WO2022268162A1 - 治疗肿瘤的方法和组合物 - Google Patents

治疗肿瘤的方法和组合物 Download PDF

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WO2022268162A1
WO2022268162A1 PCT/CN2022/100762 CN2022100762W WO2022268162A1 WO 2022268162 A1 WO2022268162 A1 WO 2022268162A1 CN 2022100762 W CN2022100762 W CN 2022100762W WO 2022268162 A1 WO2022268162 A1 WO 2022268162A1
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cells
subject
seq
day
cell
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PCT/CN2022/100762
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English (en)
French (fr)
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李宗海
汪薇
肖珺
袁岱菁
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科济生物医药（上海）有限公司
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Priority to CN202280044143.5A priority Critical patent/CN117561281A/zh
Publication of WO2022268162A1 publication Critical patent/WO2022268162A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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

Definitions

  • This application belongs to the field of immunotherapy; specifically, it relates to immune cell therapy that targets and recognizes tumor antigens, triggers the activation of immune effector cells, and exerts anti-tumor effects.
  • the present application provides a method for treating a subject with or suspected of having a CLD18-positive biliary tract tumor, comprising administering to the subject a cell targeting an exogenous receptor of CLD18.
  • the present application also provides a use of a cell targeting the exogenous receptor of CLD18 for preparing a medicament for treating a subject with or suspected of having a CLD18-positive biliary tract tumor.
  • the present application also provides a use of a cell expressing an exogenous receptor targeting CLD18 and chemical drugs or other biological drugs or radiotherapy for preparing a drug for treating a subject with or suspected of having a CLD18-positive biliary tract tumor.
  • the present application also provides a use of a cell targeting an exogenous receptor of CLD18 for treating a subject with or suspected of having a CLD18-positive biliary tract tumor.
  • the present application also provides a use of cells expressing exogenous receptors targeting CLD18 and chemical drugs or other biological drugs or radiotherapy for treating subjects with or suspected of having CLD18-positive biliary tract tumors.
  • the CLD18 is CLD18.2.
  • the biliary tract tumor includes gallbladder cancer and cholangiocarcinoma.
  • the cells comprise immune effector cells.
  • the immune effector cells are selected from T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells, stem cell-derived immune effector cells or combinations thereof.
  • the immune effector cells are derived from natural T cells and/or T cells induced by pluripotent stem cells.
  • the immune effector cells are autologous or allogeneic T cells, or primary T cells.
  • the T cells include 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, ⁇ T cells, or combinations thereof.
  • the exogenous receptor is selected from chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC), antibody- TCR chimera or combination thereof; the antigen binding domain of the exogenous receptor specifically binds to CLD18.2.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TCP T cell fusion protein
  • TAC T cell antigen coupler
  • antibody- TCR chimera or combination thereof; the antigen binding domain of the exogenous receptor specifically binds to CLD18.2.
  • the chimeric antigen receptor includes:
  • the subject is given at least one cycle of the cells for treatment; preferably, the subject is given 1-3 cycles of the cells for treatment.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x10 9 cells/kg body weight of the subject, 2x10 8 cells/kg body weight of the subject, or 2x10 7 cells/kg The weight of the subject; or the dose of cells in the cell therapy product does not exceed about 1x1011 cells/subject, 1x1010 cells/subject, 5x109 cells/subject, 2x109 cells/subject or 1x109 cells/subject.
  • the dose of cells in the cell therapy product administered per cycle is about 1x10 5 cells/kg subject body weight to 2x10 7 cells/kg subject body weight, or about 1x10 6 cells/kg Subject body weight up to 2x107 cells/kg subject body weight; or
  • the dose of cells in the cell therapy product administered per cycle is about 1x107 cells to 5x109 cells/subject, about 1x107 cells to 2x109 cells/subject, or about 1x107 cells to 1x10 9 cells/subject; or
  • the dose of cells in the cell therapy product administered per cycle is about 1x108 cells to 5x109 cells/subject, about 1x108 cells to 2x109 cells/subject, or about 1x108 cells to 1x10 9 cells/; or
  • the dose of cells in the cell therapy product administered per cycle is about 2.5x108 cells to 5x108 cells/subject.
  • pretreatment is carried out before administering the cell therapy product in each cycle, and the pretreatment includes administering chemical drugs, biological drugs (other biological drugs, herein other biological drugs are different from the cells targeting exogenous receptors of CLD18), radiotherapy or a combination thereof.
  • the pretreatment is implemented 1-8 days before the administration of the cell therapy product; preferably, it is implemented 2-6 days before the administration of the cell therapy product; preferably, each chemical drug, biological drug (other biological Drugs), radiotherapy or a combination thereof for no more than 4 consecutive days.
  • the chemical drug is selected from any one or at least two of the following: cyclophosphamide, fludarabine, tubulin inhibitors, pyrimidine antineoplastic drugs; or the chemical drug includes cyclophosphamide amide and fludarabine; or the chemotherapeutics include cyclophosphamide, fludarabine, and tubulin inhibitors.
  • the tubulin inhibitor includes taxane compounds; or the tubulin inhibitor includes paclitaxel, nab-paclitaxel, docetaxel; or the tubulin inhibitor It is albumin-bound paclitaxel.
  • the dosage of fludarabine is about 10-50 mg/m 2 /day, or about 15-40 mg/m 2 /day, or about 15-30 mg/m 2 /day, or about 20- 30 mg/m 2 /day; or about 25 mg/m 2 /day, or about 30-60 mg/day, or about 30-50 mg/day, or about 35-45 mg/day;
  • the dosage of cyclophosphamide is about 200-400mg/m 2 /day, or about 200-300mg/m 2 /day, or about 250mg/m 2 /day, or about 300-1000mg/day, or about 300- 550mg/day, or about 300-500mg/day;
  • the dosage of taxane compounds is not higher than about 300 mg/day, or not higher than about 200 mg/day, or about 90-120 mg/day.
  • the cyclophosphamide is administered 2-3 times; or the fludarabine is administered 1-2 times; or the taxane compound is administered once.
  • the antigen-binding domain or the antibody has: the scFv sequence shown in SEQ ID NO: 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33; or
  • the antigen binding domain or the antibody has HCDR1 shown in SEQ ID NO: 14, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 16, LCDR1 shown in SEQ ID NO: 17, LCDR2 set forth in SEQ ID NO: 18, LCDR3 set forth in SEQ ID NO: 19; or
  • the antigen-binding domain or the antibody has a heavy chain variable region shown in SEQ ID NO: 10, and a light chain variable region shown in SEQ ID NO: 12.
  • the chimeric antigen receptor has the scFv sequence shown in SEQ ID NO: 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 and SEQ ID NO: ID NO: 34, 35 or 36 sequentially connected sequences; or the chimeric antigen receptor has the nucleotide sequence shown in SEQ ID NO: 37, 38 or 39.
  • the subject's cytokines indicating CRS, cytokines indicating neurotoxicity, indicators indicating tumor burden, and/or indicators indicating host anti-CAR Serum levels of immune response factors were evaluated.
  • the subject after the administration of the cell therapy product, the subject does not show severe CRS, or does not show neurotoxicity exceeding grade 3.
  • the subsequent cycle of treatment is given after the cells in the previous cycle are undetectable in vivo after treatment.
  • the dose of cells administered in the subsequent cycle is sufficient to stabilize or reduce the number of cells in the subject's tumor burden.
  • the cells in each cycle are divided into 1-5 times of administration, preferably, divided into 1-3 times of administration.
  • the subject when the treatment in the later period is given, the subject has any of the following characteristics:
  • the subject does not exhibit a detectable humoral or cell-mediated immune response to the cell therapy product.
  • the level of cytokines is reduced by at least 50%, preferably by at least 20%, compared with the peak level of cytokines after administration of the previous cycle of cell therapy products, More preferably, a reduction of at least 5%.
  • the CRS level is equivalent to the CRS level before administration of the previous cycle of cell therapy products.
  • pretreatment is performed before administering the cells in each cycle, and the pretreatment includes administering chemical drugs and/or radiotherapy to the subject.
  • the radiotherapy includes whole body radiation therapy or partial radiation therapy.
  • the pretreatment is carried out 1-8 days before the administration of the cells; preferably, it is carried out 2-6 days before the administration of the cells.
  • the chemical drug is selected from any one or at least two of the following: cyclophosphamide, fludarabine, tubulin inhibitors, and pyrimidine antineoplastic drugs.
  • the chemical drugs are cyclophosphamide and fludarabine; or cyclophosphamide, fludarabine and tubulin inhibitors.
  • the tubulin inhibitor is a taxane compound
  • the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, and docetaxel;
  • the taxane compound is albumin-bound paclitaxel.
  • the pyrimidine antineoplastic drug is selected from 5-fluorouracil, bisfururacil, carmofur, doxifluridine, and capecitabine.
  • each chemical drug is used continuously for no more than 4 days.
  • the cyclophosphamide is administered 2-3 times; or the fludarabine is administered 1-2 times.
  • the taxane compound is administered once.
  • Figures 2A and 2B are measurements of tumor volume and tumor weight.
  • the inventors unexpectedly found that the immune effector cells (for example, T cells) expressing CLDN18.
  • the methods, immune effector cells and compositions provide or achieve an improved or longer-lasting response or efficacy and/or a reduced risk of toxicity or other side effects compared to the prior art.
  • the method treats a specific subject group by administering a specified or relative amount of cells, thereby significantly improving the efficacy against biliary tract tumors.
  • the range indicated by "about” covers ⁇ 1% of the given value, ⁇ 2% of the given value, ⁇ 3% of the given value, ⁇ 4% of the given value, ⁇ 5% of the given value, ⁇ 6% of the given value, Given value ⁇ 7%, given value ⁇ 8%, given value ⁇ 9%, given value ⁇ 10%, given value ⁇ 11%, given value ⁇ 12%, given value ⁇ 13%, given value Set value ⁇ 14%, given value ⁇ 15%, given value ⁇ 16%, given value ⁇ 17%, given value ⁇ 18%, given value ⁇ 19%, given value ⁇ 20%, given value Value ⁇ 25%, given value ⁇ 30%.
  • the term may mean within an order of magnitude of a value, such as within about 5 times or within about 2 times a value.
  • Ranges The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the application. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range.
  • amino acid or nucleic acid sequence described in this application has a specific sequence means that the amino acid or nucleic acid sequence has more than 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85% of the specific sequence. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Sequence identity can be measured by sequence analysis software (eg, programs such as BLAST, BESTFIT, GAP, PILEUP/PRETTYBOX, etc.).
  • Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and/or other modifications.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine acid lysine, arginine; and phenylalanine, tyrosine.
  • the BLAST program can be used, wherein a probability score between e-3 and e-100 is indicative of closely related sequences.
  • exogenous refers to a nucleic acid molecule or polypeptide that is not endogenously present in the cell, or is not expressed at a level sufficient to function when overexpressed; encompasses any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as an exogenous , heterologous and overexpressed nucleic acid molecules or polypeptides.
  • exogenous receptors include chimeric receptors, which refer to fusion molecules formed by linking DNA fragments from different sources or corresponding cDNA or polypeptide fragments of proteins using gene recombination technology, including extracellular domains, transmembrane domains and cellular receptors. inner domain.
  • Exogenous receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor (TCR), T Cell Antigen Coupler (TAC), T Cell Fusion Protein (TFP).
  • CAR includes an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • the extracellular antigen binding domain of the CAR comprises a scFv.
  • the intracellular signaling domain comprises a functional signaling domain of a stimulatory molecule and/or a co-stimulatory molecule; or comprises the entire intracellular portion of a stimulatory molecule and/or a co-stimulatory molecule, or the entire native intracellular signaling domain, or Its functional fragments or derivatives.
  • T cell receptor mediates T cell recognition of specific major histocompatibility complex (MHC)-restricted peptide antigens, including native TCR receptors and modified TCR receptors.
  • MHC major histocompatibility complex
  • the natural TCR receptor consists of two peptide chains ⁇ and ⁇ , and each peptide chain can be divided into a variable region (V region), a constant region (C region), a transmembrane region and a cytoplasmic region, etc., and its antigen-specific Sex exists in the V zone.
  • V region variable region
  • C region constant region
  • T cell fusion proteins T cell fusion proteins
  • antibody-TCR chimeras include, but are not limited to, chimeric T cell receptors (TCRs), T cell antigen couplers (TACs), T cell fusion proteins (TFPs), antibody-TCR chimeras.
  • Specific binding means that a polypeptide or fragment thereof recognizes and binds to a biomolecule of interest (eg, a polypeptide) but does not substantially recognize and bind to other molecules in a sample. Binding affinity can be determined using standard binding assays.
  • antigenic domain refers to molecules that specifically bind an antigenic determinant, including immunoglobulin molecules and immunologically active portions of immunological molecules, i.e., molecules that contain an antigen binding site that specifically binds ("immunoreacts") with an antigen .
  • antibody includes not only intact antibody molecules but also fragments of antibody molecules that retain antigen-binding ability.
  • antibody is used interchangeably with the term “immunoglobulin” and "antigenic domain” in this application.
  • an antibody comprises at least two heavy (H) chains and two light (L) chains linked by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • CH consists of three structural domains CH1, CH2, and CH3.
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • CL consists of one domain.
  • VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains include binding domains that interact with antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, immune effector cells) and the first component (Clq) of the classical complement system.
  • An antigenic domain "specifically binds" or is "immunogenic" to an antigen if the antigenic domain binds the antigen with greater affinity (or avidity) than other reference antigens (including polypeptid
  • composition refers to a mixture of two or more substances. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.
  • activation and “activation” are used interchangeably and can refer to the process by which a cell transitions from a quiescent state to an active state.
  • the process can include responses to antigenic, phenotypic or genetic changes in migration and/or functional activity status.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form, including any nucleic acid molecule encoding a polypeptide of interest or a fragment thereof.
  • the nucleic acid molecule only needs to maintain basic identity with the endogenous nucleic acid sequence, and does not need to have 100% homology or identity with the endogenous nucleic acid sequence.
  • a polynucleotide having "substantial identity" to an endogenous sequence will generally hybridize to at least one strand of a double-stranded nucleic acid molecule.
  • substantially identity or “substantial homology” refers to a polypeptide or nucleic acid molecule that exhibits at least about 50% homology or identity to a reference amino acid sequence or nucleic acid sequence.
  • peptide refers to a compound consisting of amino acid residues covalently linked by peptide bonds.
  • the exogenous receptor in this application refers to a fusion molecule formed by linking DNA fragments from different sources or corresponding cDNAs of proteins by gene recombination technology, including the extracellular domain, transmembrane domain and intracellular domain that specifically bind to CLD18; including but not Limited to: chimeric antigen receptor (CAR), chimeric T cell receptor (TCR), T cell antigen coupler (TAC), T cell fusion protein (TFP), antibody-TCR chimera.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TAC T cell antigen coupler
  • T cell fusion protein T cell fusion protein
  • the present application provides an exogenous receptor that specifically binds a CLD18.2 polypeptide.
  • the exogenous receptor binds to the extracellular domain of the CLD18.2 polypeptide.
  • the present application provides a CAR that specifically binds to a CLD18.2 polypeptide.
  • CLD18 polypeptide refers to any variant, derivative or isoform of the CLD18 gene or encoded protein, including splice variant 1 (Claudin 18.1, CLD18.1, CLDN18.1): NP_057453 , NM016369, and splice variant 2 (Claudin 18.2, CLD18.2, CLDN18.2): NP_001002026, NM_001002026.
  • the CLD18.2 polypeptide is a human CLD18.2 polypeptide, comprising at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% of the sequence shown in SEQ ID NO:9 , 99% or 100% homology or identity amino acid sequences or fragments thereof, and/or may optionally include up to one or up to two or up to three conservative amino acid substitutions.
  • the CLD18.2 polypeptide is strongly expressed in several cancer types, including gallbladder and cholangiocarcinomas.
  • the exogenous receptor included in the present application is a CAR that specifically binds to the CLD18 polypeptide.
  • the CAR specifically binds the CLD18.2 polypeptide.
  • the CAR specifically binds to the extracellular domain of the CLD18.2 polypeptide.
  • the various domains of the CAR polypeptide are in the same polypeptide chain, eg, expressed as a single polypeptide chain.
  • the individual domains of the CAR polypeptide are not contiguous to each other, e.g., are in different polypeptide chains.
  • the antigenic domain comprises an antibody. In one example, the antigenic domain comprises scFv. In one example, the antigenic domain comprises an antibody heavy chain variable region (VH) and/or light chain variable region (VL); or comprises a cross-linked Fab; or comprises F(ab) 2 . In one example, the antigenic domain comprises antibody VH and VL, forming a variable fragment (Fv).
  • the CAR includes an antibody that specifically binds to the CLD18.2 polypeptide.
  • the antigenic domain of the CAR includes an Fv that specifically binds the CLD18.2 polypeptide.
  • the antigen binding domain of the CAR comprises: HCDR1 shown in SEQ ID NO: 14, HCDR2 shown in SEQ ID NO: 15, HCDR3 shown in SEQ ID NO: 16, HCDR3 shown in SEQ ID NO: 17 LCDR1, LCDR2 shown in SEQ ID NO: 18, LCDR3 shown in SEQ ID NO: 19.
  • the antigen-binding domain of the CAR comprises: VH shown in SEQ ID NO: 10, VL shown in SEQ ID NO: 12.
  • the antigen-binding domain of the CAR comprises: the scFv sequence shown in SEQ ID NO: 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33.
  • the present application contemplates modification of the amino acid sequence of the starting antibody (eg, VH or VL) to yield a functionally equivalent molecule.
  • the CAR can be modified to include at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% of the VH or VL that specifically binds to the antigenic domain of the CLD18.2 polypeptide , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% identity sequences.
  • the antibodies of the application can be further modified such that they have changes in amino acid sequence (eg, relative to wild type) but no change in the desired activity.
  • additional nucleotide substitutions can be made to the protein, resulting in amino acid substitutions at "non-essential" amino acid residues.
  • a non-essential amino acid residue in a molecule may be replaced by another amino acid residue from the same side chain family.
  • amino acid stretches may be substituted with amino acid stretches that are structurally similar but differ in sequence and/or composition from members of the side chain family, for example, conservative substitutions may be made wherein amino acid residues are replaced by amino acids with similar side chains residue replaced.
  • the antigenic domain is linked directly to the transmembrane domain or via a hinge.
  • the hinge comprises a CD8 hinge, for example, a sequence having 95-100% identity to the sequence shown in SEQ ID NO:3.
  • a nucleic acid molecule encoding a CAR includes a polynucleotide encoding a signal peptide upstream.
  • the sequence of the signal peptide has the sequence shown in SEQ ID NO:1.
  • the transmembrane domain can anchor the CAR to the cell membrane.
  • the transmembrane domain of the CAR molecule of the present application includes a transmembrane domain selected from the following: T cell receptor ⁇ , ⁇ , or ⁇ transmembrane region, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16 , CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGA
  • the CAR comprises a CD8 transmembrane region, a sequence having 95-100% identity to the sequence shown in SEQ ID NO:4. In one example, the CAR comprises a CD28 transmembrane region, a sequence having 90-100% identity to the sequence shown in SEQ ID NO:5.
  • the CAR provided herein includes an intracellular signaling domain.
  • the intracellular signaling domain includes a primary signaling domain.
  • the intracellular signaling domain includes a co-stimulatory signaling domain.
  • the intracellular signaling domain includes a primary signaling domain and a co-stimulatory signaling domain.
  • the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, FcyRIIa, DAP10, and DAP12.
  • the primary signaling domain includes the intracellular domain of CD3 ⁇ , a sequence having 90-100% identity to the sequence shown in SEQ ID NO:8.
  • the co-stimulatory signaling domain includes a functional signaling domain selected from one or more of the following proteins: CD27, CD28, CD137, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-related antigen -1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA- 1.
  • a functional signaling domain selected from one or more of the following proteins: CD27, CD28, CD137, OX
  • the co-stimulatory signaling domain includes the intracellular domain of CD137, a sequence having 90-100% identity to the sequence shown in SEQ ID NO:6.
  • the intracellular signaling domain of the CAR comprises the human CD3 ⁇ intracellular domain. In one example, the intracellular signaling domain of the CAR includes a human CD3 ⁇ intracellular domain and a CD28 intracellular domain. In one example, the intracellular signaling domain of the CAR includes a human CD3 ⁇ intracellular domain and a CD137 intracellular domain. In one example, the intracellular signaling domain of the CAR includes a CD3 ⁇ intracellular domain, a CD28 intracellular domain, and a CD137 intracellular domain.
