-Ser)n，其中n是等于或大于1的整数。例如，n＝1，n＝2，n＝3，n＝4，n＝5，n＝6，n＝7，n＝8，n＝9，n＝10。在一实施方案 中，柔性多肽接头包括但不限于，(Gly4Ser)4或(Gly4Ser)3。在一实施方案中，接头包括(Gly2Ser)、(GlySer)、或(Gly3Ser)的多个重复。在一实施方案中，所述接头还包括带电残基，例如赖氨酸和/或谷氨酸，其可提高可溶性。在一实施方案中，所述接头还包括一个或多个脯氨酸。
术语“抗体重链”指，天然构象的抗体分子中存在的两类多肽链中的较大者，其通常决定抗体的类别。
术语“抗体轻链”指，天然构象的抗体分子中存在的两类多肽链中的较小者。К和λ轻链指两种主要的抗体轻链同种型。
抗体的“分类”是指其重链所具有的恒定结构域或恒定区的类型。主要有五类抗体：IgA、IgD、IgE、IgG和IgM，并且这些中的一些可以被进一步划分成亚类(同种异型)，例如，IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。对应于不同的类型的免疫球蛋白的重链恒定结构域分别被称为α，δ，ε，γ，和μ。
术语“可变区或可变结构域”是指参与抗体抗原结合的抗体重链或轻链的结构域。天然抗体的重链和轻链可变结构域(分别为VH和VL)通常具有相似的结构，其中各结构域包含四个保守的FR和三个CDR。。单个VH或VL结构域可足以给予抗原结合特异性。此外，结合特定抗原的抗体可以分别使用来自于所述抗原结合的抗体的VH或VL结构域筛选互补VL或VH结构域的文库来分离。。
术语“高变区”或“互补决定区”或“CDR”是指抗体可变结构域中序列高变和/或形成结构确定的环(“高变环”)和/或含有与抗原接触的残基(“抗原触点”)的各区域，抗体可变区内非连续的氨基酸序列，其赋予抗体特异性和/或结合亲和性。在某些实施方式中，CDR可通过选自如下的编号系统确定：Kabat、Chothia、IMGT、Gelfand、Aho和AbM。例如，可通过Kabat编号系统确定。例如，抗体可以包含六个CDR：VH中的三个(HCDR1，HCDR2，HCDR3)和VL中的三个(LCDR1，LCDR2，LCDR3)。
术语“Fc区”或“Fc”被用于限定含有恒定区的至少一部分的免疫球蛋白重链的C-端区域。该术语包括天然序列Fc区和变体Fc区。
“框架(FR)”是指不同于高变区(CDR)残基的可变结构域残基。可变结构域的FR通常由四个FR结构域组成：FR1，FR2，FR3和FR4。因此，在VH(或VL)中CDR和FR序列通常按以下顺序出现：
FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
除非另外指出，在本文中，CDR残基和可变结构域中的其他残基(例如，FR残基)根据以上Kabat等编号。
术语“天然抗体”是指天然存在的具有多种结构的免疫球蛋白分子。例如，天然IgG抗体是约150,000道尔顿的异源四聚糖蛋白，由通过二硫键键合的两个相同的轻链和两个相同的重链组成。从N端到C端，各重链具有可变区(VH)，其也被称为可变重链结构域或重链可变结构域，之后是三个恒定结构域(CH1，CH2和CH3)。类似地，从N端到C端，各轻链具有可变区(VL)，其也被称为可变轻链结构域或轻链可变结构域，之后是轻链恒定(CL)结构 域。抗体的轻链基于其恒定结构域的氨基酸序列可以被分配至两种类型之一，被称为κ(κ)和λ(λ)。
术语“全抗”、“全长抗体”、“完整抗体”可交换使用，是指具有与天然抗体结构基本类似的结构或具有含有如本文中所限定的Fc区的重链或包括具有抗原结合区域的完整的全长抗体。
术语“单域抗体(Single domain antibody,sdAb)”是指缺失抗体轻链而只有重链可变区的一类抗体，因其分子量小，也被称为纳米抗体(Nanobody)。
术语“单结构域抗体”是指包含抗体的全部或部分的重链可变结构域或全部或部分的轻链可变结构域。在某些实施方案中，单结构域抗体是人单结构域抗体。
术语“单克隆抗体”、“单抗”是指获自基本上同源的抗体的群体的抗体，即，包括所述群体的个体抗体是相同的和/或结合相同的表位，除可能的变体抗体以外，例如，含天然存在的突变或在单克隆抗体制剂的制备过程中产生，此种变体通常少量存在。对比于多克隆抗体制剂(通常包括针对不同的决定簇(表位)的不同抗体)，单克隆抗体制剂中的各单克隆抗体是针对抗原上的单个决定簇。因此，定于“单克隆”指示抗体的性质为获得自基本上同源的抗体群体，并且不视为要求通过任何特定的方法制备所述抗体。例如，可以通过多种技术制备，包括但不限于杂交瘤方法、重组DNA法、噬菌体展示法，以及利用含有所有或部分的人免疫球蛋白基因座的转基因动物的方法。
术语“嵌合抗体”是指抗体重链和/或轻链的一部分来源于特定来源或物种，而重链和/或轻链的剩余部分来源于不同的来源或物种的抗体。在某些实施方案中，嵌合抗体包含非人可变区(例如，来源于小鼠、大鼠、仓鼠、兔或非人灵长类动物如猴的可变区)和人恒定区。在另外的实施方案中，嵌合抗体是“类型转换”抗体，其中类型或亚类已经由亲本抗体的类型或亚类改变。嵌合抗体包括其抗原结合片段。在某些实施方案中，嵌合抗体是“人源化抗体”。
术语“人源化”用于非人抗体，例如啮齿动物或灵长类动物等，是含有来源于非人免疫球蛋白的最小序列的杂合免疫球蛋白，免疫球蛋白链或其片段。“人源化抗体”是指一种嵌合抗体，其包含来自非人CDR的氨基酸残基和来自人FR的氨基酸残基。在某些实施例中，人源化抗体将包含基本上全部的至少一个(一般为两个)可变结构域，其中所有或基本上所有的CDR对应于非人抗体的CDR，并且所有或基本上所有的FR对应于人抗体的FR。人源化抗体任选地可以包含来源于人抗体的抗体恒定区的至少一部分。本领域熟知的多种用于将抗体或抗体片段人源化的技术，基本是用啮齿类动物CDR或CDR序列替代相应的人抗体序列，即CDR嫁接。人源化抗体常是人抗体，其中一些CDR残基和可能的一些FR残基被来自啮齿动物抗体中类似位点的残基替代。
在一些实施方案中，“人源化抗体”可包括突变，例如通过体外随机或定点诱变或通过体内体细胞突变引入的突变。在一些实施方案中，可以选择待用于制备人源化抗体的人结构域，以降低抗原性。根据所谓的“最佳匹配“(best-fit)方法，相对于已知人可变结构域序列的完整文库，筛选啮齿类抗体的可变结构域序列。然后，解决啮齿类的人序列可以被接受为人 FR用于人源化抗体。在一些实施方案中，使用源自共有序列的特定FR，其中所述共有序列为具有特定亚组的轻链或重链的所有人抗体的共有序列。同一FR可以用于几种不同的人源化抗体。在一些实施方式中，人源化抗体的一些FR残基被来自非人抗体(例如，CDR残基所衍生自的抗体)的相应残基取代，例如，为了恢复或改善抗体特异性或亲和性。或是指将非人动物抗体的VH和VL的CDR的氨基酸序列移植至人抗体的VH和VL的对应的CDR处而得到的抗体。VH和VL的CDR以外的区域被称为框架区(以下记为FR)；在一实例中，构建编码由非人动物抗体的VH的CDR的氨基酸序列和任意的人抗体的VH的FR的氨基酸序列构成的VH的氨基酸序列的cDNA、以及编码由非人动物抗体的VL的CDR的氨基酸序列和任意的人抗体的VL的FR的氨基酸序列构成的VL的氨基酸序列的cDNA，构建人源化抗体表达载体，导入至动物或真菌，细菌细胞中，由此使其进行表达。
鼠源的杂交瘤抗体由于恒定区可被人体免疫系统识别而产生免疫副反应，诱发人抗鼠抗体反应(HAMA)。通过人源化改造，可消除或降低鼠源抗体免疫原性。人源化抗体具有更低的免疫原性，更接近人体内天然的抗体，可以提高药效和安全性。一实例中,与具有相同特异性的非人源化抗体(例如,人源化之前的小鼠抗体前体)相比,本公开的人源化抗体在人类受试者中具有降低的免疫原性。本发明将杂交瘤抗体SC27.105(来自WO2016073649A1)进行人源化改造。一实例中，本申请人源化抗体H1、P1、P2、P3、P4、M1、M2等，在人类受试者中具有低免疫原性，具体地，本申请抗体H1、P1、P2、P3、P4、M1、M2低于杂交瘤抗体SC27.105。低或降低的免疫原性的特征在于能够以可测量的症状缓解和低和/或可接受的毒性长期治疗患者。低或可接受的免疫原性和/或高亲和力,以及其他合适的特性,可有助于实现治疗结果。“低或降低的免疫原性”在本文中定义为HAHA、HACA或HAMA反应的比例低于90％,例如低于80％、低于70％、低于60％、低于50％、低于40％、低于30％、低于20％、低于10％的比例的患者。
术语“重组抗体”指，使用重组DNA技术产生的抗体，例如，通过噬菌体或酵母表达系统或哺乳动物细胞表达系统表达的抗体或抗体片段。该术语也应理解为指，通过如下方式产生的抗体：合成编码该抗体的DNA分子和该DNA分子表达抗体蛋白；或合成该抗体的氨基酸序列，其中该DNA或氨基酸序列通过使用本领域可得和熟知的重组DNA或氨基酸序列技术而获得。
本发明的抗体可以通过筛选具有所需一种或多种活性的抗体的组合文库来分离。例如，本领域中已知多种方法用于生成噬菌体展示文库并且针对具有所需结合性质的抗体筛选所述文库。此种方法被综述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，Human Press,Totowa,NJ,2001)中并被进一步描述于例如McCafferty等，Nature 348：552-554；Clackson等，Nature 352：624-628(1991)；Marks等，J.Mol.Biol.222:581-597(1992)；Marks,Meth.Mol.Biol.,248:161-175(Lo,ed.,Human Press,Totowa,NJ,2003)；Sidhu等,J.Mol.Biol.338(2):299-310(2004)；Lee等，J.Mol.Biol.340(5):1073-1093(2004)；Fellouse,Proc.Natl.Acad.Sci.USA101(34):12467-12472(2004)；Lee等，J.Immunol.Methods 284(1-2):119-132(2004)中。
在某些噬菌体展示方法中，VH和VL基因库通过聚合酶链式反应(PCR)分别克隆并且在噬菌体文库中随机重组，然后针对结合抗原的噬菌体筛选所述文库，如Winter等，Ann.Rev.Immunol.12:433-455(1994)中所述。噬菌体典型地将抗体片段展示为单链Fv(scFv)片段或Fab片段。来自被免疫的来源的文库向免疫原提供高亲和力抗体而不需要构建杂交瘤。备选地，可以克隆天然库(例如，由人)从而向多种非自身抗原以及自身抗原提供单一来源的抗体而不需要进行任何免疫，如Griffiths等，EMBO J,12:725-734(1993)所述。最后，也可以通过以下方式合成制备天然文库：自干细胞克隆未重排的V-基因区段，并且使用含随机序列的PCR引物以编码高变CDR3区并且在体外实现重排，如Hoogenboom，J.Mol.Biol.227:381-388(1992)所述。在某些实施方案中，本文中提供抗体的氨基酸序列变体。术语“亲本抗体”指本申请所提供的抗体或依据本申请所提供的抗体进行突变、或亲和力成熟等处理后所得的抗体。所述亲本抗体可以是天然存在的抗体，或者天然存在的抗体的变体或改造版本。亲本抗体可以指抗体本身，包含所述亲本抗体的组合物，或其编码氨基酸序列。
术语“亲和力成熟”抗体是指相比于亲本抗体在一个或多个高变区(CDR)中具有一个或多个改变的抗体，此种改变导致抗体对抗原的亲和力提高。
术语“变体”指与本申请所提供的抗体的序列具有基本上相同氨基酸序列或由基本上相同的核苷酸序列编码的一种或多种活性的多肽。所述变体与本申请实施例中所提供的抗体具有相同或相似的活性。例如，变体可以是基于本申请提供的抗体的氨基酸序列的变体抗体或抗体变体。
术语“变体抗体”或“抗体变体”包括由于相比亲本的至少一个氨基酸修饰，而不同于亲本抗体序列的抗体序列。本文中的变体抗体序列优选的具有与亲本抗体序列至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％％、91％、92％、93％、94％、95％％、96％、97％、98％、99％的氨基酸序列同一性。抗体变体可以指抗体本身，也可以指包含所述抗体变体的组合物。抗体的氨基酸序列变体可以通过向编码所述抗体的核苷酸序列引入合适的修饰或通过肽合成来制备。术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”、“氨基酸置换”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸，“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸，“氨基酸缺失”意指去除亲本多肽序列中特定位置上的氨基酸。可以进行缺失、插入和取代的任意组合以获得最终的构建体，前提是最终的构建体具有所需的特征，例如：结合抗原。
术语“修饰”一词是指本发明的蛋白或多肽的状态或结构的改变。修饰的方式可以是化学的、结构的和功能上的。
术语“保守修饰”或“保守序列修饰”意指不显著影响或改变含有所述氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。此类保守修饰包括氨基酸替代、插入和缺失。可通过本领域已知的标准技术将修饰导入本发明的抗体或抗体片段中，例如定点诱变、PCR介导的诱变。保守氨基酸替代是氨基酸残基被具有相似侧链的另一氨基酸残基替代。本领域已经定义了具有相似侧链的氨基酸残基家族，如表1所示。
表1：具有相似侧链的氨基酸残基家族
因而，可以用其他相同侧链家族的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基，并可以测试所改变的抗体(变体抗体)保留的功能。
非保守置换需要将这些组之一的成员换成另一组的成员。
一种置换变体包括置换亲本抗体(例如，人源化或人抗体)的一个或多个高变区或FR残基。通常，被选择用于进一步研究的所得的变体相对于亲本抗体将具有某些生物学性质(例如，增加的亲和力、减小的免疫原性)的改变(例如，提高)和/或将基本上保持亲本抗体的某些生物学性质。示例性替换变体是亲和力成熟抗体，其可以例如，使用基于噬菌体展示的亲和力成熟技术(如本文中描述的那些)常规制备。简言之，将一个或多个CDR或FR残基突变并将变体抗体展示在噬菌体上并且筛选特定的生物学活性(例如，结合亲和力)。
改变(例如，置换)可以在CDR区进行，例如，以提高抗体亲和力。此种改变可以在CDR“热点”中进行，所述“热点”即在体细胞成熟过程期间以高频率进行突变的密码子编码的残基(参见，例如，Chowdhury,Methods Mol.Biol.207:179-196(2008)，和/或解除抗原的残基，测试所得的变体VH或VL的结合亲和力。通过构建二级文库并自其进行再选择的亲和力成熟已被描述于例如Hoogenboom等，Methods in Molecular Biology 178:1-37(0’Brien等，ed.,Human press,Totowa,NJ,(2001))中。在亲和力成熟的一些实施方案中，通过多种方法中任一种(例如，易错PCR、链改组或寡核苷酸定向诱变)将多样性引入选择用于成熟的可变基因种。然后产生二级文库。然后筛选所述文库以鉴定任何具有所需亲和力的抗体变体。引入多样性的另一种方法包括CDR定向方法，其中若干CDR残基(例如，同时4-6个残基)被随机化。
在某些实施方案中，置换、插入或缺失可以发生在一个或多个CDR内，只要这样的改变不显著减小抗体结合抗原的能力。例如，不显著减小结合亲和力的保守改变(例如，本文中所述的保守修饰)可以在CDR中进行。此种改变可以例如在CDR中接触抗原的残基的外部。在上述提供的变体VH和VL序列的某些实施方案中，各CDR是未改变的，或含不超过一个、两个或三个氨基酸置换。
在某些实施方案中，氨基酸序列插入包括氨基和/或羧基末端融合，长度范围从一个残基到含一百或更多残基的多肽，以及序列内插入单个或多个氨基酸残基。末端插入的实例包括具有N末端甲硫氨酰基残基的抗体。所述抗体分子的其它插入变体包括，将所述抗体的N末端或C末端融合至酶或多肽，这增加所述抗体的血清半衰期。
术语“抗CLDN6抗体”、“结合CLDN6的抗体”、“CLDN6抗体”、“识别CLDN6的抗体”是指能够以足够的亲和力结合CLDN6的抗体，所述抗体可用于靶向CLDN6的诊断剂和/或治疗剂。在一个实施方案中，抗CLDN6抗体与不相关的、非CLDN6蛋白(例如CLDN4、CLDN9)的结合程度小于所述抗体与CLDN6的约20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％，如通过酶联免疫吸附实验(ELISA)测定。在一个实施方案中，抗CLDN6抗体与CLDN4、或与CLDN9的结合程度，与阴性对照组检测的结合水平相当。在某些实施方案中，抗CLDN6抗体结合CLDN6的表位，所述表位在来源于不同物种的CLDN6之间是保守的。