  • the present application contemplates modification of the entire CAR molecule, eg, modification of one or more amino acid sequences of each domain of the CAR molecule, in order to generate a functionally equivalent molecule.
  • the modifiable CAR molecule retains at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% of the starting CAR molecule , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % identity.
  • Exemplary exogenous receptors such as the sequence comprising CAR has the scFv sequence shown in SEQ ID NO: 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 and SEQ ID NO: NO: 34, 35 or 36 sequentially linked sequences.
  • the sequence shown in SEQ ID NO: 2 is connected in sequence with the sequence shown in SEQ ID NO: 34, or the sequence shown in SEQ ID NO: 2 is connected in sequence with the sequence shown in SEQ ID NO: 35, and SEQ ID NO: 2
  • the sequence shown is sequentially linked with the sequence shown in SEQ ID NO: 36; the sequence shown in SEQ ID NO: 23 is sequentially linked with the sequence shown in SEQ ID NO: 34, or the sequence shown in SEQ ID NO: 23 is connected with the sequence shown in SEQ ID NO:
  • the sequence shown in 35 is connected sequentially, the sequence shown in SEQ ID NO: 23 is connected in sequence with the sequence shown in SEQ ID NO: 36; the sequence shown in SEQ ID NO: 24 is connected in sequence with the sequence shown in SEQ ID
  • modified TCR receptor specific binding CLD18.2 polypeptides including but not limited to: chimeric T cell receptor, T cell fusion protein (TFP), T cell antigen coupler (TAC), antibody-TCR chimera .
  • the antigen binding domain of the modified TCR receptor has at least about 70% of the sequence set forth in SEQ ID NO: 2, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the host cell provided in the present application is a cell expressing a foreign receptor that specifically binds to the CLD18.2 polypeptide.
  • the cells can be activated after the exogenous receptor is combined with the CLD18.2 polypeptide.
  • host cells include stem cells.
  • Stem cells include human pluripotent stem cells (including human induced pluripotent stem cells (iPSC) and human embryonic stem cells).
  • host cells include immune effector cells (also known as immune cells).
  • the host cell is a primary cell.
  • the present application provides a cell therapy product comprising said host cell.
  • a host cell may refer to a human or non-human cell of animal origin.
  • the immune effector cells of the present application include cells of the lymphoid lineage.
  • the lymphoid lineage including B, T, and natural killer (NK) cells provide for antibody production, regulation of the cellular immune system, detection of exogenous agents in the blood, detection of foreign cells to the host, etc.
  • Non-limiting examples of immune effector cells of the lymphoid lineage include T cells, natural killer T (NKT) cells and their precursors, including embryonic stem cells and pluripotent stem cells (eg, stem cells that differentiate into lymphoid cells or pluripotent stem cells).
  • T cells include lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system.
  • T cells include any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-like memory T cells (or stem-like memory T cells), and both effector memory T cells: eg TEM cells and TEMRA cells), regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, ⁇ T cells or ⁇ T cells.
  • cytotoxic T cells are T lymphocytes capable of inducing the death of infected somatic or tumor cells.
  • the cells of the present application are selected from T cells, NK cells, cytotoxic T cells, NKT cells, macrophages, CIK cells, and stem cell-derived immune effector cells or combinations thereof.
  • the immune effector cells are T cells.
  • T cells include CD4+ T cells and/or CD8+ T cells.
  • immune effector cells include CD3+ T cells.
  • the cells in the composition of the present application include a cell population collected from PBMC cells stimulated by CD3 magnetic beads.
  • the immune effector cells are selected from T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells, stem cell-derived immune effector cells, or combinations thereof.
  • the immune effector cells are derived from natural T cells and/or T cells induced by pluripotent stem cells.
  • the immune effector cells are autologous or allogeneic T cells, or primary T cells.
  • the T cells include 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, ⁇ T cells, or combinations thereof.
  • the cells (eg, T cells) of the present application can be autologous, non-autologous (eg, allogeneic), or derived in vitro from engineered progenitor or stem cells. It can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMC peripheral blood mononuclear cells
  • T cells can be obtained from a blood sample collected from a subject using any number of techniques known to those of skill in the art, such as the Ficoll TM separation technique.
  • the cells from the circulating blood of the individual are obtained by apheresis.
  • Apheresis products usually contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or culture medium for subsequent processing steps. Multiple rounds of selection can also be used in the context of the present application. In some aspects, it may be desirable to perform a selection procedure and use "unselected" cells during activation and expansion. "Unselected" cells can also undergo additional rounds of selection.
  • the cells of the present application are capable of modulating the tumor microenvironment.
  • the source of unpurified CTLs can be any source known in the art, such as bone marrow, fetal, neonatal or adult or other source of hematopoietic cells, such as fetal liver, peripheral blood or umbilical cord blood.
  • Cells can be isolated using various techniques. For example, negative selection can initially remove non-CTLs.
  • mAbs are particularly useful for identifying markers associated with specific cell lineages and/or differentiation stages of positive and negative selection.
  • Most of the terminally differentiated cells can be removed initially by relatively rough dissection.
  • magnetic bead separation can be used initially to remove large numbers of irrelevant cells.
  • at least about 80%, usually at least about 70%, of the total hematopoietic cells will be removed prior to isolating the cells.
  • Separation procedures include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that alter cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; agents, including but not limited to complement and cytotoxins; and panning with antibodies attached to a solid substrate (eg, plate, chip, elutriation) or any other convenient technique.
  • a solid substrate eg, plate, chip, elutriation
  • Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, such as multiple color channels, low- and obtuse-angle light-scattering detection channels, impedance channels.
  • Cells can be selected for dead cells by using dyes associated with dead cells, such as propidium iodide (PI).
  • PI propidium iodide
  • cells are harvested in medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA), or any other suitable, eg, sterile isotonic medium.
  • FCS fetal calf serum
  • BSA bovine serum albumin
  • Vectors herein comprise isolated nucleic acids and are useful for delivering isolated nucleic acids to compositions inside cells, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • Vectors include autonomously replicating plasmids or viruses.
  • Vectors also include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like.
  • viral vectors AAV, retroviruses, or lentiviruses
  • AAV AAV, retroviruses, or lentiviruses
  • CAR foreign receptor
  • Non-viral vectors can also be used.
  • Transduction can use any suitable viral vector or non-viral delivery system.
  • CARs can be constructed with accessory molecules (eg, cytokines) in a single polycistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors.
  • elements for generating polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF- ⁇ B IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, abaculovirus IRES, picornavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers ( For example 2A peptides such as P2A, T2A, E2A and F2A peptides).
  • IRES viral and non-viral internal ribosome entry sites
  • cleavable linkers For example 2A peptides such as P2A, T2A, E2A and F2A peptides.
  • viral vectors that may be used include, for example, adenovirus, lentivirus and adeno-associated viral vectors, vaccinia virus, bovine papilloma virus or herpes viruses such as Epstein-Barr virus.
  • Non-viral methods can also be used for the genetic modification of immune effector cells.
  • nucleic acid molecules can be introduced into immune effector cells by microinjection under lipofection, asialomucoid-polylysine conjugation, or surgical conditions.
  • Other non-viral methods of gene transfer include in vitro transfection using liposomes, calcium phosphate, DEAE-dextran, electroporation and protoplast fusion. It is also possible to first transfer the nucleic acid molecule into a cell type that can be cultured in vitro (for example, an autologous or allogeneic primary cell or its progeny), and then inject the cell (or its progeny) modified by the nucleic acid molecule into Subject target tissue or systemic injection.
  • the composition of the present application includes said host cell or said cell therapy product.
  • the composition is provided systemically or directly to a subject to induce and/or enhance an immune response to the CLD18.2 polypeptide and/or treat and/or prevent biliary tract tumors.
  • the composition is injected directly into the organ of interest (eg, an organ affected by a tumor).
  • the composition is provided to the organ of interest indirectly, eg, by administration to the circulatory system (eg, vein, tumor vasculature).
  • Expansion and differentiation agents can be provided prior to, concurrently with or following administration to increase expansion of T cells, NKT cells or CTL cells in vitro or in vivo.
  • Immune effector cells in the compositions of the present application may include purified cell populations.
  • One skilled in the art can readily determine the percentage of immune effector cells of the application in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Suitable ranges for purity are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70% in a population comprising the immune effector cells of the present application. In one example, the purity is from about 70% to about 75%, from about 75% to about 80%, or from about 80% to about 85%. In one example, the purity is from about 85% to about 90%, from about 90% to about 95%, and from about 95% to about 100%.
  • Cells can be introduced by injection, catheter, and the like.
  • the composition of the present application may be a pharmaceutical composition comprising the immune effector cells or their progenitor cells of the present application and a pharmaceutically acceptable carrier.
  • Administration can be autologous or allogeneic.
  • immune effector cells or progenitor cells can be obtained from one subject and administered to the same subject or to a different compatible subject.
  • Peripheral blood-derived immune effector cells or their progeny eg, in vivo, ex vivo, or in vitro sources
  • they may be formulated in unit dose injectable forms (solutions, suspensions, emulsions, etc.).
  • compositions comprising the present application are provided in the form of sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered to a selected pH.
  • sterile liquid preparations such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which may be buffered to a selected pH.
  • Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection.
  • viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with a particular tissue.
  • Liquid or viscous compositions can include a carrier, which can be a solvent or dispersion medium including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable suitable ones. mixture.
  • a carrier which can be a solvent or dispersion medium including, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable suitable ones. mixture.
  • Sterile injectable solutions can be prepared by incorporating the immune effector cells in the composition of the present application into the required amount of an appropriate solvent, and incorporating different amounts of other ingredients as needed.
  • Such compositions can be mixed with suitable carriers, diluents or excipients such as sterile water, physiological saline, glucose, dextrose and the like.
  • Compositions can also be lyophilized.
  • the composition may include auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-increasing agents, preservatives, flavoring agents, pigments, etc., This depends on the route of administration and formulation desired.
  • additives can be added to enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffering agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical forms can be brought about by the use of agents which delay absorption, for example, aluminum monostearate and gelatin. However, any vehicle, diluent or additive used will have to be compatible with the genetically modified immune effector cells or their progenitors.
  • compositions may be isotonic, ie they may have the same osmotic pressure as blood and/or tear fluid.
  • the desired isotonicity of the compositions can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • Sodium chloride may be particularly useful for buffers containing sodium ions.
  • a pharmaceutically acceptable thickening agent can be used to maintain the viscosity of the composition at a selected level.
  • methylcellulose is readily and economically available and easy to use.
  • suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like.
  • concentration of the thickener can depend on the agent chosen. It is important to use the amount that will achieve the chosen viscosity.
  • suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is formulated as a solution, suspension, gel, or other liquid form, e.g. time-release or liquid-filled form).
  • the number of cells in the composition to be administered will vary for the subject being treated. More potent cells can be administered in smaller numbers.
  • the precise determination of an effective dose can be determined according to each subject's individual factors, including its size, age, sex, weight and the condition of the subject. Dosages can be readily determined by those skilled in the art from this application and knowledge in the art.
  • any additives are present in 0.001% to 50% by weight solution in phosphate-buffered saline, and the active ingredient is present in micrograms to The order of milligrams is present, for example from about 0.0001 wt% to about 5 wt%, from about 0.0001 wt% to about 1 wt%, from about 0.0001 wt% to about 0.05 wt%, or from about 0.001 wt% to about 20 wt%, from about 0.01 wt% to about 10 wt% % or from about 0.05 wt% to about 5 wt%.
  • toxicity for example by determining the lethal dose (LD) and LD50 in a suitable animal model, e.g. rodents such as mice; the dose of the composition, wherein The concentration of the components and the time of application of the composition elicit an appropriate response.
  • LD lethal dose
  • LD50 LD50
  • suitable animal model e.g. rodents such as mice
  • the present application provides a method of treating a subject with or suspected of having a CLD18-positive biliary tract tumor.
  • Biliary tract tumors include gallbladder cancer and bile duct cancer.
  • the method treats a subject who is "positive" for the CLD18.2 polypeptide of the biliary tract tumor cells, such as by immunological detection (eg, flow cytometry, immunohistochemistry, etc.) of the CLD18.2 polypeptide of the biliary tract tumor cells.
  • the expression level of is significantly higher than the control using isotype matching, and/or substantially similar to the level of cells known to be positive for CLD18.2 polypeptides, and/or significantly higher than that of cells known to be negative for CLD18.2 polypeptides Level; the subject can obtain clinical benefit after being treated with the method.
  • the method treats a subject whose biliary tract tumor cells are "negative" for the CLD18.2 polypeptide, e.g., when stained with an antibody that specifically binds the CLD18.2 polypeptide, said staining being immunologically detectable Detected at a level that is significantly lower than the level of staining detected when the same procedure is performed under the same conditions using an isotype-matched control, and/or significantly lower than the level of staining known to be effective for the CLD18.2 polypeptide
  • Clinical benefit means that the subject responds to the method or has a therapeutic effect.
  • "Response” in this article refers to complete remission CR (Complete Response, all target lesions disappear, and the short diameter of all pathological lymph nodes must be reduced to ⁇ 10mm) according to the Response Evaluation Criteria for Solid Tumors Version 1.1 (RECIST1.1); or partial Remission of PR: the sum of the diameters of target lesions is reduced by at least 30% compared with the baseline level; or stable disease (SD): the degree of reduction of target lesions does not reach the level of PR, and the degree of increase does not reach the level of PD, in between, the study The minimum value of the sum of the diameters can be used as a reference.
  • PD progressive disease
  • OS overall survival
  • a response includes CR or PR following treatment with the methods of treatment of the present application.
  • the disease burden is reduced by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%.
  • treatment by the methods provided herein reduces the probability of relapse compared to other methods.
  • the probability of recurrence or progression after treatment is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than About 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%.
  • Subject and “patient” are used interchangeably herein and refer to an individual, such as a human or other animal, usually a human, who receives treatment with a cell product.
  • Treatment herein refers to interventions in an attempt to modify the course of a disease, to alleviate or reduce the disease or symptoms associated therewith, in whole or in part.
  • Said therapeutic effects include, but are not limited to, preventing occurrence or recurrence of biliary tract tumors, relieving symptoms, reducing any direct or indirect pathological consequences of biliary tract tumors, preventing metastasis, slowing down the rate of progression of biliary tract tumors, improving or alleviating the state of biliary tract tumors, and Alleviate or improve the prognosis, or delay the development of biliary tract tumors.
  • Said "delaying the development of biliary tract tumors” means postponing, hindering, slowing down, slowing down, stabilizing, curbing and/or delaying the development of biliary tract tumors. This delay can be of varying lengths of time, depending on the disease history and/or the individual being treated. It will be appreciated by those skilled in the art that a sufficient or significant delay may encompass prophylaxis (for individuals who do not develop biliary tract tumors).
  • the method comprises administering to a subject a cell therapy product or composition comprising a host cell described herein.
  • a cell therapy product or composition comprising a host cell described herein.
  • an effective amount or a therapeutically effective amount of the host cell, cell therapy product or composition described herein is administered to the subject.
  • Effective amount refers to a dose sufficient to prevent or treat biliary tract tumors in an individual. Effective doses for therapeutic or prophylactic use depend on the stage and severity of the biliary tract tumor being treated, the age, weight and general health of the subject, and the judgment of the prescribing physician. The size of the dose will also depend on the active substance chosen, the method of administration, the time and frequency of administration, the presence, nature and extent of adverse side effects that may accompany the administration of the particular active substance, and the desired physiological effect. According to the judgment of the prescribing physician or those skilled in the art, it may be necessary to administer the host cells, cell therapy products or compositions described in the present application for one or more cycles.
  • Tumor burden in this article includes tumor volume or degree of differentiation, or type and stage of metastasis, and/or appearance and disappearance of common complications of advanced cancer, such as cancerous pleural effusion, and/or appearance or disappearance of tumor markers Changes in expression levels, and/or likelihood or incidence of toxic outcomes in subjects, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity and/or against administered cells and/or chimerism Host immune response to antigen receptors.
  • the size of the tumor is measured with PET (Positron Emission Tomography) and CT (Computed Tomography) built-in scales.
  • Tumor markers refer to substances characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or substances produced by the host in response to tumor stimulation, and can reflect tumor occurrence and development, and monitor tumor response to treatment a class of substances.
  • Tumor markers exist in the tissues, body fluids and excreta of tumor patients, and can be detected by immunological, biological and chemical methods, exemplary, including alpha-fetoprotein (AFP), CA125, CA15-3, squamous Cellular carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.
  • AFP alpha-fetoprotein
  • SCC squamous Cellular carcinoma antigen
  • CYFRA21-1 soluble fragment of cytokeratin 19
  • CEA carcinoembryonic antigen
  • Cycle in this article refers to the period from the baseline period before treatment (i.e. the evaluation period) to the baseline period before the next treatment for each cell therapy; Cycle begins prior to pretreatment drug therapy. In some embodiments, each cycle of administration of the cell therapy product requires pretreatment.
  • Pretreatment refers to giving other drugs or treatments to the subject before administering the host cells, cell therapy products or compositions, so that the subject's physical condition is more suitable for infusion of the host cells, cell therapy products or compositions.
  • Cell therapy product or composition includes administering to the subject a chemotherapeutic drug, a biologic drug, radiation therapy, or a combination thereof.
  • chemical drugs are given for pretreatment, more preferably, the chemical drugs are chemotherapeutic drugs.
  • the subject has about 50%, 55%, 60%, 65% or 70% fewer lymphocytes after receiving preconditioning than before preconditioning.
  • “Chemical drugs” in this article refer to anticancer drugs prepared by chemical synthesis, including traditional chemotherapeutic drugs, such as alkylating agents, antimetabolites, anticancer antibiotics, etc., as well as targeted drugs, such as afatinib , Alectinib, etc.
  • chemical drugs include but are not limited to alkylating agents, anti-metabolites, antitumor antibiotics, plant anticancer drugs, hormones, immune agents, etc.; for example, diterpene alkaloids (such as taxanes), Cyclophosphamide, fludarabine, cyclosporine, rapamycin, melphalan, bendamustine, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, multi Ruubicin, fluorouracil, hydroxyurea, methotrexate, rituximab, vinblastine and/or vincristine, etc.
  • diterpene alkaloids such as taxanes
  • Cyclophosphamide fludarabine
  • cyclosporine cyclosporine
  • rapamycin rapamycin
  • melphalan bendamustine
  • bendamustine asparaginase
  • busulfan carboplatin
  • cisplatin daunorubi
  • anti-metabolites include, but are not limited to, carmofur, tegafur, pentostatin, doxifluridine, trimesat, fludarabine, gimeracil, oteracil potassium, bismuth Furacil, carmofur, capecitabine, galocitabine, cytarabine cytadecyl phosphate sodium, fosteabine sodium hydrate, raltitrexed, paltitrexid, pyretidine, thiazofurin ), noratrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylenecytidine, 2'-fluoromethylene-2'-deoxycytidine, N- [5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2( E),
  • alkylating agents include, but are not limited to, dacarbazine, melphalan, cyclophosphamide, temozolomide, chlorambucil, busulfan, nitrogen mustard, nitrosourea, and the like.
  • biological drugs ie other biological drugs
  • tubulin inhibitor herein includes tubulin polymerization accelerators and tubulin polymerization inhibitors.
  • Tubulin inhibitors include, but are not limited to, taxanes, epothilones, spongolactones, and laulimalide.
  • taxanes drug refers to a drug whose main component contains a taxane compound, and the taxane compound has a bridged methylene benzocyclodecene core similar to a taxane structure.
  • the bridged methylene benzocyclodecene core structure of the taxane compound contains unsaturated bonds or does not contain unsaturated bonds.
  • the carbon atoms on the methylene benzocyclodecene core structure of the taxanes are replaced by heteroatoms selected from N, O, S, and P.
  • the taxane compound is administered by injection.
  • the pretreatment includes administering to the subject any one or at least two of cyclophosphamide, fludarabine, tubulin inhibitors, pyrimidine antineoplastic drugs or a combination thereof.
  • the subject before administering the host cells, cell therapy products or compositions in each cycle, the subject is pretreated, and the pretreatment includes administering chemotherapeutic drugs, biological drugs and/or radiotherapy to the subject.
  • the radiation therapy is whole body radiation therapy, or localized radiation therapy.
  • dose herein is meant a dose calculated on a weight basis, or a dose calculated on a body surface area (BSA) basis, or a dose calculated on an individual person basis.
  • the weight-based dosage is the dosage calculated based on the subject's body weight, for example, mg/kg of the subject's or patient's body weight, cell number/kg of the subject's or patient's body weight, and the like.