术语“嵌合T细胞受体”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。嵌合T细胞受体由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该嵌合T细胞受体整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：(1)抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；(2)胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；(3)跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。
术语“跨膜结构域”与胞外抗原结合结构域连接。跨膜结构域可以来源于天然或合成的。当跨膜结构域是天然来源时，其可以源自任何膜结合蛋白或跨膜蛋白。在一方面，每当CAR与靶标结合，跨膜结构域能够将信号传导至胞内结构域。跨膜结构域包括：T细胞受体的α、β、γ、δ或ζ链；CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154；IL2受体p55(α链)、p75(β链)或γ链；FcγRIII。当跨膜结构域是合成来源时，可以包含疏水残基例如亮氨酸和缬氨酸。一方面，在合成的跨膜结构域的两端可以存在苯丙氨酸、色氨酸和缬氨酸三联体。
一些情况下，跨膜结构域可以包括与该跨膜区相邻的一个或多个额外的氨基酸，例如与该跨膜结构域所源自的蛋白质的胞外区相关的一个或多个氨基酸，和/或与该跨膜结构域所源自的蛋白质的胞内区相关的一个或多个氨基酸。一些情况下，跨膜结构域可以通过“铰链区”与CAR的胞外区连接。使用“铰链区”向细胞外抗原结合结构域提供更多的柔性和可接近性。铰链区可以包含至多达300个氨基酸，优选10-100个氨基酸，最优选 25至50个氨基酸。铰链区可以来源于天然存在的分子如CD8、CD4、CD28、FcγRIII或IgG1、IgG4的所有或部分的细胞外区域，或来自抗体恒定区的所有或部分。铰链区还可以是合成的序列。
术语“胞内信号区”或“胞内信号传导结构域”指CAR分子的胞内部分，负责激活已经引入了CAR的免疫细胞的至少一种正常效应子功能。胞内信号传导结构域产生可以促进含CAR的细胞(例如CAR-T细胞)的细胞裂解活性和辅助活性，包括分泌细胞因子。“信号传导结构域”指转导效应子信号功能信号并引导细胞执行特异性功能的蛋白质部分。用于CAR的信号传导结构域可以是T细胞受体的细胞质序列，和在抗原受体接合之后协同引发信号转导的共受体的细胞质序列，和这些序列的任意衍生物或变体，以及具有相同功能的任意合成序列。
胞内信号传导结构域可以包括刺激分子(也可称作刺激性分子、初级信号分子)和/或共刺激性分子的功能信号传导结构域。在一实施方案中，胞内信号传导结构域可以包含初级信号分子，示例性，初级信号分子源自负责第一刺激或抗原依赖性刺激的分子的那些结构域。在一实施方案中，通过TCR单独产生的信号不足以完全激活T细胞，还需要第二和/或共刺激信号。胞内信号传导结构域可以包含共刺激胞内信号传导结构域，示例性，共刺激胞内信号传导结构域包括源自负责共刺激信号或抗原非依赖性刺激的分子的那些结构域。胞内信号传导结构域可以包含其所源自的分子的整个胞内部分、或该分子的整个天然胞内信号传导结构域、或其功能片段。例如，刺激性分子可以是与T细胞受体复合体结合的ζ链；例如，细胞质信号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28的胞内序列。
在本发明中，一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自刺激分子的功能信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域、和胞内信号传导结构域，所述胞内信号传导结构域含有源自共刺激分子的功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR包含嵌合融合蛋白，所述蛋白包含胞外抗原识别结构域、跨膜结构域和胞内传导结构域，所述胞内信号传导结构域包含源自一个或多个共刺激分子的至少两个功能性信号传导结构域和源自刺激分子的功能性信号传导结构域。一方面，CAR在CAR融合蛋白的氨基酸(ND端)包含可选的“前导序列”。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)切下。
术语“前导序列”(leader sequence)(也称为信号肽)可操作地连接在CAR核酸序列上，并定位为使得新合成的多肽引导到细胞的分泌途径中。通常将前导序列定位在编码多肽的核酸序列的5’端。信号肽包括CAR天然存在的蛋白质的信号序列或合成的非天然存在的信号序列。在一些实施方案中，信号肽选自CD8α、GM-CSF受体α和IgG1重 链。
术语“初级信号分子”或“刺激分子”以刺激性方式调节TCR复合物的初始活化。通常，初级信号由例如TCR/CD3复合物与加载了肽的MHC分子的结合而引发，由此介导T细胞反应(包括但不限于，增殖、活化、分化等)。以刺激性方式起作用的初级信号分子可以包含免疫受体酪氨酸激活基序或ITAM的信号传导基序。在本发明中尤其有用的包含ITAM的初级信号分子的功能信号传导结构域(初级信号域)的例子包括但不限于，源自CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)、CD66d、DAP10和DAP12的序列，在本发明CAR中，在任何一个或多个本发明CAR中胞内信号传导结构域包含胞内信号传导序列，例如CD3ζ的初级信号域。
术语“共刺激信号域”或“共刺激分子”通常是指能够与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号的共刺激分子的胞内结构域。共刺激分子通常为T细胞上的关连结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，例如，包括但不限于增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激胞内信号传导结构域可以源自共刺激分子的胞内部分。共刺激分子可以出现在以下蛋白质家族中：TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)和激活性NK细胞受体。共刺激分子包括但不限于，MHC I类分子、BTLA和Toll配体受体、以及OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、4-1BB(CD137)、GITR、CD30、CD40、ICOS、BAFFR、HVEM、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160、B7-H3、和特异结合CD83的配体等。
术语“CD3ζ(也称为CD3Zeta)”或“TCRζ”或“ζ链”定义为GenBan登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3ζ结构域”定义为来自ξ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。CD3ζ的胞质结构域包含GenBan登录号BAG36664.1的残基52至164、其功能性直向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。示例性，CD3ζ的氨基酸序列如SEQ ID NO：24所示。
术语“4-1BB(也称CD137)”指TNFR超家族的成员，其具有GenBank Acc.No.AAA62478.2的氨基酸序列、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“4-1BB共刺激结构域”定义为GenBank Acc.No.AAA62478.2的氨基酸残基214-255，或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。示例性，4-1BB共刺激结构域的氨基酸序列如SEQ ID NO：26所示。
术语“趋化因子”是一种分子量为8～10kDa的多肽，是最大的细胞因子家族，主要功能是招募血液中的单核细胞、中性粒细胞、淋巴细胞等进入特定的淋巴器官和组织以及感染发生的部位。
术语“趋化因子受体”是一类介导趋化因子行使功能的七次跨膜G蛋白偶联受体 (GPCR)，通常表达于免疫细胞、中性粒细胞、内皮细胞等细胞膜上。趋化因子及趋化因子受体在介导细胞迁移、增殖和抵御病原体入侵过程中发挥重要作用，并且与免疫环境中炎症和癌症的发生发展密切相关。
术语“安全开关”是为了提高CAR-T疗法的安全性，为CAR-T细胞设计一个快速且可逆的“关闭”或“开启”安全开关，最大限度地减少与治疗相关的毒性。尽管CAR-T细胞疗法具有出色的临床特征，但当肿瘤负荷不可预测且T细胞的活性不受控制时，引发严重的CRS等潜在致命副作用。为了控制毒性，严重的CRS需要使用CRS分级系统作为指导进行适当监测，并使用基于小分子的安全开关进行精确调节。
发明详述
抗体
在本文中，描述了利用本领域常规人源化抗体制备技术获得了人源化抗CLDN6特异性抗体，并通过CDR区随机化突变技术和噬菌体筛选技术获得人源化抗CLDN6特异性抗体的突变体。这些分子展示了精细的特异性。例如，该抗体仅识别CLDN6，及表达CLDN6的293T细胞、OVCAR3细胞和OV90细胞，不识别表达CLDN4、CLDN9或其组合的细胞。本发明中如果没有特别说明，本文CLDN6指人的CLDN6。
在一些实施方案中，本发明包括具有scFv序列的抗体，所述scFv序列与一个或多个重链恒定区域融合以形成具有人免疫球蛋白Fc区的抗体以产生双价蛋白，从而增加抗体的总体亲和力和稳定性。此外，Fc部分允许将其他分子(包括但不限于荧光染料、细胞毒素、放射性同位素等)与例如用于抗原定量研究中的抗体直接缀合，以便固定抗体用于亲和力测量、用于定向递送治疗药、使用免疫效应细胞测试Fc介导的细胞毒性和许多其它应用。
本文提供的结果突出本发明抗体在靶向CLDN6时的特异性、灵敏性和效用。
本发明的抗体或抗体片段基于使用人源化抗体制备技术和噬菌体筛选技术获得单链抗体片段(scFv)，所述scFv的氨基酸序列赋予抗体或抗体片段针对CLDN6的特异性并且形成本公开的全部抗体的基础。因此，所述scFv可以用来设计一系列不同“抗体或抗体片段”，包括例如全长抗体、其片段如F(ab’)2、融合蛋白、多价抗体，即，具有针对相同抗原或不同抗原的多于一种特异性的抗体，例如，双特异性T细胞结合抗体(BiTE)、三抗体等(见Cuesta等人，Multivalent antibodies:when design surpasses evolution,Trends in Biotechnology 28:355-362,2010)。
在具体实施例中，本发明提供全长抗体，其重链和轻链可以是全长(例如，抗体可以包括至少一条，优选地两条，完整重链，和至少一条，优选地两条，完整轻链)或可以包括抗原结合部分(Fab、F(ab’)2、Fv或scFv)。在其他实施方案中，抗体重链恒定区选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE。抗体类型的选择将取决于所设计的抗体欲引发的免疫效应子功能。在构建重组免疫球蛋白时，各种免疫球蛋白同种型的恒定区的适宜氨基酸序列和用于产生广泛种类抗体的方法是本领域技术人员已知的。
本发明提供了识别CLDN6的抗体或抗原结合片段，包含重链可变区，所述重链可变区包含GYYMN(SEQ ID NO：35)所示的HCDR1；和/或EINPATGSTTYNQKFKA(SEQ ID  NO：36)所示的HCDR2；和/或RDYYX1GSX2X3YAX4DY(SEQ ID NO:52)所示的HCDR3，其中X1是Y或L，X2是G或N，X3是F或S，X4是M或L；和/或所述抗体包含轻链可变区，所述轻链可变区包含QASQSVSNNLN(SEQ ID NO：38)所示的LCDR1；和/或GASKLED(SEQ ID NO：39)所示的LCDR2；和/或X5QHRX6X7WT(SEQ ID NO:53)所示的LCDR3，其中X5是L或Q，X6是Y或F，X7是L或M。
在某些实施方式中，所述抗体或抗原结合片段可以包含重链可变区中的至少一个CDR；和/或所述抗体或抗原结合片段可以包含轻链可变区中的至少一个CDR。在某些实施方式中，所述抗体或抗原结合片段可以包含重链可变区中的一个CDR、两个CDR或三个CDR。在某些实施方式中，所述抗体或抗原结合片段可以包含轻链可变区中的一个CDR、两个CDR或三个CDR。
在某些实施方式中，所述抗体可以包含重链可变区中的一个CDR、两个CDR或三个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR。例如，可以包含重链可变区中的一个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR；例如，可以包含重链可变区中的两个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR；例如，可以包含重链可变区中的三个CDR，还包含轻链可变区中的一个CDR、两个CDR或三个CDR；例如，可以包含重链可变区中的三个CDR，还包含轻链可变区中的三个CDR。
因此，本发明的抗体或抗原结合片段包含重链可变区，所述重链可变区的HCDR3与SEQ ID NO：41所示的HCDR3具有至少95％、96％、97％、98％或99％的氨基酸序列同一性；和/或
本发明的抗体或抗原结合片段包含轻链可变区，所述轻链可变区的LCDR3与SEQ ID NO：42、43或44所示的LCDR3具有至少60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性。
在某些实施方式中，所述抗体可以包含所述重链可变区和/或所述轻链可变区。例如，所述重链可变区可以包含重链CDR1(HCDR1)，所述HCDR1可以包含如SEQ ID NO:35所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR2(HCDR2)，所述HCDR2可以包含如SEQ ID NO:36所示的氨基酸序列。例如，所述重链可变区可以包含重链CDR3(HCDR3)，所述HCDR3可以包含如SEQ ID NO:37或41任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR3，所述HCDR1可以包含如SEQ ID NO:35所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:37或41任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1和HCDR2，所述HCDR1可以包含如SEQ ID NO:35所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:36所示的氨基酸序列。例如，所述重链可变区可以包含HCDR2和HCDR3，所述HCDR2可以包含如SEQ ID NO:36所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:37或41任一所示的氨基酸序列。例如，所述重链可变区可以包含HCDR1，HCDR2和HCDR3，所述HCDR1可以包含如SEQ ID NO:35所示的氨基酸序列，所述HCDR2可以包含如SEQ ID NO:36所示的氨基酸序列，所述HCDR3可以包含如SEQ ID NO:37或41任一所示的氨基酸序列。例如，所述轻链可变区可以包含 轻链CDR1(LCDR1)，所述LCDR1可以包含如SEQ ID NO:38所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR2(LCDR2)，所述LCDR2可以包含如SEQ ID NO:39所示的氨基酸序列。例如，所述轻链可变区可以包含轻链CDR3(LCDR3)，所述LCDR3可以包含如SEQ ID NO:40、42、43或44任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR3，所述LCDR1可以包含如SEQ ID NO:38所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:40、42、43或44任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1和LCDR2，所述LCDR1可以包含如SEQ ID NO:38所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:39所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR2和LCDR3，所述LCDR2可以包含如SEQ ID NO:39所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO:40、42、43或44任一所示的氨基酸序列。例如，所述轻链可变区可以包含LCDR1、LCDR2和LCDR3，所述LCDR1可以包含如SEQ ID NO:38所示的氨基酸序列，所述LCDR2可以包含如SEQ ID NO:39所示的氨基酸序列，所述LCDR3可以包含如SEQ ID NO40、42、43或44任一所示的氨基酸序列。