  • the dose calculated based on BSA is the dose calculated based on the surface area of the subject, such as mg/m 2 , cell number/m 2 and the like.
  • the dose calculated on an individual basis refers to the dose administered per cycle or per subject, for example, mg/subject, cell number/subject.
  • “Number of administrations” herein refers to the frequency of administration of the host cell, cell therapy product or composition or each pretreatment drug within each cycle.
  • the frequency of administration of fludarabine once; or once a day for 4, 3 or 2 consecutive days.
  • Dosing frequency of cyclophosphamide 1 time; or 1 time per day for 4, 3 or 2 consecutive days.
  • Dosing times of nab-paclitaxel once; or once a day for 4, 3 or 2 consecutive days.
  • the host cells, cell therapy products or compositions administered in each cycle are administered at one time; or divided into 2, 3, 4, 5, 6, 7, 8, 9, 10 administrations, which can be administered on consecutive days , or given at intervals of 1, 2, 3, 4, 5, 6 days.
  • the subject is administered at least one cycle of the host cell, cell therapy product or composition for treatment.
  • the dosage can be the same or different for each cycle.
  • the dose within a cycle is administered to the subject once or divided into multiple doses, and the divided doses for each administration may be the same or different.
  • the host cells, cell therapy products or compositions in one cycle are administered to the subject once, or administered to the subject in 2, 3, 4, 5 or more times.
  • the dose of the host cells, cell therapy product or composition or pretreatment drug given each time is determined by the doctor according to the specific conditions of the subject. Sure.
  • each dose is administered in ascending order. In one example, each dose administered is in decreasing order. In one example, the dose administered each time tends to increase first and then decrease. In one example, the dose administered each time tends to decrease first and then increase.
  • the multi-period administration of the host cells, cell therapy products or compositions means that there are multiple time periods, and a certain total amount of the host cells, cell therapy products or compositions are administered in each time period.
  • the time intervals of each time period are consistent.
  • the intervals of each time period are inconsistent.
  • the host cell, cell therapy product or composition is administered for at least 2 cycles, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles.
  • the dose and number of administrations administered in each cycle, and the design of the interval between different cycles are evaluated with the goal of improving one or more outcomes, for example, the outcome may be to reduce the side effects of the subject The degree or likelihood of side effects can also be improved.
  • the dose of cells (such as CLD18.2-CAR-T cells) that specifically bind to the exogenous receptor of the CLD18.2 polypeptide is administered per cycle is no more than about 2x10 9 cells/kg body weight of the subject, Or no more than about 1x1011 cells/person.
  • the dose administered per cycle does not exceed about 2x108 cells/kg body weight of the subject, or does not exceed about 1x1010 cells/person. More preferably, the dose administered per cycle does not exceed about 2x107 cells/kg body weight of the subject, or does not exceed about 5x109 cells/person, or 2x109 cells/person, or 1x109 cells/person , or 5x108 cells/person.
  • the dose administered per cycle is about 1x10 5 cells/kg subject body weight to 2x10 7 cells/kg subject body weight, or about 1x10 6 cells/kg subject body weight to 2x10 7 cells/kg subject body weight.
  • the dose administered per cycle is about 1x107 cells to 1x109 cells, 1x107 cells to 2x109 cells, 1x107 cells to 5x109 cells, or 1x107 cells to 1x10 10 cells, preferably 1x108 cells to 1x109 cells, 1x108 cells to 2x109 cells, 1x108 cells to 5x109 cells, more preferably 1x108 cells to 5x108 cells cells, or 2.5x10 8 cells to 5x10 8 cells, 3.75x10 8 cells to 5x10 8 cells.
  • the pretreatment comprises administering to the subject cyclophosphamide and fludarabine; or cyclophosphamide, fludarabine and a tubulin inhibitor.
  • the tubulin inhibitor is a taxane compound, preferably, the taxane compound is selected from paclitaxel, nab-paclitaxel, docetaxel, more preferably, the taxane The alkane compound is albumin-bound paclitaxel.
  • fludarabine, cyclophosphamide, and nab-paclitaxel can be administered on the same day or on different days. If fludarabine, cyclophosphamide, and nab-paclitaxel are given on the same day, cyclophosphamide and/or nab-paclitaxel can be given before or after fludarabine; or before or after cyclophosphamide Administration of fludarabine and/or nab-paclitaxel; or administration of cyclophosphamide and/or fludarabine before or after nab-paclitaxel.
  • fludarabine, cyclophosphamide, and nab-paclitaxel may be administered simultaneously or sequentially.
  • cyclophosphamide is administered prior to fludarabine.
  • cyclophosphamide is administered after fludarabine is administered.
  • nab-paclitaxel is administered prior to fludarabine.
  • nab-paclitaxel is administered after fludarabine is administered.
  • nab-paclitaxel is administered prior to administration of cyclophosphamide.
  • nab-paclitaxel is administered after cyclophosphamide.
  • the administered amount of fludarabine is about 20-60 mg/day, or about 30-55 mg/day, or about 30-50 mg/day. In one example, the administration amount of fludarabine is about 10-50 mg/m 2 /day, or about 15-40 mg/m 2 /day, or about 15-30 mg/m 2 /day, or about 20-30 mg /m 2 /day; or about 25 mg/m 2 /day. In one example, the administration amount of cyclophosphamide is about 200-1000 mg/day, or about 300-600 mg/day, or about 300-560 mg/day, or about 300-550 mg/day, or about 300-500 mg/day .
  • cyclophosphamide is administered in an amount of about 200-400 mg/m 2 /day, or about 200-300 mg/m 2 /day, or about 250 mg/m 2 /day.
  • the dosage of the taxane compound is not higher than about 300 mg/day, or not higher than about 200 mg/day, preferably, the dosage of the taxane compound is about 90-200 mg/day, More preferably, the dosage of the taxane compound is about 90-120 mg/day.
  • each pretreatment drug is used continuously for no more than 4 days.
  • cyclophosphamide is administered 2-3 times.
  • fludarabine is administered 1-2 times.
  • the taxane is administered once.
  • fludarabine is administered on days -6 and -5. In one example, fludarabine is administered on days -5 and -4. In one example, fludarabine is administered on days -4 and -3. In one example, fludarabine is administered on days -6, -5, -4 and -3. In one example, fludarabine is administered on days -7, -6, -5 and -4. In one example, cyclophosphamide is administered on days -6, -5, -4 and -3. In one example, cyclophosphamide is administered on days -6, -5 and -4.
  • cyclophosphamide is administered on days -5, -4 and -2. In one example, cyclophosphamide is administered on days -5, -4 and -3. In one example, cyclophosphamide is administered on days -4, -3 and -2. In one example, cyclophosphamide is administered on days -4 and -3. In one example, cyclophosphamide is administered on days -6 and -5. In one example, cyclophosphamide is administered on days -5 and -4. In one example, a taxane compound (such as nab-paclitaxel) is administered on day -2, day -3, day -4, day -5, or day -6.
  • a taxane compound such as nab-paclitaxel
  • the use or method described herein further includes monitoring the degree or risk of the subject's toxic response to the host cell, cell therapy product or composition, and determining the subsequent divided doses according to the degree of toxicity or risk.
  • the degree of toxicity or risk includes, but is not limited to, CRS, neurotoxicity, macrophage activation syndrome, tumor lysis syndrome, and the like.
  • the toxic reaction or its symptoms or biochemical indicators include one or more of fever, hypotension, hypoxia, neurological disorders, inflammatory cytokines, and serum levels of C-reactive protein (CRP) .
  • the acceptable level of risk of the toxic reaction or its symptoms or biochemical indicators refers to 95%, 90%, 85%, 80%, 75% of the peak level after giving the previous cycle of treatment.
  • the serum level of a factor indicative of cytokine-release syndrome (CRS) in the subject is no more than the serum level of the subject prior to administration of the previous cycle 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 times more often the treatment given in the later cycle.
  • the tumor load stabilized or decreased, before the adaptive host immune response (that is, the body's immune rejection of CAR-T cells)
  • one or more Post cycle treatment under such conditions, immune surveillance, eradication, or prevention of proliferation or metastasis of residual tumor cells can be safely and effectively provided in the later cycle.
  • the latter cycle is a disease consolidating dose.
  • the subsequent cycle of treatment is administered at least 28, 29, 30, 31, 32, 33, 34, 35 days after administration of the prior cycle of the cell therapy product.
  • the host cell, cell therapy product or composition is administered to the subject by autologous reinfusion.
  • the cells such as CLD18.2-CAR-T cells
  • the cells that specifically bind to the exogenous receptor of the CLD18.2 polypeptide are derived from a subject who needs treatment by this method, and are reinfused after being genetically modified in vitro .
  • the cells such as CLD18.2-CAR-T cells
  • the cells that specifically bind to the exogenous receptor of the CLD18.2 polypeptide are administered to the subject by means of allogeneic reinfusion.
  • the cells (such as CLD18.2-CAR-T cells) that specifically bind to the exogenous receptor of the CLD18.2 polypeptide are derived from a healthy donor, and the donor is different from the subject in which the cells are reinfused.
  • the host cells, cell therapy products or compositions may be administered by any suitable means, for example, by injection, for example, intravenous or subcutaneous injection, intraperitoneal injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, Intraseptal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival injection, subconjunctival injection, episcleral injection, retrobulbar injection, periocular injection or peribulbar delivery.
  • Extraperitoneal infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • fludarabine, cyclophosphamide, and nab-paclitaxel may be administered by any route, including intravenous (IV).
  • the host cell, cell therapy product or composition can be administered by any route, including intravenous (IV) injection.
  • the CAR-T cells are administered IV at about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, About 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes Minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes.
  • cyclophosphamide and fludarabine may cause adverse events in subjects after administration. It is within the scope of the present application to administer compositions to subjects to reduce some of these adverse events.
  • administration of adjuvants and excipients is included. For example, mesna (sodium 2-mercaptoethanesulfonate).
  • administration of exogenous cytokines is involved.
  • the biological activity of the administered cell population can be measured by any of a number of known methods. Parameters assessed include: specific binding of cells to antigen, either in vivo (eg, by imaging) or ex vivo (eg, by ELISA or flow cytometry). In one example, the biological activity of the cells is measured by measuring the expression and/or secretion of cytokines, such as CD107a, IFNy, IL-2 and TNF. In one example, biological activity is assessed by clinical outcome, such as reduction in tumor burden. In one example, a reduction in tumor markers is assessed. In one example, the cells are assessed for toxic outcome, persistence and/or proliferation, and/or the presence or absence of a host immune response.