在某些实施方式中，所述抗体可以包含所述重链可变区和所述轻链可变区。例如，所述抗体，其包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：40所示的LCDR3；或者所述抗体，其包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：40所示的LCDR3；或者所述抗体，其包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：42所示的LCDR3；或者所述抗体，其包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：43所示的LCDR3；或者所述抗体，其包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：42所示的LCDR3；或者所述抗体，其包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：43所示的LCDR3；或者所述抗体，其包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：44所示的LCDR3。在具体的实施方式中，本发明的抗体或抗原结合片段的HCDR2具有SEQ ID NO:36所示的氨基酸序列。
其中，所述CDR可通过选自如下的编号系统确定：Kabat、Chothia、IMGT、Gelfand、Aho和AbM。本发明所提供的识别CLDN6的抗体可以包含通过上述任意编号系统确定的某个CDR序列或其组合，组成抗体的重链和/或轻链中的CDR不必是通过相同的编号确定的， 例如，抗体中的一个或一些CDR可通过Kabat系统确定，其他CDR可通过上述编号系统的任意一种或其组合确定。在某些实施方式中，所述抗体的CDR可以是通过一种编号系统确定的，例如，可通过Kabat编号系统确定；例如，可通过Chothia编号系统确定；例如，可通过IMGT编号系统确定；例如，可通过Gelfand编号系统确定；例如，可通过Aho编号系统确定；例如，可通过AbM编号系统确定。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:1或5中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述轻链可变区包含如SEQ ID NO:3、7、9或11中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:1或5中任一项所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:3、7、9或11中任一项所示的氨基酸序列或其变体。例如，所述重链可变区包含如SEQ ID NO:1所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:3、7、9或11中任一项所示的氨基酸序列或其变体；例如，所述重链可变区包含如SEQ ID NO:5所示的氨基酸序列或其变体，且所述轻链可变区包含如SEQ ID NO:3、7、9或11中任一项所示的氨基酸序列或其变体。
在某些实施方式中，所述重链可变区包含如SEQ ID NO:1所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:3所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:1所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:7所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:1所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:9所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:5所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:3所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:5所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:7所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:5所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:9所示的氨基酸序列。例如，所述重链可变区包含如SEQ ID NO:1所示的氨基酸序列，且所述轻链可变区包含如SEQ ID NO:11所示的氨基酸序列
在某些实施方式中，本发明提供了识别CLDN6的抗体，包含重链可变区，所述重链可变区包含SEQ ID NO:1或5任一所示的氨基酸序列、或上述序列的变体。
在某些实施方式中，本发明提供了识别CLDN6的抗体，包含轻链可变区，该轻链可变区包含SEQ ID NO:3、7、9或11任一所示的氨基酸序列、或上述序列的变体。
在某些实施方式中，本发明提供了识别CLDN6的抗体，包含上述重链可变区及轻链可变区的抗体。
考虑到这些重链可变区和轻链可变区序列各自可以结合CLDN6，可以“混合和匹配”重链和轻链可变区序列来产生本发明的抗CLDN6的结合分子。
在某些实施方式中，本发明提供了结合CLDN6的抗体的变体或其片段的变体。这样的变体包含重链可变区，所述重链可变区的HCDR1、HCDR2和HCDR3与SEQ ID NO：35、36和37所示的HCDR1、HCDR2和HCDR3具有至少85％、90％、91％、92％、93％、94％、 95％、96％、97％、98％或99％的氨基酸序列同一性。这样的变体还可以包含轻链可变区，所述轻链可变区的LCDR1、LCDR2和LCDR3与SEQ ID NO：38、39和40所示的LCDR1、LCDR2和LCDR3具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性。
进一步地，本发明的抗体的变体或其片段的变体包含与本发明抗体的重链或轻链可变区序列具有至少80％相同的重链和/或轻链可变区。优选的，重链和/或轻链可变区的氨基酸序列同一性是至少80％，优选至少85％，更优选至少90％，最优选至少95％，特别是96％，更特别97％，甚至更特别98％，最特别99％，包括例如80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％和100％。变体可以以本申请所述的抗体为母本抗体，通过酵母库筛选、噬菌体库筛选、点突变等方法得到。
在某些实施方式中，本发明提供了特异性结合CLDN6的抗体，所述抗体是全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段或多功能抗体。
在某些实施方式中，所述抗体是杂交瘤抗体、嵌合抗体、人源化抗体或全人源抗体。
在某些实施方式中，所述抗体是单克隆抗体。
抗体测定
通过本领域已知的多种测定可以鉴定、筛选本文中提供的抗CLDN6抗体或表征其物理/化学性质和/或生物学活性。包括例如ELISA、biacore、Western印迹和流式细胞仪分析。合适的测定详细描述在实施例中。
术语“亲和力”是指分子(例如：抗体)的单个结合位点及其结合配体(例如：抗原)之间非共价相互作用的力量总和。除非另外指出，如本文中使用的“结合亲和力”是指固有结合亲和力，其反应结合对的成员(例如：抗体和抗原)之间1：1相互作用。分子X对其配体Y的亲和力通常可以由解离常数(Kd)代表。亲和力可以通过本领域中已知的常规方法测量，所述方法包括利用Biacore测定抗体的亲和力。例如，本文中的抗体对CLDN6的“亲和力”以抗体的KD表示。抗体的KD是指抗体——抗原相互作用的平衡解离常数。抗体结合其抗原的KD值越大，其对所述具体抗原的结合亲和力越弱。例如，本文中的抗体对CLDN抗原的“亲和力”以抗体的EC50表示。
术语“EC50”，半最大效应浓度(concentration for 50％of maximal effect,EC50)是指能引起50％最大效应的浓度。
本领域已知多种确定抗体结合亲和力的方法。在一实施方案中，确定结合亲和力的方法使用表面等离子体共振。表面等离子体共振是一种光学现象，利用该现象，通过例如使用Biacore系统检测生物芯片基质中蛋白浓度的变化，可以分析实时的生物特异性相互作用。
“不显著结合”的特征在于抗体和抗原结合的亲和力降低、解离速率较快和/或结合信号较低，特别是解离速率较快和/或结合信号较低。在一实例中，“不显著结合”，指抗体结合抗原蛋白或多肽的结合水平，在统计学上不显著高于背景；所谓背景，是在不存在抗体时检测到的结合水平、或存在阴性对照蛋白(例如，同种型对照抗体)的情况下检测 到的结合水平。例如，使用生物传感器分析(例如Biacore)检测抗体结合抗原蛋白或多肽的结合水平，或检测抗体结合表达在细胞表面的抗原蛋白或多肽的结合水平。示例的，利用PBS替代抗体来检测抗原结合水平。在一实例中，本发明的抗体结合CLDN6。在一实例中，本发明的抗体不显著结合CLDN4或CLDN9。
抗原
术语“抗原”或“Ag”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞(immunologically-competent cells)的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽，都可以作为抗原。此外，抗原可以源自重组DNA或基因组DNA。包含编码可以引起免疫翻译的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA，均可以编码抗原。本领域技术人员明了，抗原不必仅由基因的全长核苷酸序列编码。包括但不限于，使用一个以上基因的部分核苷酸序列，这些核苷酸序列可以以各种组合方式排列以编码可以引起期望免疫反应的多肽。此外，抗原也可以无需由基因编码。抗原可以合成产生、或可以源自生物样品、或可以是除多肽之外的大分子。所述生物样品可以包括但不限于组织样品、肿瘤样品、细胞或液体以及其它生物学成分。
术语“表位”指可被抗体、B细胞、T细胞或工程化细胞识别的抗原或部分抗原。例如，表位可以是被抗体识别的肿瘤表位或病原体表位；抗体识别抗原内的多个表位。表位也可以突变。
术语“抗原决定部位”又称“抗原表位”或“表位”或“抗原决定簇”，包括任何能够被抗体结合的决定簇或区域。抗原表位是抗原中被靶向所述抗原的抗体结合的区域，包括与抗体直接接触的特定氨基酸。示例性，抗原表位可以由CLDN6蛋白序列的连续序列组成，也可以由CLDN6蛋白序列不连续的三维结构组成。示例性，本文中使用的抗原是人的CLDN6。
免疫缀合物
本发明还提供了免疫缀合物，所述的免疫缀合物包括本文所述的抗体，以及与之连接的一种或多种功能性分子。本发明提供的所述抗体已在前文描述，本发明提供的偶联物包含其全部技术方案。所述抗体与所述功能性分子可以通过共价连接、偶联、附着、交联等方式构成免疫缀合物。
“连接”或“融合”在本文中可互换使用。其通常是指通过包括化学缀合或重组方法的任何手段将两个以上化学元件或组件连接在一起。“框内融合”是指以维持原始开放阅读框(ORF)的正确阅读框的方式连接两个或更多个ORF以形式连续的较长的ORF。因此，所得到的重组融合蛋白是含有两个或更多个片段的单一蛋白质，这些片段对应于由原始ORF编码的多肽(这些片段在自然状态通常不是如此连接)。尽管阅读框因此在整个融合片段中是连续的，但这些片段可以在物理上或空间上通过例如框内连接序列(例如“flexon”)分开。
所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫 细胞的表面标志物的分子或可检测标记物。在一些实施方案中，所述的靶向肿瘤表面标志物的分子可以是结合肿瘤表面标志物的抗体或配体，可以与本发明的抗体协同作用，更精准地靶向肿瘤细胞。
在一些实施方案中，所述的抑制肿瘤的分子包括细胞毒剂，所述细胞毒剂包括但不限于：放射性同位素、化疗剂、生长抑制剂、酶及其片段、抗生素、毒素。在一些实施方案中，所述的抑制肿瘤的分子是抗肿瘤的细胞因子，细胞因子包括但不限于：IL-2、IL-7、IL-12、IL-15、I型IFN、TNF-α。
在一些实施方案中，所述的靶向免疫细胞的表面标志物的分子是结合免疫细胞表面标志物的抗体或配体，能够识别免疫细胞，其携带本发明的抗体到达免疫细胞，同时本发明的抗体可将免疫细胞靶向于肿瘤细胞，从而引发免疫细胞特异性杀伤肿瘤细胞。所述的免疫细胞表面标志物选自CD3、CD6、CD28、NKG2A、NKG2C、NKG2D、CD94、CD159a、CD159c、CD158、CD56、LIR/ILT2、CD244、CD226、CD2、CD16、CD161，所述免疫细胞选自T细胞、NK细胞、NKT细胞。在一实施方案中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与本发明所述的抗体形成T细胞参与的双功能抗体。
在一些实施方案中，所述的可检测标记物包括但不限于：荧光标记物、显色标记物；例如：酶、辅基、荧光材料、发光材料、生物发光材料、放射性材料、正电子发射金属以及非放射性顺磁性金属离子。为了检测和/或分析和/或诊断目的用于标记抗体的标记依赖于使用的特定检测/分析/诊断技术和/或方法例如免疫组织化学染色(组织)样品、流式细胞计量术等。
所述免疫缀合物包括抗体-药物偶联物(ADC)，其中，抗体偶联至一种或多种药物，包括但不限于美登木素、奥瑞他汀例如单甲基奥瑞他汀药物部分DE和DF(MMAE和MMAF)、多拉司他汀、卡奇霉素或其衍生物、蒽环类抗生素(例如道诺霉素或阿霉素)、甲氨蝶呤、长春地辛、紫杉烷(例如多西他赛、紫杉醇、莱龙太素、泰斯它赛(Tesetaxel)和奥它塔赛(Oxtataxel))、单端孢霉烯、CC1065。本发明另一方面提供了编码本发明免疫缀合物的核酸分子。一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。
嵌合受体
本发明还提供了嵌合受体，其通常是指用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子的表达产物，可以包括胞外域、跨膜域和胞内域。其中，所述胞外域包含抗原结合结构域。在一实施方案中，所述胞外域包含本发明提供的抗体，所述抗体已在前文详细描述，本发明提供的嵌合受体包含其全部技术方案。所述嵌合受体包 括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)、合成多肽受体(synNotch)。
在一实施方案中，可以通过将特异性结合目的抗原的抗原结合结构域工程化掺入CAR中，使表达CAR的细胞靶向结合目的抗原。
在一些实施方案中，本发明的嵌合受体是嵌合抗原受体(CAR)。所述嵌合抗原受体通常包含胞外抗原结合区或者抗体。在一些实施例中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区可以是人源化的。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施例中，所述胞外抗原结合区可以是非人的。
在某些实施方式中，所述嵌合抗原受体还可以设计为包含多种抗原结合区，包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与结合其同源受体的自然配体。在一些实施例中，胞外抗原结合区可以包含scFv、Fab或天然配体，以及它们的任何衍生物。胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。胞外抗原结合区，例如scFv、Fab或天然配体，可以是确定抗原特异性的CAR的一部分。胞外抗原结合区可以结合任何互补靶。胞外抗原结合区可以衍生自已知可变区序列的抗体。胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得胞外抗原结合区。
在一些实施例中，CAR的胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR和/或重链CDR来确定。在一些实施例中，CAR的胞外抗原结合区的结合特异性可以通过轻链可变区和/或重链可变区来确定。
在一些实施例中，CAR的胞外区包括铰链或间隔区，铰链和间隔区可以互换使用。铰链可以被认为是用于向胞外抗原结合区提供柔性的CAR的一部分。在一些实施例中，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测胞外抗原结合区的抗体不起作用或不可用时。在一些实施例中，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。可以根据所使用的胞外抗原结合区来调节铰链。铰链可以是任何长度的。在一些实施例中，铰链可以是IgG1、IgG4的天然铰链区，或其突变的铰链区，或铰链区之外的其它结构部分。例如，所述铰链可以包含如SEQ ID NO:21、27、28、29或30所示的氨基酸序列。
CAR的跨膜结构域(或称为结构区)可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号：NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8 多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号：NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些实施例中，跨膜部分可以包含CD8α区。例如，所述跨膜结构域可以包含如SEQ ID NO:22或25所示的氨基酸序列。
CAR的(细)胞内信号传导区可以负责活化包含所述CAR的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌，如IL-2，TNF-α,γ-IFN等。因此，术语细胞内信号传导区是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区，但是在许多情况下，不必使用信号结构域的整个链。在一些实施例中，使用细胞内信号传导区的截短部分。在一些实施例中，术语细胞内信号传导区因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。
在CAR中使用的信号结构域(或称为结构区)的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。
含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3ζ结构域，也称为T细胞受体CD3ζ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面起重要作用。如本文所用，CD3ζ主要是指人类CD3ζ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，不需要全T细胞受体CD3ζ链，并且其包含T细胞受体CD3ζ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。例如，所述CD3ζ链的信号结构域可以包含如SEQ ID NO:24所示的氨基酸序列。
在某些实施方式中，CAR的细胞内信号传导结构域(或称为结构区)可以选自表2的任何一个共刺激结构域。在一些实施例中，可以修饰结构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表1的任何一个结构域，使得修饰形式可以包含约50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或至多约100％的同一性。
在某些实施方式中，CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如ζ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。例如，所述CD28的共刺激结构域可以包含如SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可 以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。在本领域众所周知的，在幼稚T细胞中，T细胞受体的单独结合不足以诱导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
在一些实施方案中，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一些实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。
表2.共刺激结构域
本发明提供经工程化而表达CAR的细胞(例如T细胞)，其中该表达CAR的细胞(例如CAR-T细胞)表现出抗肿瘤性质。一方面，用CAR转导细胞，CAR在细胞表面上表达。在一些实施案例中，用编码CAR的病毒载体转导细胞(例如T细胞)。在一些实施案例中，病毒载体是逆转录载体。在一些实施案例中，病毒载体是慢病毒载体。一些实施方案中，细胞可以稳定地表达CAR。在一些实施方案中，用编码CAR的核酸，例如mRNA、cDNA、DNA转染细胞(例如T细胞)。
在某些实施方式中，CAR包含靶向CLDN6的抗原结合结构域。在某些实施方式中，CAR的CLDN6结合部分是scFv，该抗体片段是功能性的，与其所来自的IgG抗体相比，其保持相当的结合亲和力，例如其以相当的功效结合抗原；其由此提供生物化学反应，例如激活免疫反应、抑制从其靶抗原的信号传导起始、抑制激酶活性等。示例性，CAR的抗CLDN6抗原结合域包含如SEQ ID NO：13、14、15、16、17、18或19任一所示的scFv序列。
在某些实施方式中，CAR的抗CLDN6抗原结合结构域是人源化抗体或其片段。在某些实施方式中，CAR的抗CLDN6抗原结合结构域是全人抗体或其片段。在某些实施方式中，CAR的抗CLDN6抗原结合结构域是鼠源抗体或其片段。
在某些实施方式中，本发明CAR将特定抗体的抗原结合结构域和胞内信号传导分子组合在一起。例如，胞内信号传导分子包括但不限于，CD3ζ链、4-1BB和CD28信号传导模块及其组合。
在某些实施方式中，CLDN6-CAR包含至少一个胞内信号传导结构域，其选自CD137(4-1BB)信号传导结构域、CD28信号传导结构域、CD3ζ信号结构域，及其任何组合。一方面，CLDN6-CAR包含至少一个胞内信号传导结构域，其来自一个或多个非CD137(4-1BB)或CD28的共刺激分子。
作为示例性的，CLDN6-CAR的序列包含：具有SEQ ID NO:13所示的胞外域、SEQ ID NO：21所示的铰链域、SEQID NO：22所示的跨膜域、SEQ ID NO：23所示的共刺激信号域、以及SEQ ID NO：24所示的初级信号域(H1-28Z)；或具有SEQ ID NO:17所示的胞外域、SEQ ID NO：21所示的铰链域、SEQID NO：22所示的跨膜域、SEQ ID NO：23所示的共刺激信号域、以及SEQ ID NO：24所示的初级信号域(P4-28Z)。
示例性的，嵌合抗原受体的氨基酸序列包含如SEQ ID NO：13、14、15、16、17、18或19任一所示的序列分别与SEQ ID NO：45、46或47任一所示的序列连接。例如，嵌合抗原受体的氨基酸序列包含：如SEQ ID NO：13所示的序列与SEQ ID NO：45所示的序列连接；或如SEQ ID NO：13所示的序列与SEQ ID NO：46所示的序列连接；或如SEQ ID NO：13所示的序列与SEQ ID NO：47所示的序列连接；或如SEQ ID NO：14所示的序列与SEQ ID NO：45所示的序列连接；或如SEQ ID NO：14所示的序列与SEQ ID NO：46所示的序列连接；或如SEQ ID NO：14所示的序列与SEQ ID NO：47所示的序列连接；或如SEQ ID NO：15所示的序列与SEQ ID NO：45所示的序列连接；或如SEQ ID NO：15所示的序列与SEQ ID NO：46所示的序列连接；或如SEQ ID NO：15所示的序列与SEQ ID NO：47 所示的序列连接；或如SEQ ID NO：16所示的序列与SEQ ID NO：45所示的序列连接；或如SEQ ID NO：16所示的序列与SEQ ID NO：46所示的序列连接；或如SEQ ID NO：16所示的序列与SEQ ID NO：47所示的序列连接；或如SEQ ID NO：17所示的序列与SEQ ID NO：45所示的序列连接；或如SEQ ID NO：17所示的序列与SEQ ID NO：46所示的序列连接；或如SEQ ID NO：17所示的序列与SEQ ID NO：47所示的序列连接；或如SEQ ID NO：18所示的序列与SEQ ID NO：45所示的序列连接；或如SEQ ID NO：18所示的序列与SEQ ID NO：46所示的序列连接；或如SEQ ID NO：18所示的序列与SEQ ID NO：47所示的序列连接；或如SEQ ID NO：19所示的序列与SEQ ID NO：45所示的序列连接；或如SEQ ID NO：19所示的序列与SEQ ID NO：46所示的序列连接；或如SEQ ID NO：19所示的序列与SEQ ID NO：47所示的序列连接。
上述嵌合抗原受体的跨膜域和胞内域，本领域技术人员可以选择常规的跨膜域和胞内域进行替换，且均落入本申请的保护范围。
核酸、载体、病毒、宿主细胞
术语“核酸”或“多核苷酸”指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)和其聚合物、寡核苷酸、由PCR生成的片段、由连接、断链、核酸内切酶作用和外切核酸酶作用中的任一种生成的片段。核酸序列包含天然核苷酸序列，也隐含地包括其保守修饰的变体(例如简并密码子替代)、等位基因、直向同源物、SNP和互补序列。修饰的核苷酸可以在糖部分和/或嘧啶或嘌呤碱部分中具有改变。例如，糖修饰包括将一个或多个羟基替换成卤素、烷基基团、胺和叠氮基团，或者糖可以被醚或酯官能化。
术语“密码子“指在mRNA上(或在DNA分子的有义链上)的三个核苷酸，其通过核糖体翻译成一个氨基酸残基。
术语“密码子优化”指同义密码子(编码相同氨基酸的密码子)在编码DNA中的出现频率在不同物种中存在偏好，给定物种的高度表达的基因中通常罕见的密码子被这种物种中高度表达的基因的常见密码子置换。密码子简并性允许多个核苷酸序列编码相同的多肽。简并密码子替代可以通过如下实现：产生序列，在该序列中一个或多个选定的(或所有)密码子的第三位被混合碱基和/或脱氧肌苷残基替代。
术语“编码”指多核苷酸例如基因、DNA或mRNA中特定核苷酸序列的固有性质，即所述序列可以充当模板，以在生物学过程中合成具有规定的核苷酸序列(例如rRNA、tRNA和mRNA)或规定的氨基酸序列的其它聚合物和大分子以及由此导致的生物学性质。因此，当相应于基因的mRNA在细胞或其它生物学系统中的转录和翻译导致蛋白质产生时，则该基因、cDNA、或RNA编码该蛋白。编码链(其核苷酸序列与mRNA序列相同，通常在序列表中提供)和非编码链(用作基因或cDNA转录的模板)两者都可以被称作编码蛋白、或该基因或cDNA的其它产物。
术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链 的氨基酸的顺序。因此，核酸序列编码氨基酸序列。“编码氨基酸序列的核苷酸序列”包括彼此为简并形式的编码相同氨基酸序列的所有核苷酸序列。当用于指核苷酸序列时，本文所用的术语“序列”包括DNA或RNA，并且可以是单链或双链。
术语“靶序列”是指与指导序列具有互补性的序列，靶序列与指导序列之间互补配对促进CRISPR复合物的形成。一个靶序列可以包含任何多核苷酸，如DNA或RNA多核苷酸。在一些实施例中，靶序列位于细胞的细胞核或细胞质中。
术语“同一性”或“同源性”指两个核酸分子之间、或两个多肽分子之间的亚单位序列同一性。当一个亚单位位置在两个分子中被相同单体亚单位占据时，例如当两个DNA分子在一个位置上都被腺苷占据时，则它们在该位置是同源的或同一的。两个序列之间的同源性是匹配的或同源的位置数目除以序列中的位置总数目，并将结果乘以100，以产生序列同一性的百分比。例如，当两个序列中80％位置(例如在长10个bp的核酸分子中8个bp)是同源的，则这两个序列同源性为80％。
术语“转染”或“转导”或“转化”是指将外源核酸引入宿主细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。“转染的”或“转化的”或“转导的”细胞是已经用外源性核酸转染、转化或转导的细胞。该细胞包括原代受试者细胞及其子代。
术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，如质粒、病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。所述质粒载体也可以包含选择标记物，该选择标记物提供接收所述载体的细胞的识别和/或筛选。
术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。还可以包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。载体可以与任何细胞渗透技术(如声孔效应或电穿孔或其衍生技术)相关联或相结合。载体的选择主要取决于待插入载体的核酸的大小和待用载体转发的特定宿主细胞。根据载体的功能(异源多核苷酸的扩增和/或表达)以及载体与其所在的特定宿主细胞的相容性，每个载体含有不同的组件。载体组件通常包括但不限于：复制起点、选择性标记基因、启动子、核糖体结合位点(RBS)、信号序列、异源核酸插入物和转录终止序列。将载体引入细胞的物理方法包括磷酸钙沉淀、脂质体转染、粒子轰击、显微注射、电穿孔等。将载体引入细胞的化学方法包括胶体分散体系，如大分子复合物、纳米胶囊、微球体、珠粒和基于脂质的体系(包括水包油乳液、胶束、混合胶束和脂质体)。
术语“慢病毒”是指逆转录病毒科的属。逆转录病毒在能够感染非分裂细胞方面是逆 转录病毒中独特的；它们可以将大量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV、SIV和FIV都是慢病毒的实例。源自慢病毒的载体提供了在体内实现显著水平的基因转移的手段。“整合慢病毒载体(LV)”是指作为非限制的实例的这些载体，其能整合靶细胞的基因组。“非整合慢病毒载体(NILV)”相对地是指有效基因递送载体，这些载体不通过病毒整合酶的作用整合靶细胞的基因组。
术语“内源”是指来自生物体、细胞、组织或系统自身的任何物质，或在生物体、细胞、组织或系统中产生的任何物质，例如核酸分子或多肽。
术语“外源”指的是生物体、细胞、组织或系统引入的，或在生物体、细胞、组织或系统之外产生的任何物质，例如核酸分子、多肽、细胞。
术语“外源蛋白”可以是识别靶抗原的外源转入细胞的蛋白，如外源受体(即本文中前述“嵌合受体”)。
术语“宿主”是指接受移植物移植的受体，在一些实施方式中，可以是接受外源细胞植入的个体，如人。
术语“分离的”是指从天然状态改变或移出。例如，天然存在于活的动物中的核酸或肽不是“分离的”，但与其天然状态下的共存物部分地或完全地分开的该核酸或肽是“分离的”。分离的核酸或蛋白可以以基本上纯的形式存在，或可以存在于非天然环境例如宿主细胞中。也可以通过人工组装的方法，例如通过化学合成或重组表达，从而提供“分离的”物质。
术语“表达”指启动子驱动的特定核苷酸序列的转录和/或翻译。
术语“启动子”(promoter)是RNA聚合酶识别、结合和开始转录的一段DNA序列，是基因的一个重要组成部分，主要功能是调控基因转录的起始时间和表达程度。在一些实施方案中，编码CAR的核酸可操作地连接至启动子。
本发明提供了编码识别CLDN6的抗体或其片段的分离的核酸、载体以及包含所述核酸或载体的宿主细胞。核酸可位于完整细胞中、细胞裂解液中或者以部分纯化的或基本纯化的形式。
可以使用标准的分子生物学技术获得本发明的核酸，例如可以通过标准的PCR扩增或cDNA克隆技术，获得编码抗体的轻链和重链或者编码VH和VL区段的cDNA。对于从免疫球蛋白基因文库获得的抗体(例如，使用噬菌体展示技术)，可以从文库回收编码抗体的一种或多种核酸。向宿主细胞中导入外源核酸的方法是本领域普遍已知的，并可随所使用的宿主细胞而变化。
优选的，本发明核酸分子是选自编码重链可变区的SEQ ID NO:2或6，和/或选自编码轻链可变区的SEQ ID NO:4、8、10或12。优选的，是这样的核酸分子，所述核酸分子包含SEQ ID NO:2的重链可变区序列，和包含SEQ ID NO:4的轻链可变区序列；或者包含SEQ ID NO:2的重链可变区序列，和包含SEQ ID NO:8的轻链可变区序列；或者包含SEQ ID NO:2的重链可变区序列，和包含SEQ ID NO:10的轻链可变区序列；或者包含SEQ ID NO:6的 重链可变区序列，和包含SEQ ID NO:4的轻链可变区序列；或者包含SEQ ID NO:6的重链可变区序列，和包含SEQ ID NO:8的轻链可变区序列；或者包含SEQ ID NO:6的重链可变区序列，和包含SEQ ID NO:10的轻链可变区序列；或者包含SEQ ID NO:2的重链可变区序列，和包含SEQ ID NO:12的轻链可变区序列。