  • treatment in a later cycle may be administered, if desired.
  • whether to administer the treatment in the later cycle, when to administer the treatment in the later cycle, and/or the dose of the cell therapy product administered in the later cycle is based on the expression of the cells in the previous cycle in the subject. The presence, absence or extent of an immune response or a detectable immune response to the chimeric antigen receptor is determined.
  • a CRS-associated outcome e.g., a serum factor associated with or indicative of CRS
  • clinical signs or symptoms thereof such as fever, hypoxia, hypotension, or neurological disturbance
  • the treatment of the later cycle was given after the decline started after the administration of the previous cycle.
  • a later cycle of treatment is administered when a host adaptive immune response has not been detected, has not yet been established, or has not yet reached a certain level, extent or stage. In some aspects, the later cycle of treatment is administered before the subject develops an anamnestic immune response.
  • a detectable immune response refers to an amount detectable by any of a number of known methods for assessing specific immune responses to particular antigens and cells.
  • an ELISPOT, ELISA, or cell-based antibody detection method e.g., by flow cytometry
  • the presence of an antibody that binds to an epitope of a chimeric antigen receptor, such as a CAR to detect a particular type of immune response.
  • the isotype of the detected antibody is determined and may indicate the type of response and/or whether the response is a memory response.
  • T cells were prepared from peripheral blood mononuclear cells (PBMCs) of human subjects with cancer based on "mononuclearization", and encoded with a chimeric antigen receptor (CAR)
  • PBMCs peripheral blood mononuclear cells
  • CAR chimeric antigen receptor
  • Cells were cryopreserved in the infusion medium in individual flexible freezing bags. Each contains a single unit dose of cells, which is about 1 x 106 cells to 5 x 107 cells.
  • No more than about 1 x 1012 cells preferably no more than about 1 x 1011 cells, more preferably no more than about 1 x 1010 cells infused per subject in the previous cycle Or about 5x109 cells or about 2x109 cells. Cells are maintained at a temperature of about less than -130°C or about less than -175°C prior to infusion.
  • cytokine release syndrome CRS
  • TNF ⁇ tumor necrosis factor alpha
  • IFN ⁇ interferon gamma
  • IL-6 IL-6
  • Tumor burden can optionally be assessed by measuring the size or mass of the solid tumor, eg, by PET or CT scan, before initiation of treatment.
  • Resuscitation is performed by warming to approximately 38°C, and subjects are administered cells from previous cycles by multiple infusions. Each infusion is given intravenously (IV) as a continuous infusion over a period of about 3-30 minutes.
  • IV intravenously
  • subjects undergo a physical examination and are monitored for any toxicity or symptoms of toxic consequences, such as fever, hypotension, hypoxia, neurological disturbances, or serum levels of inflammatory cytokines or C-reactive protein (CRP) raised.
  • blood is obtained from the subject on one or more occasions following administration of the previous cycle and assessed for levels of serum factors indicative of CRS by methods of ELISA and/or MSD and/or CBA.
  • the levels of serum factors are compared to the levels of serum factors obtained just before administration of the previous cycle. If necessary, give anti-IL6 or other CRS therapy to reduce the symptoms of CRS.
  • the presence or absence of an anti-CAR immune response in the subject is optionally detected, e.g., by qPCR, ELISA, ELISPOT, after administration of the previous cycle, e.g., 1, 2, 3 and/or 4 weeks after initiation of dosing , cell-based antibody assays and/or mixed lymphocyte reactions.
  • the percent reduction in tumor burden achieved by the previous cycle can optionally be measured one or more times after administration of the previous cycle in patients with solid tumors by scanning (e.g., PET and CT scans), and/or by quantification in the blood or tumor tumor-positive cells.
  • scanning e.g., PET and CT scans
  • Subjects were monitored periodically beginning with the first dose and continuing for up to several years. During follow-up, measure tumor burden, and/or detect CAR-expressing cells by flow cytometry and quantitative polymerase chain reaction (qPCR) to measure in vivo proliferation and persistence of administered cells, and/or assess resistance to CAR Development of an immune response.
  • qPCR quantitative polymerase chain reaction
  • Each subject has infused no more than about 1 x 1012 cells, preferably no more than about 1 x 1011 cells, preferably no more than about 1 x 1010 cells, Or not more than about 5 ⁇ 10 9 cells, or not more than 2 ⁇ 10 9 cells.
  • Cells were kept at a temperature below -175°C prior to infusion. During the infusion, raise the temperature to about 38°C for resuscitation.
  • Dosing in later cycles was subject-specific and based on tumor burden, presence of anti-CAR immune response, and level of CRS-related outcomes.
  • the dose administered in the later period is not higher than about 1 ⁇ 10 12 cells, preferably not higher than about 1 ⁇ 10 11 cells, more preferably not higher than about 1 ⁇ 10 10 cells, more preferably , not more than about 5 x 10 9 cells or not more than about 2 x 10 9 cells.
  • kits for treating and/or preventing biliary tract tumors in a subject includes an effective amount of the host cell, cell therapy product, composition group or pharmaceutical composition of the present application.
  • the kit comprises sterile containers; including boxes, ampoules, bottles, vials, tubes, bags, sachets, blister packs or other suitable container forms known in the art. Containers include plastic, glass, laminated paper, foil, or other materials suitable for containing the drug.
  • the kit includes a nucleic acid molecule encoding the CAR of the present application, which recognizes the CLD18.2 polypeptide in an expressible form, and may optionally be included in one or more vectors.
  • the composition and/or nucleic acid molecule of the present application and administering the composition or nucleic acid molecule to a subject suffering from a tumor or a pathogen or an immune disease or developing a tumor or a pathogen or an immune disease supplied with the instruction manual.
  • the instructions generally include information about the use of the composition in the treatment and/or prophylaxis of tumors or pathogenic infections.
  • the instructions include at least one of the following: a description of the therapeutic agent; a dosage form and administration for the treatment or prevention of tumors, pathogenic infections, or immune diseases or symptoms thereof; precautions; warnings; indications; incompatibility ; Medication Information; Adverse Reactions; Animal Pharmacology; Clinical Studies; and/or References.
  • These instructions may be printed directly on the container, or as a label affixed to the container, or provided within or with the container as separate sheets, booklets, cards or file folders.
  • This application significantly improves the efficacy of the treatment of biliary tract tumors (including gallbladder cancer and cholangiocarcinoma) by using immune effector cells expressing chimeric antigen receptors: the number or size of tumors is reduced, and/or the number and/or degree of metastasis are reduced , and/or a decrease in tumor markers, and/or the disappearance or weakening of common complications of advanced cancer.
  • the anti-tumor effect on solid tumors is remarkable, and it provides a safe and effective treatment that can stabilize or even cure CLA18.2-positive middle and advanced biliary tract tumors (including gallbladder cancer and cholangiocarcinoma).
  • a pretreatment composition for improving the treatment of immune effector cells against biliary tract tumors including gallbladder cancer, cholangiocarcinoma), and administer fludarabine, cyclophosphamide, and albumin-bound paclitaxel compositions before administering immune effector cells , can significantly promote the anti-tumor efficacy of immune effector cells, greatly reduce the tumor burden of the subject, improve the quality of life of the subject, and prolong the survival period.
  • Exemplary antigen receptors of the present application including CAR, and methods for engineering and introducing receptors into cells, refer to, for example, Chinese Patent Application Publication Nos. CN106554414A ⁇ CN105331585A ⁇ CN106397593A ⁇ CN106467573A ⁇ CN104140974A ⁇ CN 108884459A ⁇ CN107893052A ⁇ CN108866003A ⁇ CN108853144A ⁇ CN109385403A ⁇ CN109385400A ⁇ CN109468279A ⁇ CN109503715A ⁇ CN 109908176A ⁇ CN109880803A ⁇ CN 110055275A ⁇ CN110123837A ⁇ CN 110438082A ⁇ CN 110468105A ⁇ WO2017186121A1 ⁇ WO2018006882A1 ⁇ WO2015172339A8 ⁇ WO2018/018958A1 ⁇ WO2014180306A1 ⁇ WO2015197016A1 ⁇ WO2016008405A1 ⁇ WO2016086813A1 ⁇ WO2016150400A1 ⁇ WO2017032293A1
  • the CAR-T cells expressing and specifically recognizing CLD18.2 were prepared by conventional molecular biology methods in the field. Using PRRLSIN as the carrier, insert the chimeric antigen receptor sequence shown in Table 1 to construct the lentiviral vector expressing the chimeric antigen receptor of CLD18.2 antibody, respectively infect the activated T cells and culture and expand to the required Quantity, get CLD18.2-BBZ CAR-T cells, CLD18.2-28Z CAR-T cells, CLD18.2-28BBZ CAR-T cells.
  • CD8 ⁇ signal peptide SEQ ID NO:1; CLDN18.2scFV (SEQ ID NO:2); CD8 hinge region (SEQ ID NO:3); CD8 transmembrane region (SEQ ID NO:4); CD28 transmembrane Region (SEQ ID NO:5); CD137 intracellular domain (SEQ ID NO:6); CD28 intracellular domain (SEQ ID NO:7); CD3 ⁇ intracellular domain (SEQ ID NO:8).
  • Target cells CLD18.2-negative human cholangiocarcinoma cells RBE and human gallbladder cancer cells GBC-SD; RBE-CLD18.2 cells and GBC-SD-CLD18.2 cells exogenously expressing human CLD18.2 .
  • CLD18.2-28Z CAR-T kills CLD18.2-positive cells
  • CLD18.2-28Z CAR-T basically does not kill CLD18.2-negative cells RBE and GBC-SD.
  • GBC-SD-CLD18.2 cells were inoculated subcutaneously in the right axilla of female NPG mice (purchased from Beijing Weitongda Biotechnology Co., Ltd.) at the age of 5-6 weeks, and the tumor inoculation diary was D0. On D15 after inoculation, the tumor volume grew to about 150mm 3 , and they were randomly divided into 4 groups, namely UTD and CLD18.2-28Z CAR-T groups.
  • the CLD18.2-28Z CAR-T group included CAR-T1 (5*10 ⁇ 5 cells/mouse), CAR-T2 (1*10 ⁇ 6 cells/mouse), CAR-T3 (3*10 ⁇ 6 cells/mouse).