在一个实施方案中，提供一种或多种包含上述核酸的载体(例如，表达载体)。
术语“细胞”指人或非人动物来源的细胞。
术语“宿主细胞”指被引入外源核酸的细胞，包括此种细胞的后代。宿主细胞包括“转化体”和“转化的细胞”，其包括转化的原代细胞及来源于其的后代(不考虑传代次数)。后代的核酸内容可以与亲本细胞不完全相同，并且可以含有突变。本文中包括具有与对于原始转化的细胞中筛选或选择的相同的功能或生物学活性的突变体后代。
术语称细胞或细胞群针对具体标志物呈“阳性”指，细胞上或细胞中具体标志物(通常是表面标志物)的可检测的存在。当述及表面标志物时，该术语指，通过流式细胞术检测到存在表面表达，例如，通过用特异性地结合至所述标志物的抗体染色，并检测所述抗体，其中，所述染色可被流式细胞术以一定水平检测到，所述水平显著高于采用同种型匹配的对照在其它条件相同的情况下进行相同步骤时检测到的染色的水平，和/或基本相似于已知对所述标志物呈阳性的细胞的水平，和/或显著高于已知对所述标志物呈阴性的细胞的水平。
术语称细胞或细胞群针对具体标志物呈“阴性”指，所述细胞上或细胞中具有具体标志物(通常是表面标志物)的基本可检测的不存在。当述及表面标志物时，该术语指，通过流式细胞术检测到存在表面表达，例如，通过用特异性地结合至所述标志物的抗体染色，并检测所述抗体，其中，所述染色可被流式细胞术以一定水平检测到，所述水平显著高于采用同种型匹配的对照在其它条件相同的情况下进行相同步骤时检测到的染色的水平，和/或显著低于已知对所述标志物呈阳性的细胞的水平，和/或基本相似于已知对所述标志物呈阴性的细胞的水平。
术语“CLDN6阳性宿主细胞”是指在细胞表面上表达CLDN6的宿主细胞，这些细胞可以通过例如使用抗体的流式细胞术来检测，这些抗体特异性识别CLDN6上的表位。
在一些实施方案中，所述宿主细胞是免疫细胞。
术语“免疫细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞、嗜中性粒细胞、嗜酸性粒细胞、和/或嗜碱性粒细胞等。在一些实施方案中，所述的免疫细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。“免疫效应功能或免疫效应应答”是指免疫细胞，例如增强或促进靶细胞的免疫攻击的功能或反应。例如，免疫功能或应答是指促进靶细胞的杀伤或者抑制生长或增殖的T细胞或NK细胞的属性。
术语“经人工改造的具有免疫细胞功能的细胞”是指不具有免疫效应的细胞或细胞系经 人工改造或接受刺激物刺激后，该细胞获得了免疫细胞功能。如293T细胞，经人工改造，使其具有免疫细胞的功能；如干细胞，经体外诱导，使其分化成免疫细胞。干细胞可以是成体干细胞、非人类胚胎干细胞，更具体的是非人类干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导的多能干细胞、全能干细胞或造血干细胞。
在一些情况下，细胞一般是原代细胞，如直接从个体分离和/或从个体分离并冷冻的那些细胞。在一些实施方式中，细胞包括T细胞或其它细胞类型的一个或多个亚组，如全T细胞群、CD4+细胞、CD8+细胞及其亚群，如由功能、活化状态、成熟、分化潜力、扩增、再循环、定位，和/或持久能力、抗原特异性、抗原受体类型、特定器官或隔室中的存在、标志物或细胞因子分泌情况，和/或分化程度限定的那些。对于待治疗的对象，细胞可以是同种异体和/或自体的。
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)、效应记忆T细胞(Tem)、原初T(TN)细胞、肿瘤-浸润性淋巴细胞(TIL)、不成熟T细胞、成熟T细胞、辅助性T细胞(例如TH1细胞、TH2细胞、TH3细胞、TH17细胞、TH9细胞、TH22细胞、滤泡辅助性T细胞)、细胞毒性T细胞、和/或粘膜相关的非变体T(MAIT)细胞。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如αβT细胞、γδT细胞。
T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。在一个实施方案中，可以从健康供体，或来自诊断患有肿瘤的患者衍生细胞获得T细胞。采用本领域常规制备CAR-T的方法，采用由PBMC细胞经抗CD3和CD28抗体的磁珠活化后继续培养得到的T细胞，经慢病毒感染后得到CAR-T细胞。
术语“外周血单个核细胞”(peripheral blood mononuclear cell,PBMC)是指外周血中具有单个核的细胞，包含淋巴细胞、单核细胞等。
术语“激活”和“活化”可互换使用，可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指NK细胞、T细胞逐步活化的过程。
术语“T细胞活化”或“T细胞激活”指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。
“遗传工程改造”一般涉及将编码所述重组或经工程改造的部分的核酸导入细胞，例如，通过逆转录病毒或慢病毒转导、转染或转化进行，或通过转座子、电穿孔进行。一些实施方式中，通过如下方式进行：首先，刺激细胞，例如，通过将其与刺激物合并，所述刺激物诱导响应，例如增殖、存活和/或活化，例如，通过细胞因子或活化标志物的表达来检测，然后转导该活化的细胞，并在培养物中扩增至足以供于临床应用的数量。
在另一实施方案中，提供包含本文前述核酸的宿主细胞。宿主细胞包含(例如，转导有)：(1)载体，所述载体包含核酸，所述核酸编码包含抗体VL的氨基酸序列和包含抗体VH的氨基酸序列，或(2)包含编码包含抗体VL的氨基酸序列的核酸的第一载体，和包含编码包含抗体VH的氨基酸序列的核酸的第二载体。在一个实施方案中，宿主细胞是真核的，例如，293T细胞。
在另一实施方案中，所述宿主细胞表达本发明所述的嵌合受体。
在另一实施方案中，所述宿主细胞包括T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、调节性T细胞、NK92细胞、和/或干细胞衍生的免疫细胞。
在另一实施方案中，所述T细胞为来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞；优选地，所述T细胞为自体/同种异体T细胞；优选地，所述T细胞为原代T细胞；优选地，所述T细胞来源于人的自体T细胞。
在另一实施方案中，所述T细胞包含记忆性干细胞样T细胞(Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef)、调节性T细胞(Tregs)，效应记忆T细胞(Tem)、γδT细胞或其组合。
在另一实施方案中，所述宿主细胞结合表达CLDN6的细胞，不显著结合表达CLDN4、CLDN9或其组合的细胞。
在另一实施方案中，所述宿主细胞还携带外源的细胞因子的编码序列。
在另一实施方案中，所述宿主细胞还可以表达除了上述结合抗原的受体以外的另一种嵌合受体。
在另一实施方案中，所述宿主细胞还可以表达趋化因子受体。
在另一实施方案中，所述宿主细胞还可以表达安全开关。
在一个实施例中，提供制备抗CLDN6抗体的方法，其中所述方法包括在适合于表达如上所述的抗体的条件下培养包含编码所述抗体的核酸的宿主细胞，和任选地从宿主细胞(或宿主细胞培养基)回收抗体。
为了表达蛋白质，可以将编码本发明抗体的核酸整合到表达载体中。多种表达载体可用于蛋白质表达。表达载体可包括自我复制的染色体外载体，或整合到宿主基因组中的载体。用于本发明的表达载体包括但不限于使蛋白质能够在哺乳动物细胞、细菌、昆虫细胞、酵母和体外系统中表达的那些。如本领域已知的，多种表达载体是可商业或其他方式获得的。可用于本发明中来表达抗体。
在一优选例中，所述宿主细胞与增强其功能的药剂组合施用，优选地，与化疗药物联 用；和/或所述宿主细胞与改善其相关的一种或多种副作用的药剂联合施用；和/或所述宿主细胞与表达靶向CLDN6之外的嵌合抗原受体的宿主细胞联合施用。
药物组合物、组合治疗
本发明的抗体、包含该抗体的免疫辍合物、嵌合受体、宿主细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述抗体、免疫辍合物、嵌合受体、核酸或宿主细胞，还可包含药学上可接受的载体。
术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖、甘露糖、蔗糖、葡聚糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；磷酸盐缓冲液；佐剂例如氢氧化铝；缓冲剂；稀释剂；稳定剂或赋形剂等。
本文所述的药物组合物可包含一种或多种药学可接受的盐。“药学可接受的盐”指这样一种盐，其保留亲本化合物的期望生物学活性且不产生任何不利的毒物学效果。此类盐的例子包括酸加成盐和碱加成盐。
酸加成盐包括衍生自无毒无机酸，诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等的盐，以及衍生自无毒有机酸，诸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳香族酸、脂肪族和芳香族磺酸等的盐。碱加成盐包括衍生自碱土金属(诸如钠、钾、镁、钙等)的盐，以及衍生自无毒有机胺的盐，诸如N,N'-二苄乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等。
本文所述的药物组合物还可包含抗氧化剂。抗氧化剂的实例包括但不限于:水溶性抗氧化剂，诸如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠等；油溶性抗氧化剂，诸如抗坏血酸棕榈酸酯、丁基化羟基茴香醚(BHA)、丁基化羟基甲苯(BHT)，卵磷脂、没食子酸丙酯、α-生育酚等；和金属螯合剂，诸如柠檬酸、乙二胺四乙酸(EDTA)、山梨醇、酒石酸、磷酸等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。还可以经动脉、皮内、肿瘤内、结节内、髓内、腹膜内施用给患者。
在一些实施方案中，组合物可以是等渗的，即它们可以具有与血液和泪液相同的渗透压。本发明组合物的期望等渗性可以使用氯化钠或其它药学上可接受的试剂如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。如果需要，组合物的粘度 可以使用药学上可接受的增稠剂维持在选定的水平。合适的增稠剂包括，例如，甲基纤维素、黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的优选浓度将取决于所选择的试剂。显然，合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型。
在一实施方案中，本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒与其它已知活性剂或治疗组合施用。“组合施用”指两种或两种以上不同治疗施用给个体。在一实施方案中，一种治疗在第二治疗施用开始时仍然在继续，由此在施用上存在重叠。在一实施方案中，一种治疗施用结束后，开始施用第二治疗。在一实施方案中，组合施用使治疗更有效。在一些实施方案中，两种治疗的效果可以是部分相加的、完全相加的、或大于加和的。所述“组合施用”的治疗包括但不限于以下的一种或多种：手术、化疗、放射、免疫抑制剂(例如环孢素(cyclosporin)、硫唑嘌呤(azathioprine)、甲氨蝶呤(m e t h o t r e xa t e)、霉酚酸酯(m y c o p h e n o la t e)和F K 50 6)、抗体或其它免疫清除剂(immunoablative agents)例如CAMPATH、抗CD3抗体或其它抗体治疗、环磷酰胺(cytoxan)、氟达拉滨(fludarabine)、雷帕霉素(rapamycin)、霉酚酸(mycophenolic acid)、类固醇(steroids)、FR901228、细胞因子和辐射。所述“组合施用”的治疗还包括免疫调节剂，例如干扰素α、干扰素β、TGF-β2肽抑制剂、或poly-ICLC。
在一些实施方案中，所述组合物包含另一治疗剂。在一些实施方案中，本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒与增强其功能的药剂组合施用。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些和/或其药学上可接受的盐或类似物。示例性，化疗剂包括烷化剂、基于铂的药剂、血管发生抑制剂(例如VEGF途径抑制剂、酪氨酸激酶抑制剂EGF途径抑制剂)、mTOR抑制剂。在一些实施方案中，所述治疗剂包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述另外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。在一些实施方案中，所述另外的治疗剂包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂等。
在一实施方案中，本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细 胞、药物组合物或试剂盒与抑制性分子的抑制剂组合施用。抑制性分子包括PD1、PD-L1、CTLA-4、TIM3、LAG3、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4和TGFRβ。抑制性核酸例如dsRNA，例如siRNA或shRNA，可以用于抑制抑制性分子在CAR表达细胞中的表达。在一个实施方案中，抑制剂是shRNA。在一个实施方案中，CAR表达细胞中抑制性分子被抑制。在这些实施方案中，抑制抑制性分子表达的dsRNA分子可以与编码CAR的成分(例如所有成分)的核酸连接。一个实施方案中，抑制性信号的抑制剂可以是例如与抑制性分子结合的抗体或抗体片段。例如，该活性剂可以是与PD1,PD-L1,PD-L2或CTLA4结合的抗体或抗体片段(例如伊匹单抗(ipilimumab)、Tremelimumab)。
在一实施方案中，本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒与改善其相关的一种或多种副作用的药剂联合施用。所述副作用包括但不限于CRS。CRS的症状包括高热、恶心、短暂性低血压、缺氧等。本发明提供的联合施用的药剂可以管理由抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒引起的可溶性因子的升高水平。在个体中升高的可溶性因子包括IFN-γ、TNFα、IL-2和/或IL-6。施用改善副作用的药剂可以是中和这些可溶性因子之一或多个的活性剂。此类活性剂包括但不限于类固醇、TNFα抑制剂和/或IL-6抑制剂。TNFα抑制剂包括但不限于依那西普(Etanercept)。IL-6抑制剂包括但不限于托珠单抗(Tocilizumab)(toc)。
在一实施方案中，本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒与表达靶向CLDN6之外的嵌合抗原受体的细胞联合施用。在一实施方案中，与治疗表达CLDN6相关疾病的药剂联合施用。在一实施方案中，所述药剂包括抗体、细胞、RNA、疫苗、溶瘤病毒、检查点抑制剂、BKT抑制剂、化学药、放射治疗剂、激素治疗剂、毒素、免疫治疗剂或其组合。
本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒和另一治疗剂可以同时地、在相同的组合物或分开的组合物中施用，或顺序地施用。在一实施方案中，可以首先施用本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒，再施用另一治疗剂。在一实施方案中，可以先施用另一治疗剂，再施用本发明的抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。
试剂盒