  • CLD18.2-positive biliary tract tumor subjects were treated with CLD18A2-targeted CAR-T cells, and the expression of CLD18.2 was detected by using tumor tissue sections from previous surgery or biopsy.
  • the subjects received the apheresis technique of "removing mononuclear cells" and underwent pretreatment treatment. Through apheresis, PBMCs were obtained from the subject, and CAR-T cells were obtained by transduction and amplification of a viral vector encoding an anti-CLD18A2 CAR (an exemplary CAR uses CLD18.2-28Z CAR).
  • pretreatment fludarabine, cyclophosphamide and nab-paclitaxel.
  • tumor burden can optionally be assessed by measuring the size or character of the solid tumor by, for example, PET or CT scan, and can also be assessed by detecting tumor markers and/or observing the occurrence and severity of tumor complications .
  • the administration of CAR-T in the latter cycle shall be given 4 weeks after the completion of the administration of CAR T in the previous cycle.
  • the administration of CAR-T in each cycle can be administered once or divided into two or more intravenous (IV) infusions.
  • the cells administered each time are infused within about 3-30 minutes, preferably 5-25 minutes. Minutes to complete the infusion.
  • the subject will undergo a physical examination and be monitored for any symptoms of toxicity or toxic outcomes, such as fever, hypotension, hypoxia, neurological disorders, or inflammatory cytokines or C-reactive protein Serum levels of (CRP) were elevated.
  • the examination may be to obtain blood from the subject and evaluate the level of cytokines indicative of CRS by ELISA, and/or MSD, and/or CBA. If necessary, give anti-IL-6 therapy, or give other CRS treatments to reduce the symptoms of CRS.
  • the CAR-T in the subject can be evaluated by qPCR, ELISA, ELISPOT, antibody assay, etc. quantity.
  • Reduction in tumor burden after each cycle of treatment can be obtained by scans (eg, PET and CT scans), and/or by quantification of cells positive for antigens (eg, claudin18.2) in the blood or at the tumor site.
  • scans eg, PET and CT scans
  • antigens eg, claudin18.2
  • subject 1 male, 65 years old, 44 kg, poorly differentiated adenocarcinoma of the gallbladder, CLD18.2 expression was +++ (strong positive staining), and the percentage of stained tumor cells was 90%.
  • Pretreatment before CAR-T treatment fludarabine 35mg/d on day 1-2, cyclophosphamide 350mg/d on day 1-3, nab-paclitaxel 100mg/d on day 2.
  • Received 2 cycles of CAR-T infusion the infusion dose of cycle 1 was 3.75 ⁇ 10 8 cells, and the infusion dose of cycle 2 was 2.5 ⁇ 10 8 cells, all of which were reinfused once.
  • Subject 2 female, 63 years old, 59kg, high-grade well-differentiated adenocarcinoma of the gallbladder, CLD18.2 expression was +++ (strong positive staining), and the percentage of stained tumor cells was 80%.
  • Pretreatment before CAR-T treatment Fludarabine 38.75 mg/d on the first 2 days, cyclophosphamide 387.5 mg/d on the 1st to 3rd days, nab-paclitaxel 100 mg/d on the 2nd day.
  • Subject 3 male, 57 years old, 65kg, extrahepatic cholangiocarcinoma of moderate to poor differentiation, CLD18.2 expression was +++ (strong positive staining), and the percentage of stained tumor cells was 80%.
  • Pretreatment before CAR-T treatment fludarabine at 42.25 mg/d on day 1-2, cyclophosphamide at 422.5 mg/d on day 1-3, nab-paclitaxel at 100 mg/d on day 2.
  • Subject 1 was in PR after treatment, PFS was 4.2 months, and OS was about 10 months.
  • Subjects 2 and 3 reached SD after treatment, and the progression-free survival periods were about 9.3 months and 7.5 months, respectively, and they were still in a stable stage.
  • the amplified copy number D5 in subject 1 reached a peak of 6713 copies/ ⁇ g gDNA, which persisted for at least 62 days; subject 2 reached a peak of 8237 copies/ ⁇ g gDNA on D7, and persisted. At least 85 days; subject 3 peaked on D14, 5698 copies/ ⁇ g gDNA, and persisted for at least 28 days.

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Abstract

一种给予患有或怀疑患CLD18阳性胆道肿瘤个体的免疫效应细胞的治疗方法，包括给予个体特异性识别CLD18.2的免疫效应细胞。先进行预处理后再给予本申请的免疫效应细胞时，能够显著提高免疫效应细胞的肿瘤治疗疗效。

Description

治疗肿瘤的方法和组合物
优先权信息
本申请要求：于2021年6月23日提交的中国专利申请CN202110701218.X、于2022年4月8日提交的中国专利申请CN202210378612.9，它们的全部内容通过整体引用并入本文。
同时提交的序列表文件
下列ASCII码文本文件的全部内容通过整体引用并入本文：计算机可读格式(CRF)的序列表(FF00616PCT-sequence listing.txt,日期：2022年6月23日，大小：51.7kb)
技术领域
本申请属于免疫治疗领域；具体地，涉及靶向识别肿瘤抗原、引发免疫效应细胞活化且发挥抗肿瘤效应的免疫细胞治疗。
背景技术
目前已发表临床研究结果显示，既往接受过一线标准治疗的胆道肿瘤的无进展生存期均低于约6个月。因此，急需开发新的治疗胆道肿瘤或延长患者生存期的治疗手段。
发明内容
本申请提供了一种治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的方法，包括向所述受试者给予靶向CLD18的外源受体的细胞。
本申请还提供了一种靶向CLD18的外源受体的细胞用于制备治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的药物的用途。
本申请还提供一种表达靶向CLD18的外源受体的细胞和化学药物或其他生物药物或放疗用于制备治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的药物的用途。
本申请还提供了一种靶向CLD18的外源受体的细胞用于治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的用途。
本申请还提供了一种表达靶向CLD18的外源受体的细胞和化学药物或其他生物药物或放疗用于治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的用途。
在一优选例中，所述CLD18为CLD18.2。
在一优选例中，所述胆道肿瘤包括胆囊癌和胆管癌。
在一优选例中，所述细胞包含免疫效应细胞。
在一优选例中，所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、肥大细胞、巨噬细胞、树突细胞、CIK细胞、干细胞衍生的免疫效应细胞或其组合。
在一优选例中，所述免疫效应细胞来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞。
在一优选例中，所述免疫效应细胞为自体或同种异体T细胞、或原代T细胞。
在一优选例中，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、αβT细胞或其组合。
在一优选例中，所述外源受体选自嵌合抗原受体(CAR)、T细胞受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、抗体-TCR嵌合物或其组合；所述外源受体的抗原结合域特异性结合CLD18.2。
在一优选例中，所述嵌合抗原受体包括：
(i)特异性结合CLD18.2的抗体、CD8或CD28的跨膜区和CD3ζ胞内域；
(ii)特异性结合CLD18.2的抗体、CD8或CD28的跨膜区、CD137胞内域和CD3ζ胞内域；
(iii)特异性结合CLD18.2的抗体、CD28或CD8的跨膜区、CD28胞内域和CD3ζ胞内域；或
(iv)特异性结合CLD18.2的抗体、CD8或CD28的跨膜区、CD28胞内域、CD137胞内域和CD3ζ胞内域。