本发明还提供了包含本文所述的抗体、免疫缀合物、嵌合受体、核酸或宿主细胞的试剂盒。在一些实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的本文所述的抗体、嵌合受体、核酸或宿主细胞的治疗或预防组合物。在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。在一些实施方案中，所述试剂盒包含本文所述的抗体、 免疫辍合物、嵌合受体、核酸或宿主细胞，以及将本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞给予个体的说明书。说明书中通常包含使用本文所述的抗体、免疫辍合物、嵌合受体、核酸或宿主细胞来治疗或预防癌症或肿瘤的方法。在一些实施方案中，试剂盒包含本文所述的宿主细胞，并且可以包括约1×104个细胞至约1×106个细胞。在一些实施方案中，试剂盒可以包括至少约1×105个细胞，至少约1×106个细胞，至少约1×107个细胞，至少约4×107个细胞，至少约5×107个细胞，至少约6×107个细胞，至少约6×107个细胞，8×107个细胞，至少约9×107个细胞，至少约1×108个细胞，至少约2×108个细胞，至少约3×108个细胞，至少约4×108个细胞，至少约5×108个细胞，至少约6×108个细胞，至少约6×108细胞，至少约8×108个细胞，至少约9×108细胞，至少约1×109个细胞，至少约2×109个细胞，至少约3×109个细胞，至少约4×109个细胞，至少约5×109个细胞，至少约6×109个细胞，至少约8×109个细胞，至少约9×109个细胞，至少约1×1010个细胞，至少约2×1010个细胞，至少约3×1010个细胞，至少约4×1010个细胞，至少约5×1010个细胞，至少约6×1010个细胞，至少约7×1010个细胞、至少约8×1010个细胞、至少约9×1010个细胞，至少约1×1011个细胞，至少约2×1011个细胞，至少约3×1011个细胞，至少约4×1011个细胞，至少约5×1011个细胞，至少约8×1011个细胞，至少约9×1011个细胞，或至少约1×1012个细胞。例如，可以在试剂盒中包括大约5×1010个细胞。在另一个实例中，试剂盒可以包括3×106个细胞；细胞可以扩增至约5×1010个细胞并施用于受试者。
在一些实施方案中，试剂盒可以包括同种异体细胞。在一些实施方案中，试剂盒可以包括可以包含基因组修饰的细胞。在一些实施方案中，试剂盒可以包含“现成的”细胞。在一些实施方案中，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。
在一些实施方案中，说明书包括以下中的至少一个：治疗剂的描述；用于治疗或预防肿瘤或其症状的剂量方案和给药；预防措施、警示、禁忌症、过量信息、不良反应、动物药理学、临床研究、和/或引用文献。说明书可以直接打印在容器上(如果有的话)，或作为容器上的标签，或作为容器内或容器中提供的单独的纸张、小册子、卡片或文件夹打印。在一些实施方案中，说明书提供施用本发明所述的抗体用于治疗或预防肿瘤的方法。在某些情况下，说明书提供了施用化学治疗剂之前、之后或同时给与本发明的抗体的方法。
用于诊断/检测/治疗的方法
术语“调控”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。在一具体实施方式中，是指负向改变。
术语“治疗”是指在试图改变疾病过程的干预措施，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少疾病任何直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。该术语不表示完全治愈疾病、或完全消除任何症状、或对所有症状或结果具有效果。
术语“抗肿瘤效应”指生物学效应，其可以表现为多种形式，包括但不限于，例如肿 瘤体积的缩小、肿瘤细胞数量的减少、转移瘤数量的减少、寿命的增加、肿瘤细胞增殖的减少、肿瘤细胞存活的减少、或各种关于癌症病症相关的生理症状的改善。“抗肿瘤效应”也可以表现为本发明肽、多核苷酸、细胞和抗体从一开始就防止肿瘤发生的能力。
术语“预防”是指对于疾病在倾向于发展疾病但尚未被诊断患病的对象中的发生或复发提供预防方法，或在试图在疾病(如细胞移植产生的排斥反应)产生前进行的干预措施，对疾病或疾病状态的防止或预防性治疗。
术语“自体”指，从个体获得任何物质，并且之后重新引入到该同一个体中。
术语“同种异体”指，引入个体的任何物质来自于与个体相同物种的不同个体。当两个或两个以上个体在一个或多个基因座上的基因不同时，这些个体被称为是同种异基因的。在一些方面，来自相同物种的个体的同种异体物质可以在遗传上足够不同以发送抗原性上的相互作用。
术语“异种”指，源自不同物种的个体的移植物。
术语“肿瘤”或“癌症”指，特征在于快速且不受控的异常细胞生长的疾病。肿瘤细胞或癌症细胞可以局部地或通过血流和淋巴系统而扩散到身体的其他部分。
术语“与CLDN6表达相关的疾病”或“表达CLDN6相关的疾病”包括但不限于，与CLDN6表达相关的疾病、或与表达CLDN6的细胞相关的病症，例如卵巢癌、乳腺癌、宫颈癌、胃癌、肺癌、睾丸癌、生殖细胞和胚胎肿瘤、卵巢上皮癌、非小细胞肺癌，非鳞状非小细胞肺癌、子宫内膜癌等。
术语“检测”包括定量或定性检测。本发明的抗体可用于检测生物样品中CLDN6的存在，所述生物样品包含血液、血清、细胞或组织。
术语“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本发明的过度增生性病症是指肿瘤。
本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7 相关的(TEM7R)；Claudin 6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P4501B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。优选的，所述肿瘤抗原为CS1、Claudin18.2、GPC3、BCMA或者CD19。
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。
术语“个体”旨在包括可以引起免疫反应的活的生物体，是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
术语“有效量”是指提供治疗或预防益处的量，在一定剂量下且作用必要的时间长度时，有效实现所需治疗或预防结果。可以由医师根据患者(个体)在年龄、体重、肿瘤大小、感染程度、转移程度等个体状况差异而确定。
本文中提供的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒都可以用于治疗方法中。
本发明提供的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒通过引起针对CLDN6的抗原特异性反应，提供以下一项或多项：靶向和破坏CLDN6表达肿瘤细胞、减少或消除肿瘤、促进免疫细胞向肿瘤部位的浸润、和增强/延长抗肿瘤反应。由于CLDN6在正常(即非癌)组织中以不可检测的水平表达，故本发明提供的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒被认为可以避免靶向/破坏正常组织和细胞。
在一个方面，提供用作药物的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在另一方面，提供用于治疗疾病的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，提供用于治疗方法的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在某些实施方案中，本发明提供任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒，其用于治疗患有疾病的个体的方法，所述方法包括向个体施用有效量的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本发明提供任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒在制备或配制药物中的用途。在一个实施方案中，所述药物用于治疗疾病。在另一个实施方案中，所述药物用于治疗疾病的方法，所述方法包括向患病个体施用有效量的药物。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本发明提供用于治疗疾病的方法。在一个实施方案中，所述方法包括向患有表达CLDN6的疾病的个体施用有效量的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒。在一个实施方案中，所述方法还包括向个体施用有效量的至少一种另外的治疗剂。所述“个体”优选是人。
在另一个方面，本发明提供例如用于任一上述治疗方法的包含本文中提供的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒的药物制剂。在一个实施方案中，所述药物制剂包含本文中提供的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒和药用载体。在另一个实施方案中，所述药物制剂包含本文中提供的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒和至少一种另外的治疗剂。
在另一个方面，所述药物制剂用于治疗疾病。在一个实施方案中，向患病个体施用所述药物制剂。根据任一以上实施方案的“个体”优选是人。
在另一个方面，本发明提供用于制备药物或药物制剂的方法，所述方法包括将本文中提供的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒与药用载体混合，例如，以用于任一上述治疗方法。在一个实施方案中，用于制备药物或药物制剂的方法还包括添加至少一种另外的治疗剂至药物或药物制剂。
本发明的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒可以单独地用于治疗或与其他试剂组合地用于治疗。或者，本发明的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒可以与至少一种另外的治疗剂共同施用。
上述的此种组合治疗包括组合施用(其中两种以上治疗剂被包含在同一或分开的制剂中)和分开施用，在此种情况中，本发明的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒的施用可以发生在另外的治疗剂或试剂的施用之前、同时、和/或之后。在一个实施方案中，本发明的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒的施用和另外的治疗剂的施用彼此发生在约一个月以内、或在约一周、两周或三周以内，或在约一天、两天、三天、四天、五天或六天以内。
本发明的任一抗CLDN6抗体、免疫缀合物、嵌合受体修饰的宿主细胞、药物组合物或试剂盒(以及任意另外的治疗剂)可以通过任何合适的手段施用，包括肠胃外施用、肺内施用或鼻内施用，以及，如果具备治疗需要，病变内施用。肠胃外输注包括肌肉内施用、静脉内施用、动脉内施用、腹膜内施用或皮下施用。用药可以是通过任何合适的途径，例如，通过注射，如静脉内或皮下注射，这部分取决于施用是短暂的还是长期的。本文中考虑多种用药方案，包括但不限于单次施用或在多个时间点的多次施用、推注施用，和脉冲注入。
给予个体的包含免疫反应性细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个免疫反应性细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常，施用包含约1×104至约1×1010个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×105至约1×109个免疫反应性细胞、约5×105至约5×108个免疫反应性细胞、或约1×106至约1×107个免疫反应性细胞。然而，根据肿瘤的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。
在一些实施方案中，使用嵌合受体来刺激宿主细胞介导的免疫应答。例如，T细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一些实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。
在某些情况下，治疗患有肿瘤的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本发明所述的宿主细胞。所述宿主细胞可结合肿瘤靶分子并诱导癌细胞死亡。如前文所述，本发明还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本发明的宿主。
本发明的免疫反应性细胞的给药频率将根据包括所治疗疾病的因素、特定免疫反应性细胞的元件和给药方式。例如可以每日给药4次、3次、2次或每日一次、每隔一天、 每三天、每四天、每五天、每六天一次、每周一次、每八天一次、每九天一次、每十天、每周一次、或者每月两次给药。如本文所述，由于本申请的免疫应答细胞具有改善的活力，从而可以不仅以与类似的但不表达外源性I型干扰素的免疫应答细胞更低的治疗有效的量给药，并且可以以更低的频率给药，以获得至少类似、并且优选更加显著的疗效。
本发明的优点：
本发明提供了特异性识别CLDN6的人源化抗体，由该抗体制备的CAR T细胞在体内外表现出对靶细胞较好的杀伤效果。
应认识到，为清楚起见在单独实施方案的上下文中描述的本发明的某些特征也可组合提供于单个实施方案中。相反地，为简明起见在单个实施方案的上下文中描述的本发明的各种特征也可单独地或以任何合适的子组合提供。关于本发明的实施方案的所有组合被明确地包括在本发明中并公开于本文中，就如同每个和每一个组合被个别地且明确地公开。此外，各种实施方案的所有子组合及其元素也被明确地包括在本发明中并在本文公开，就如同每个和每一个此类子组合被个别地且清楚地在本文公开。
实施例1.CLDN6人源化抗体的制备
本实施例采用鼠抗SC27.105(来自WO2016073649A1)为母本抗体，将母本抗体序列与IMGT数据库的germline序列进行比较，筛选SC27.105抗体重链CDR移植模板1，和轻链CDR移植模板2。用SC27.105抗体的LCDR区替换掉抗体模板2的CDR区，构成人源化抗体H1的轻链可变区VL(氨基酸序列见SEQ ID NO:3)。将SC27.105抗体的重链CDR2中第5位丝氨酸(Serine，S)突变成丙氨酸(Alanine，A)，后将母本抗体HCDR1，突变后HCDR2，HCDR3替换掉移植模板2的CDR区，构成人源化抗体H1的重链可变区VH(氨基酸序列见SEQ ID NO:1)。人源化抗体H1的VH氨基酸序列中框架区为SEQ ID NO:1中第1-30，36-49，67-98，113-123位氨基酸；人源化抗体H1的VL氨基酸序列中框架区为SEQ ID NO:3中第1-23，35-49，57-88，97-106位氨基酸。
H1重链可变区(SEQ ID NO:1)，CDR区以下划线标出，框架区为除CDR以外的序列。
H1轻链可变区(SEQ ID NO:3)CDR区以下划线标出，框架区为除CDR以外的序列。
实施例2.人源化抗体H1的特异性和亲和力检测
1、构建细胞系
利用常规分子生物学技术通过慢病毒将人CLDN6(SEQ ID NO:32)，CLDN4(SEQ ID NO:31)和CLDN9(SEQ ID NO:33)分别转染293T细胞，通过有限稀释法挑选阳性克隆，构建293T-CLDN6、293T-CLDN4、293T-CLDN9稳转细胞系(图1)。
2、检测抗体H1与细胞系的结合EC50
取上述293T-CLDN6、293T-CLDN4、293T-CLDN9细胞2×105cells/孔于96孔圆底 培养板中，PBS清洗；加入抗体H1，4℃孵育离心，弃上清后PBS清洗；加入Goat-anti-Mouse FITC，4℃孵育；离心，弃上清后清洗，用流式细胞仪进行检测，结果用FlowJo v.X.0.7统计及GraphPad Prism8.0作图。
检测结果显示抗体H1(scFv-huFc,10ug/ml)特异性结合293T-CLDN6细胞系，EC50值为474.9nM(图2，3)，而不结合293T-CLDN4和293T-CLDN9细胞系(图2)。对照抗体C46-S(scFv-huFc,10ug/ml，来自专利WO2015150327A1)与293T-CLDN4、293T-CLDN6、293T-CLDN9细胞均有显著结合。
实施例3.抗体H1的突变体筛选和鉴定
为了提升抗体H1的亲和力，利用常规分子生物学技术对抗体H1轻链CDR3、重链CDR3分别进行了随机化突变，构建了H1-VH和H1-VL两个噬菌体库，库容均为1E+9。首先将噬菌体库与293T细胞共孵育1-2h，离心取上清与293T-CLDN6细胞共孵育1-2h，清洗；对细胞上的噬菌体进行洗脱，中和洗脱液，随后感染大肠杆菌TG1。扩大培养后进行噬菌体纯化，并用于下一轮筛选。
重复上述筛选过程2-3轮后共挑选约1000个单克隆进行ELISA检测，得到70多个与293T-CLDN6细胞结合较强的克隆，经测序，得到23个序列。将23个克隆进行原核表达、纯化得到单链抗体(scFv)，流式细胞术检测显示其中有4个可以特异性结合293T-CLDN6细胞(如图4所示，空白组(NA)一抗为PBS)，分别命名为P1、P2、P3、P4(抗体浓度为10μg/ml)。
抗体P1的VH的氨基酸序列如SEQ ID NO：5所示，VL的氨基酸序列如SEQ ID NO：3所示；HCDR3的氨基酸序列如SEQ ID NO：41所示；scFv的氨基酸序列如SEQ ID NO：14所示。
抗体P2的VH的氨基酸序列如SEQ ID NO：1所示，VL的氨基酸序列如SEQ ID NO：7所示；LCDR3的氨基酸序列如SEQ ID NO：42所示；scFv的氨基酸序列如SEQ ID NO：15所示。
抗体P3的VH的氨基酸序列如SEQ ID NO：1所示，VL的氨基酸序列如SEQ ID NO：9所示；LCDR3的氨基酸序列如SEQ ID NO：43所示；scFv的氨基酸序列如SEQ ID NO：16所示。
抗体P4的VH的氨基酸序列如SEQ ID NO：1所示，VL的氨基酸序列如SEQ ID NO：11所示；LCDR3的序列如SEQ ID NO：44所示；scFv的氨基酸序列如SEQ ID NO：17所示。
流式细胞检测结果(如图5所示)显示，抗体P1，P2、P3和P4与293T-CLDN6细胞均具有良好的细胞结合亲和力，EC50分别为62.66nM，144.2nM、554.8nM和183.2nM。
实施例4.H1突变体的特异性和亲和力检测
通过分子克隆技术，将实施例3中抗体P1的重链可变区分别与抗体P2和P3的轻链可变区进行组合，得到抗体M1和M2。原核表达纯化后，得到纯化的单链抗体(scFv)。 将M1和M2分别与293T-CLDN6，293T-CLDN4和293T-CLDN9细胞系共孵育，对结合细胞的scFv进行荧光标记，流式分析仪检测，实验数据用FlowJo分析软件计算平均荧光强度(MFI)。
检测结果如图6所示，M1和M2(scFv,5ug/ml)特异性结合293T-CLDN6细胞，不结合293T-CLDN4和293T-CLDN9细胞。
图7显示，M1和M2与293T-CLDN6细胞均有显著结合，EC50分别为23.04nM和24.71nM，与抗体H1相比亲和力提高了近20倍。
实施例5.CLDN6 CAR-T细胞的制备
1.CAR载体的构建
以PRRLSIN-cPPT.EF-1α为载体，构建表达抗体H1、P4的二代嵌合抗原受体的慢病毒质粒PRRLSIN-cPPT.EF-1α-H1-28Z、PRRLSIN-cPPT.EF-1α-P4-28Z。得到H1-28Z的氨基酸序列如SEQ ID NO：48所示；P4-28Z的氨基酸序列如SEQ ID NO：49所示。
对照组C46-S-28Z的氨基酸序列如SEQ ID NO：51所示。
2.CAR-T细胞的制备
采用本领域常规制备CAR-T的方法，采用由PBMC细胞经抗CD3和CD28抗体的磁珠活化后继续培养得到的T细胞，经慢病毒感染后得到CAR-T细胞。
用磷酸钙法进行慢病毒的包装，病毒上清用PEG8000/NaCl进行纯化，纯化后病毒按MOI值为20感染CD3/CD28磁珠活化48小时后的T细胞，得到表达H1-28Z、P4-28Z、C46-S-28Z的CAR-T细胞，未转染病毒的T细胞视为UTD。感染后第5天用FACS法检测CAR阳性率，检测一抗为Biotin-anti-F(ab')2-488(Jackson ImmunoResearch)，二抗为SA-PE(eBioscience)。结果如图8所示。
实施例6.CLDN6 CAR-T细胞对靶细胞的体外特异性杀伤
首先将靶细胞293T-CLDN4细胞，293T-CLDN6细胞，293T-CLDN9细胞，OVCAR3细胞(人卵巢癌细胞,ATCC)和OV90细胞(人卵巢癌细胞,ATCC)分别用1640培养基调整其密度至0.2x10^6/mL，96孔细胞培养板每孔加入50μl，即每孔10000个靶细胞，然后根据效靶比1：1加入50μl效应细胞(CAR-T细胞，UTD做对照)，37℃共孵育16小时后取上清采用LDH试剂盒进行检测，最后用酶标仪进行OD490读数。
结果如图9所示，H1CAR-T细胞和P4CAR-T细胞对293T-CLDN4细胞和293T-CLDN9细胞都没有杀伤作用；对于表达CLDN6的293T-CLDN6，OVCAR3和OV90细胞皆表现出明显的杀伤作用，效靶比3：1时，杀伤率约60％-90％。结果表明H1CAR-T和P4CAR-T细胞在对表达CLDN6的细胞具有特异性的体外杀伤作用。C46-S CAR-T细胞具有非特异性的细胞杀伤作用。
实施例7.RTCA检测分析CLDN6 CAR-T对卵巢癌细胞的体外杀伤
取靶细胞OV90卵巢癌细胞，1E+04 cells/pore接种于RTCA检测孔板中，20h后，按照效靶比1:1分别加入UTD、H1-28Z-CAR T、P4-28Z-CAR T细胞，共培养8h、12h、16h、24h、48h、72h、80h后，检测靶细胞裂解率。如图10所示，H1-28Z-CAR T和 P4-28Z-CAR T对OV90细胞都表现出显著杀伤，48h以后杀伤率达到90％以上，72h杀伤率接近100％。
实施例8.CLDN6-CAR T细胞对荷人卵巢癌细胞的NPG小鼠皮下移植瘤抗肿瘤效果
皮下接种5×106OV90细胞于NPG小鼠中，接种后13天肿瘤平均体积约为234mm3，将小鼠分为4组，尾静脉分别注射UTD(3x106)、H1-28Z(1x106)、H1-28Z(3x106)、P4-28Z(3x106)。结果如图11A所示，CAR-T注射后22天，与UTD相比，H1-28Z(1x106)组抑瘤率为74.11％，H1-28Z(3x106)组抑瘤率为88.26％，P4-28Z组抑瘤率为70.81％。如图11B所示，各CAR-T组的小鼠体重无明显变化。图11C显示，根据瘤重变化，H1-28Z(1x106)组抑瘤率为73.46％，H1-28Z(3x106)组抑瘤率为92.41％，P4-28Z组抑瘤率为71.39％。图11D显示，CAR-T细胞输注后11天，各组小鼠体内的外周血中人T细胞的存活情况。
序列表



Claims (37)

  1. 识别CLDN6的抗体或抗原结合片段，选自下组：
    (1)所述抗体或抗原结合片段包含重链可变区，所述重链可变区的HCDR3与SEQ ID NO：41所示的HCDR3具有至少95％、96％、97％、98％或99％的氨基酸序列同一性；
    (2)所述抗体或抗原结合片段包含重链可变区，所述重链可变区包含GYYMN(SEQ ID NO：35)所示的HCDR1；和/或
    EINPATGSTTYNQKFKA(SEQ ID NO：36)所示的HCDR2；和/或
    RDYYX1GSX2X3YAX4DY(SEQ ID NO:52)所示的HCDR3，其中X1是Y或L或是Y或L的保守性取代氨基酸残基，X2是G或N或是G或N的保守性取代氨基酸残基，X3是F或S或是F或S的保守性取代氨基酸残基，X4是M或L或是M或L的保守性取代氨基酸残基；
    (3)所述抗体或抗原结合片段包含重链可变区，所述重链可变区的HCDR1、HCDR2和HCDR3与SEQ ID NO：35、36和37所示的HCDR1、HCDR2和HCDR3具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性；
    (4)所述抗体或抗原结合片段包含轻链可变区，所述轻链可变区的LCDR3与SEQ ID NO：42、43或44所示的LCDR3具有至少60％、65％、70％、75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性；
    (5)所述抗体或抗原结合片段包含轻链可变区，所述轻链可变区包含QASQSVSNNLN(SEQ ID NO：38)所示的LCDR1；和/或
    GASKLED(SEQ ID NO：39)所示的LCDR2；和/或
    X5QHRX6X7WT(SEQ ID NO:53)所示的LCDR3，其中X5是L或Q或是L或Q的保守性取代氨基酸残基，X6是Y或F或是Y或F的保守性取代氨基酸残基，X7是L或M或是L或M的保守性取代氨基酸残基；
    (6)所述抗体或抗原结合片段包含轻链可变区，所述轻链可变区的LCDR1、LCDR2和LCDR3与SEQ ID NO：38、39和40所示的LCDR1、LCDR2和LCDR3具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列同一性；
    (7)所述抗体或抗原结合片段包含(1)～(3)中任一项所述的重链可变区及(4)～(6)中任一项所述的轻链可变区；
    (8)所述抗体或抗原结合片段是(1)～(7)中任一项所述的抗体或抗原结合片段的变体，所述变体在1、2、3、4、5或6个CDR区上共包含至少一个且不超过7、6、5、4、3或2个氨基酸改变，且具备与(1)～(7)中任一项所述的抗体或抗原结合片段相同或相似的活性。
  2. 如权利要求1所述的抗体或抗原结合片段，其特征在于，选自下组：
    (1)所述的抗体或抗原结合片段包含重链可变区，所述重链可变区包含SEQ ID NO：35所示的HCDR1，和/或SEQ ID NO：36所示的HCDR2，和/或SEQ ID NO：37或41 所示的HCDR3；
    (2)所述的抗体或抗原结合片段包含轻链可变区，所述轻链可变区包含SEQ ID NO：38所示的LCDR1，和/或SEQ ID NO：39所示的LCDR2，和/或SEQ ID NO：40、42、43或44所示的LCDR3；
    (3)所述的抗体或抗原结合片段包含(1)所述的重链可变区及(2)所述的轻链可变区；
    (4)所述的抗体或抗原结合片段是(1)～(3)中任一项所述的抗体或抗原结合片段的变体，所述变体在1、2、3、4、5或6个CDR区上共包含至少一个且不超过7、6、5、4、3或2个氨基酸改变，且具备与(1)～(3)中任一项所述的抗体或抗原结合片段相同或相似的活性。
  3. 如权利要求2所述的抗体或抗原结合片段，其特征在于，选自下组：
    (1)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：40所示的LCDR3；或者
    (2)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：40所示的LCDR3；或者
    (3)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：42所示的LCDR3；或者
    (4)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：43所示的LCDR3；或者
    (5)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：42所示的LCDR3；或者
    (6)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：41所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：43所示的LCDR3；或者
    (7)所述的抗体或抗原结合片段包含SEQ ID NO：35所示的HCDR1、SEQ ID NO：36所示的HCDR2、SEQ ID NO：37所示的HCDR3以及SEQ ID NO：38所示的LCDR1、SEQ ID NO：39所示的LCDR2、SEQ ID NO：44所示的LCDR3；或者
    (8)所述的抗体或抗原结合片段是(1)～(7)中任一项所述的抗体或抗原结合片段的变体，所述变体在1、2、3、4、5或6个CDR区上共包含至少一个且不超过7、6、5、4、3或2个氨基酸改变，且具备与(1)～(7)中任一项所述的抗体或抗原结合片段相同或相似的活性。
  4. 识别CLDN6的抗体或抗原结合片段，选自下组：
    (1)所述的抗体或抗原结合片段包含重链可变区，所述重链可变区包含SEQ ID NO:1或5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
    (2)所述的抗体或抗原结合片段包含轻链可变区，该轻链可变区包含SEQ ID NO:3、7、9或11所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；
    (3)所述的抗体或抗原结合片段包含(1)所述的重链可变区及(2)所述的轻链可变区；
    (4)所述的抗体或抗原结合片段是(1)～(3)中任一项所述的抗体或抗原结合片段的变体，所述变体在VH或VL上包含至少一个且不超过20、15、10、9、8、7、6、5、4、3或2个氨基酸改变，且具备与(1)～(3)中任一项所述的抗体或抗原结合片段相同或相似的活性。
  5. 如权利要求4所述的抗体或抗原结合片段，其特征在于，选自下组：
    (1)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:3所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
    (2)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:3所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
    (3)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:7所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
    (4)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:9所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
    (5)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:7所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
    (6)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:5所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:9所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
    (7)所述抗体或抗原结合片段的重链可变区具有SEQ ID NO:1所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；所述抗体或抗原结合片段的轻链可变区具有SEQ ID NO:11所示的氨基酸序列，或与其具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列；或者
    (8)所述的抗体或抗原结合片段是(1)～(7)中任一项所述的抗体或抗原结合片段的变体，所述变体在VH或VL上包含至少一个且不超过20、15、10、9、8、7、6、5、4、3或2个氨基酸改变，且具备与(1)～(7)中任一项所述的抗体或抗原结合片段相同或相似的活性。
  6. 识别CLDN6的抗体或抗原结合片段，包含重链可变区和轻链可变区，所述轻链可变区包含SEQ ID NO:3、7、9、11所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；和
    所述重链可变区包含SEQ ID NO:35所示的HCDR1，SEQ ID NO:36所示的HCDR2，和SEQ ID NO:37或41所示的HCDR3。
  7. 如权利要求6所述的抗体或抗原结合片段，其特征在于，所述重链可变区包含SEQ ID NO:1、5所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列。
  8. 识别CLDN6的抗体或抗原结合片段，包含重链可变区和轻链可变区，所述重链可变区包含SEQ ID NO:1、5所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列；和
    所述轻链可变区包含SEQ ID NO:38所示的LCDR1，SEQ ID NO:39所示的LCDR2，和SEQ ID NO:40、42、43或44所示的LCDR3。
  9. 如权利要求8所述的抗体或抗原结合片段，其特征在于，所述轻链可变区包含SEQ ID NO:3、7、9或11所示的序列，或与上述序列具有80％、85％、90％、91％，92％，93％，94％，95％，96％，97％，98％，99％同一性的氨基酸序列。
  10. 如权利要求1-9任一项所述的抗体或抗原结合片段，其特征在于，选自全抗、scFv、单域抗体、Fab片段、Fab’片段、Fv片段、F(ab’)2片段、Fd片段、dAb片段、多功能抗体、IgG4抗体、scFv-Fc抗体、杂交瘤抗体、嵌合抗体、人源化抗体、全人源抗体或单克隆抗体。
  11. 如权利要求10所述的抗体或抗原结合片段，其特征在于，包含SEQ ID NO：13、14、15、16、17、18或19所示的氨基酸序列，或与上述序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的氨基酸序列，或包含在上述序列中具有至少一个且不超过20、15、10、9、8、7、6、5、4、3或2个氨基酸改变。
  12. 如权利要求1-11任一所述的抗体或抗原结合片段，其特征在于，所述抗体或抗原结合片段结合CLDN6，不显著结合CLDN4或CLDN9；和/或，所述抗体或抗原结合片段结合表达CLDN6的细胞，不显著结合表达CLDN4、CLDN9或其组合的细胞。
  13. 免疫缀合物，包括：权利要求1-12任一所述的抗体或抗原结合片段，以及与之连接的功能性分子。
  14. 嵌合受体，包含胞外区，所述胞外区包含权利要求1-12任一所述的抗体或抗原结合片段；
    所述嵌合受体包括：嵌合抗原受体(CAR)、嵌合T细胞受体、T细胞抗原耦合器(TAC)、合成多肽受体(synNotch)或其组合。
  15. 如权利要求14所述嵌合受体，其特征在于，所述嵌合受体为嵌合抗原受体
    (CAR)，其包含权利要求1-12任一所述的抗体或抗原结合片段、跨膜区和胞内信号区；优选地，所述抗体或抗原结合片段通过铰链域连接到所述跨膜区。
  16. 如权利要求15所述的嵌合受体，其特征在于，所述跨膜区包含选自TCR的α、β、γ或ζ链、CD3ε、CD3ζ、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD27、CD28、CD33、CD37、CD45、CD64、CD80、CD134、CD137、CD152、CD154或PD1的跨膜区；
    优选地，所述跨膜区选自CD8或CD28的跨膜结构域；
    更优选地，所述跨膜区选自SEQ ID NO:25或22所示的序列，或与其具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
  17. 如权利要求15或16所述的嵌合受体，其特征在于，所述胞内信号区包含初级信号域；
    优选地，所述初级信号域选自TCRξ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b、CD278(也称作“ICOS”)、CD66d或CD3ζ。
  18. 如权利要求15-17任一所述的嵌合受体，其特征在于，所述胞内信号区还包含一个或多个共刺激信号域；
    优选地，所述共刺激信号域选自CARD11、CD2、CD5、CD7、CD27、CD28、CD30、CD40、CD54、CD83、OX40、CD137、CD134、CD150、CD152、CD223、CD270、PD-L2、PD-L1、CD278、DAP10、LAT、NKD2C、SLP76、TRIM、FcεRIγ、MyD88、ICAM-1、LFA-1(CD11a/CD18)、4-1BB或4-1BBL的胞内信号区或其组合；
    更优选地，所述共刺激信号域选自CD28和/或CD137的胞内信号域。
  19. 如权利要求17或18所述的嵌合受体，其特征在于，所述胞内信号区选自SEQ ID NO：24、或包括SEQ ID NO：23和24所示的序列，或包括SEQ ID NO：26和24所示的序列，或包括SEQ ID NO：23、26和24的序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
  20. 如权利要求15所述的嵌合受体，其特征在于，所述铰链区来自CD8、lgG4或lgG1；
    优选地，所述铰链区包含SEQ ID NO：21、27、28、29或30所示的序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
  21. 如权利要求14-20任一所述的嵌合受体，其特征在于，所述的嵌合受体包括：
    权利要求1-12任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区和CD3ζ；或
    权利要求1-12任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区、CD137的胞内信号区和CD3ζ；或
    权利要求1-12任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区、CD28的胞内信号区和CD3ζ；或
    权利要求1-12任一所述的抗体或抗原结合片段、CD8/CD28的跨膜区、CD28的胞内信号区、CD137和CD3ζ。
  22. 如权利要求21所述的嵌合受体，其特征在于，所述嵌合受体包含如SEQ ID NO：13、14、15、16、17、18或19任一所示的序列分别与SEQ ID NO：45、46或47任一所示的序列连接的氨基酸序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列；
    优选地，所述嵌合受体包含如SEQ ID NO：48或49所示的序列，或与上述序列具有至少80％、85％、90％、95％、96％、97％、98％、99％序列同一性的氨基酸序列。
  23. 编码权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求14-22任一所述的嵌合受体的核酸。
  24. 载体，其包含权利要求23所述的核酸。
  25. 细胞，其包含权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求14-22任一所述的嵌合受体、权利要求23所述核酸、和/或权利要求24所述的载体。
  26. 如权利要求25所述的细胞，其特征在于，所述细胞包括T细胞、自然杀伤细胞、自然杀伤T细胞、NK92细胞、细胞毒性T细胞、树突细胞、巨噬细胞、CIK细胞、多能干细胞、干细胞衍生的免疫细胞或其组合。
  27. 如权利要求26所述的细胞，其特征在于，所述T细胞包括天然的T细胞和/或经多能干细胞诱导产生的T细胞；
    优选地，所述T细胞包括自体T细胞和/或同种异体T细胞；
    优选地，所述T细胞为原代T细胞；
    优选地，所述T细胞来源于人的自体T细胞。
  28. 如权利要求25-27任一所述的细胞，其特征在于，所述细胞结合表达CLDN6的细胞，不显著结合表达CLDN4、CLDN9或其组合的细胞。
  29. 如权利要求25-28任一所述的细胞，其特征在于，其还携带外源的细胞因子的编码序列；和/或
    其还表达非靶向CLDN6的嵌合受体；和/或
    其还表达趋化因子；和/或
    其还表达趋化因子受体；和/或
    其还表达安全开关；和/或
    其还表达抑制性分子。
  30. 药物组合物，其包含权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求14-22任一所述的嵌合受体、权利要求23所述核酸、权利要求24所述的载体和/或权利要求25-29任一所述的细胞，以及药学上可接受的佐剂。
  31. 联合用药，其特征在于，权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求14-22任一所述的嵌合受体、权利要求25-29任一所述的细胞、权利要求30所述的药物组合物与增强其功能的药剂组合施用，
    优选地，与化疗药物联用；
    和/或与改善其相关的一种或多种副作用的药剂联合施用；
    和/或与表达靶向CLDN6之外的嵌合抗原受体的细胞联合施用；
    和/或与治疗表达CLDN6相关疾病的药剂联合施用；
    优选地，所述药剂包括抗体、细胞、RNA、疫苗、溶瘤病毒、检查点抑制剂、BKT抑制剂、化学药、放射治疗剂、激素治疗剂、毒素、免疫治疗剂或其组合。
  32. 一种制备权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求14-22任一所述的嵌合受体的方法，所述方法包含培养权利要求25-29任一所述的细胞，并分离出由所述细胞表达的所述抗体、免疫缀合物或嵌合受体。
  33. 一种试剂盒，其特征在于，其包括权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求14-22任一所述的嵌合受体、权利要求23所述核酸、权利要求24所述的载体、权利要求25-29任一所述的细胞和/或权利要求30所述药物组合物。
  34. 如权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求25-29任一所述的细胞、权利要求30所述的药物组合物和/或权利要求33所述的试剂盒的用途，
    (1)杀伤表达CLDN6的细胞；
    (2)抑制表达CLDN6的细胞的增殖；
    (3)介导疾病或肿瘤的缓解；
    (4)预防肿瘤形成或再形成；
    (5)抑制表达CLDN6的细胞的转移；
    (6)制备用于治疗/诊断疾病的药物；
    其特征在于，所述疾病表达CLDN6；优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤；更优选地，所述肿瘤是实体瘤；更优选地，所述肿瘤是卵巢癌、乳腺癌、宫颈癌、胃癌、肺癌、睾丸癌、生殖细胞和胚胎肿瘤、卵巢上皮癌、非小细胞肺癌，非 鳞状非小细胞肺癌、子宫内膜癌、或其组合。
  35. 一种用于以下用途的并且包含权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求25-29任一所述的细胞、权利要求30所述的药物组合物的药剂，
    (1)杀伤表达CLDN6的细胞；
    (2)抑制表达CLDN6的细胞的增殖；
    (3)介导疾病或肿瘤的缓解；
    (4)预防肿瘤形成或再形成；
    (5)抑制表达CLDN6的细胞的转移；
    (6)用于治疗/诊断疾病。
  36. 一种治疗/诊断疾病的方法，其包括向有需要的受试者给予有效量的如权利要求1-12任一所述的抗体或抗原结合片段、或如权利要求13所述的免疫缀合物、或如权利要求25-29任一所述的细胞、或如权利要求30所述的药物组合物、或如权利要求33所述的试剂盒；
    优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤；
    优选地，所述受试者是人；
    优选地，其中所述细胞对于所述受试者是自体的或同种异体的T细胞。
  37. 如权利要求1-12任一所述的抗体或抗原结合片段、权利要求13所述的免疫缀合物、权利要求25-29任一所述的细胞、权利要求30所述的药物组合物和/或权利要求33所述的试剂盒，其用于治疗/诊断疾病，所述疾病包含表达CLDN6；优选地，所述疾病选自炎性病症、感染、自身免疫性疾病和肿瘤；更优选地，所述肿瘤是实体瘤；更优选地，所述肿瘤是卵巢癌、乳腺癌、宫颈癌、胃癌、肺癌、睾丸癌、生殖细胞和胚胎肿瘤、卵巢上皮癌、非小细胞肺癌，非鳞状非小细胞肺癌、子宫内膜癌、或其组合。
PCT/CN2023/126593 2022-10-25 2023-10-25 抗体及其应用 WO2024088325A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211308680 2022-10-25
CN202211308680.4 2022-10-25

Publications (1)

Publication Number Publication Date
WO2024088325A1 true WO2024088325A1 (zh) 2024-05-02

Family

ID=90830067

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/126593 WO2024088325A1 (zh) 2022-10-25 2023-10-25 抗体及其应用

Country Status (1)

Country Link
WO (1) WO2024088325A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106146666A (zh) * 2015-03-26 2016-11-23 科济生物医药（上海）有限公司 靶向cldn6的免疫效应细胞及其制备方法和应用
CN107090043A (zh) * 2011-05-13 2017-08-25 加尼梅德药物公司 用于治疗表达密蛋白6之癌症的抗体
CN107207580A (zh) * 2014-11-05 2017-09-26 艾伯维施特姆森特克斯有限责任公司 抗‑cldn嵌合抗原受体和使用方法
CN113727720A (zh) * 2019-02-08 2021-11-30 生物技术细胞和基因治疗公司 用于治疗表达cldn6的癌症的嵌合抗原受体修饰的细胞
CN113950485A (zh) * 2019-07-10 2022-01-18 中外制药株式会社 密蛋白-6结合分子及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107090043A (zh) * 2011-05-13 2017-08-25 加尼梅德药物公司 用于治疗表达密蛋白6之癌症的抗体
CN107207580A (zh) * 2014-11-05 2017-09-26 艾伯维施特姆森特克斯有限责任公司 抗‑cldn嵌合抗原受体和使用方法
CN106146666A (zh) * 2015-03-26 2016-11-23 科济生物医药（上海）有限公司 靶向cldn6的免疫效应细胞及其制备方法和应用
CN113727720A (zh) * 2019-02-08 2021-11-30 生物技术细胞和基因治疗公司 用于治疗表达cldn6的癌症的嵌合抗原受体修饰的细胞
CN113950485A (zh) * 2019-07-10 2022-01-18 中外制药株式会社 密蛋白-6结合分子及其用途

Similar Documents

Publication Publication Date Title
US10717780B2 (en) Agents for treatment of claudin expressing cancer diseases
KR102584300B1 (ko) 암을 표적하는 키메라 항원 수용체
JP7280827B2 (ja) Ａｘｌまたはｒｏｒ２に対するキメラ抗原受容体およびその使用方法
US20200325223A1 (en) Methods of treatments using antigen-binding proteins targeting cd56
US20200115461A1 (en) Compositions and methods for adoptive cell therapies
KR20210057705A (ko) 세포 요법을 위한 다양한 항원 결합 도메인, 신규한 플랫폼 및 다른 향상
KR20190034588A (ko) 키메라 항원 수용체 및 pd-1 억제제의 조합 요법
EP3875484A1 (en) Cll1-targeting antibody and application thereof
KR20180002604A (ko) 혈액 악성종양을 표적으로 하는 키메라 항원 수용체(car), 조성물 및 이의 용도
KR20210138574A (ko) Dll3 표적화 키메라 항원 수용체 및 결합제
CN116063565A (zh) 靶向Fc受体样5的嵌合抗原受体及其用途
JP2021515558A (ja) 二量体抗原受容体（ｄａｒ）
KR102412805B1 (ko) 세포 면역 요법을 위한 조성물 및 방법
KR20230129983A (ko) 조작된 세포 요법을 위한 표적화된 사이토카인 구축물
US20210403885A1 (en) Chimeric adaptor and kinase signaling proteins and their use in immunotherapy
EP2920209B1 (en) Agents for treatment of claudin expressing cancer diseases
WO2024088325A1 (zh) 抗体及其应用
KR20220030248A (ko) 4-1bb 공동자극 도메인을 갖는 키메라 항원 수용체
WO2023143537A1 (zh) Gprc5d抗体及其应用
WO2023138661A1 (zh) Nkg2a抗体及其应用
CN117645670A (zh) 一种新型嵌合抗原受体及其用途
KR20240034205A (ko) 항EGFRviii 항체, 폴리펩타이드, 상기 폴리펩타이드를 발현하는 세포, 상기 세포를 포함하는 의약 조성물, 상기 세포의 제조 방법 및 상기 폴리펩타이드를 코딩하는 염기서열을 포함하는 폴리뉴클레오티드 또는 벡터