在一优选例中，给予所述受试者至少一个周期的所述细胞进行治疗；优选的，给予受试者1-3个周期的所述细胞进行治疗。
在一优选例中，每个周期给予的细胞治疗产品中细胞的剂量不超过约2ⅹ10 9个细胞/kg受试者体重、2ⅹ10 8个细胞/kg受试者体重、或2ⅹ10 7个细胞/kg受试者体重；或细胞治疗产品中细胞剂量不超过约1ⅹ10 11个细胞/受试者、1ⅹ10 10个细胞/受试者、5ⅹ10 9个细胞/受试者、2ⅹ10 9个细胞/受试者或1ⅹ10 9个细胞/受试者。
在一优选例中，每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 5个细胞/kg受试者体重至2ⅹ10 7个细胞/kg受试者体重，或约1ⅹ10 6个细胞/kg受试者体重至2ⅹ10 7个细胞/kg受试者体重；或者
每个周期给予的细胞治疗产品中细胞剂量为约1ⅹ10 7个细胞至5ⅹ10 9个细胞/受试者、约1ⅹ10 7个细胞至2ⅹ10 9个细胞/受试者、或约1ⅹ10 7个细胞至1ⅹ10 9个细胞/受试者；或者
每个周期给予的细胞治疗产品中细胞剂量为约1ⅹ10 8个细胞至5ⅹ10 9个细胞/受试者、约1ⅹ10 8个细胞至2ⅹ10 9个细胞/受试者、或约1ⅹ10 8个细胞至1ⅹ10 9个细胞/；或者
每个周期给予的细胞治疗产品中细胞剂量为约2.5ⅹ10 8个细胞至5ⅹ10 8个细胞/受试者。
在一优选例中，每个周期给予所述细胞治疗产品之前进行预处理，所述预处理包括给 予所述受试者化学药物、生物药物(其他生物药物，在本文中其他生物药物不同于所述靶向CLD18的外源受体的细胞)、放疗或其组合。
在一优选例中，所述预处理在给予细胞治疗产品前1-8天实施；优选的，在给予细胞治疗产品前2-6天实施；优选地，每种化学药物、生物药物(其他生物药物)、放疗或其组合连续使用不超过4天。
在一优选例中，所述化学药物选自以下任意一种或至少两种：环磷酰胺、氟达拉滨、微管蛋白抑制剂、嘧啶类抗肿瘤药物；或所述化学药物包括环磷酰胺和氟达拉滨；或者所述化学药物包括环磷酰胺、氟达拉滨以及微管蛋白抑制剂。
在一优选例中，所述微管蛋白抑制剂包括紫杉烷类化合物；或所述微管蛋白抑制剂包括紫杉醇、白蛋白结合型紫杉醇、多西他赛；或所述微管蛋白抑制剂为白蛋白结合型紫杉醇。
在一优选例中，氟达拉滨的给予量约为10-50mg/m 2/天、或约15-40mg/m 2/天、或约15-30mg/m 2/天、或约20-30mg/m 2/天；或约25mg/m 2/天、或约为30-60mg/天、或约30-50mg/天、或约35-45mg/天；
环磷酰胺的给予量约为200-400mg/m 2/天、或约200-300mg/m 2/天、或约250mg/m 2/天、或约为300-1000mg/天、或约300-550mg/天、或约300-500mg/天；
紫杉烷类化合物给予量为不高于约300mg/天、或不高于约200mg/天，或约为90-120mg/天。
在一优选例中，所述环磷酰胺给予2-3次；或所述氟达拉滨给予1-2次；或所述紫杉烷类化合物给予一次。
在一优选例中，所述抗原结合域或所述抗体具有：SEQ ID NO：2、23、24、25、26、27、28、29、30、31、32或33所示的scFv序列；或
所述抗原结合域或所述抗体具有SEQ ID NO：14所示的HCDR1、SEQ ID NO：15所示的HCDR2、SEQ ID NO：16所示的HCDR3、SEQ ID NO：17所示的LCDR1、SEQ ID NO：18所示的LCDR2、SEQ ID NO：19所示的LCDR3；或
所述抗原结合域或所述抗体具有SEQ ID NO：10所示的重链可变区，SEQ ID NO：12所示的轻链可变区。
在一优选例中，所述的嵌合抗原受体具有SEQ ID NO：2、23、24、25、26、27、28、29、30、31、32或33所示的scFv序列分别与SEQ ID NO：34、35或36顺序连接的序列；或所述的嵌合抗原受体具有SEQ ID NO：37、38或39所示的核苷酸序列。
在一优选例中，在每个周期给予细胞治疗产品之前，对所述受试者的指示CRS的细胞因子、指示神经毒性的细胞因子、指示肿瘤负荷的指标、和/或指示宿主抗-CAR免疫应答的因子的血清水平进行评价。
在一优选例中，在给予细胞治疗产品治疗后，所述受试者没有显示严重CRS，或没有显示超过3级的神经毒性。
在一优选例中，待在先周期的所述细胞治疗后在体内检测不到后，再给予后续周期的治疗。
在一优选例中，所述的后续周期给予的所述细胞剂量为足以稳定或降低所述受试者的肿瘤负荷的细胞数量。
在一优选例中，每个周期的所述细胞分为1-5次给予，优选的，分为1-3次给予。
在一优选例中，给予在后周期的治疗时，所述受试者具有以下任一特征：
(i)细胞因子释放综合征(CRS)相关的细胞因子在受试者中血清中的峰值水平比在先周期中细胞治疗产品给予后的峰值水平小；
(ii)没有显示出3级或更高的神经毒性；
(iii)CRS水平与给予在先周期的细胞治疗产品后的CRS水平相比，呈现降低；
(iv)所述受试者没有显示出针对细胞治疗产品的可检测的体液或细胞介导的免疫应答。
在一优选例中，所述(i)中，细胞因子的水平与给予在先周期的细胞治疗产品后的细胞因子的的峰值水平相比，降低至少50％，优选的，降低至少20％，更优的，降低至少5％。
在一优选例中，所述CRS水平与给予在先周期的细胞治疗产品之前的CRS水平相当。
在一优选例中，每个周期给予所述细胞之前进行预处理，所述预处理包括给予所述受试者化学药物和/或放疗。
在一优选例中，所述放疗包括全身辐射治疗或局部辐射治疗。
在一优选例中，所述预处理在给予所述细胞前1-8天实施；优选的，在给予所述细胞前2-6天实施。
在一优选例中，所述化学药物选自以下任意一种或至少两种：环磷酰胺、氟达拉滨、微管蛋白抑制剂、嘧啶类抗肿瘤药物。
在一优选例中，所述化学药物为环磷酰胺和氟达拉滨；或者环磷酰胺、氟达拉滨以及微管蛋白抑制剂。
在一优选例中，所述微管蛋白抑制剂是紫杉烷类化合物；
优选的，所述紫杉烷类化合物选自紫杉醇、白蛋白结合型紫杉醇、多西他赛；
更优选的，所述紫杉烷类化合物为白蛋白结合型紫杉醇。
在一优选例中，所述嘧啶类抗肿瘤药物选自5-氟尿嘧啶、双呋氟尿嘧啶、卡莫氟、去氧氟尿苷、卡培他滨。
在一优选例中，每种化学药物连续使用不超过4天。
在一优选例中，所述环磷酰胺给予2-3次；或所述氟达拉滨给予1-2次。
在一优选例中，所述紫杉烷类化合物给予一次。
应理解，在本申请范围内中，本申请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.CAR-T细胞的细胞毒性测定；
图2.CAR-T细胞体内杀伤实验：图2A、2B为肿瘤体积、肿瘤重量测定。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现经表达CLDN18.2-CAR的免疫效应细胞(例如，T细胞)及其组合物用于治疗胆道肿瘤(包括胆管癌、胆囊癌)的受试者的方法和用途。在此基础上完成了本申请。在一些方面，与现有技术相比，该方法、免疫效应细胞和组合物提供或实现改善的或更持久的应答或功效和/或降低的毒性风险或其他副作用。在一实例中，所述方法通过给予指定或相对数量的细胞，治疗特定受试者群体，显著提高抗胆道肿瘤疗效。
目前已发表临床研究结果显示，既往接受过一线标准治疗的胆道肿瘤的无进展生存期均低于约6个月(1.8-5.6个月)。本申请针对胆囊癌受试者，施用CAR-T细胞治疗，显示了更长的无进展生存期和总生存期，使受试者获得显著临床受益：1例胆囊癌受试者在CAR-T细胞治疗后达到PR(partial response，部分缓解)，PFS(progression-free survival，无进展生存期)为4.2个月，OS(Overall Survival，总生存期)约10个月；1例胆囊癌受试者在CAR-T细胞治疗后达到SD(stable disease，病情稳定，处于平稳状态)，PFS已达9.3个月，并且仍在持续稳定期；1例胆管癌受试者在CAR-T细胞治疗后达到SD，PFS已达7.5个月，并且仍在持续稳定期。
除非另外定义，本文使用的所有专业术语、符号和其它技术和科学术语或专有词汇旨在具有本申请所属领域技术人员通常所理解的相同含义。在一些情况中，本文出于阐明和/或便于引用的目的对具有所常规理解的含义的术语加以进一步限定，本文中包括的此类进一步限定不应理解为表示与本领域常规理解的有实质上的差异。
本申请中提及的所有出版物，包括专利文件、学术论文和数据库，均可独立地通过引用其全文纳入本文。如果本文所示的定义与通过引用纳入本文的专利、公开申请和其它出版物中所示的定义不同或有其它情况下不一致，以本文所示的定义为主。
1.定义
如本文所用，“约”可表示取决于具体情况并且由本领域技术人员已知或可知的，或表示在给定值的至多约±1％、±2％、±3％、±4％、±5％、±6％、±7％、±8％、±9％、±10％、±11％、±12％、±13％、±14％、±15％、±16％、±17％、±18％、±19％、±20％、±25％、±30％范围内变化。即“约”表示的范围涵盖给定值±1％、给定值±2％、给定值±3％、给定值±4％、给定值±5％、给定值±6％、给定值±7％、给定值±8％、给定值±9％、给定值±10％、给定值 ±11％、给定值±12％、给定值±13％、给定值±14％、给定值±15％、给定值±16％、给定值±17％、给定值±18％、给定值±19％、给定值±20％、给定值±25％、给定值±30％。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。
范围：范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本申请的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。
在描述氨基酸或者核酸序列时，本申请中“具有”特定序列应当被理解为涵盖了特定序列的变体。本申请中所述氨基酸或者核酸序列具有某特定序列是指所述氨基酸或者核酸序列与所述特定序列具有超过70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或者99％的序列同一性。序列同一性可以通过序列分析软件(例如，BLAST、BESTFIT、GAP、PILEUP/PRETTYBOX等程序)进行测量。这样的软件通过将同源性程度分配给各种取代、缺失和/或其他修饰来匹配相同或相似的序列。保守取代通常包括以下组内的取代：甘氨酸、丙氨酸；缬氨酸、异亮氨酸、亮氨酸；天冬氨酸、谷氨酸、天冬酰胺、谷氨酰胺；丝氨酸、苏氨酸赖氨酸、精氨酸；和苯丙氨酸、酪氨酸。在确定同一性程度的示例性方法中，可以使用BLAST程序，其中e-3和e-100之间的概率得分指示密切相关的序列。
术语“外源”，是指核酸分子或多肽不是内源性存在细胞中的，或表达水平不足以实现过表达时具有的功能；涵盖在细胞中表达的任何重组核酸分子或多肽，例如外源、异源和过表达的核酸分子或多肽。在一实例中，外源受体包括嵌合受体，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或多肽片段连接而成的融合分子，包括胞外域、跨膜域和胞内域。外源受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)、T细胞融合蛋白(TFP)。
术语“CAR”包括胞外抗原结合域、跨膜域和胞内信号传导域。在一实例中，CAR的胞外抗原结合域包含scFv。胞内信号传导域包括刺激性分子和/或共刺激性分子的功能信号传导域；或包括刺激性分子和/或共刺激性分子的全部细胞内部分、或全部天然胞内信号传导域、或其功能片段或衍生物。
术语“T细胞受体(TCR)”介导T细胞对特异性主要组织相容性复合物(MHC)-限制性肽抗原进行识别，包括天然TCR受体和修饰的TCR受体。天然TCR受体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等，其抗原特异性存在于V区。天然TCR改造后获得能特异性结合CLD18.2多肽的修饰TCR受体。本文中修饰TCR受体包括但不限于嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)、T细胞融合蛋白(TFP)、抗体-TCR嵌合物。
“特异性结合”是指多肽或其片段识别并结合目的生物分子(例如多肽)但基本上不识别并结合样品中其他分子。结合亲和力可以采用标准结合测定法测定。
术语“抗原结构域”是指特异性结合抗原决定簇的分子，包括免疫球蛋白分子和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。术语“抗体”不仅包括完整的抗体分子，也包括保留抗原结合能力的抗体分子的片段。本申请中术语“抗体”与术语“免疫球蛋白”“抗原结构域”可互换使用。抗体，包括但不限于单克隆抗体、多克隆抗体、天然抗体、双特异性抗体、嵌合抗体、Fv、Fab、Fab’、Fab’-SH、F(ab’)2、线性抗体、单链抗体分子(例如scFv)、单域抗体。在一实例中，抗体包括通过二硫键连接的至少两个重(H)链和两个轻(L)链。每条重链由重链可变区(VH)和重链恒定区(CH)组成。CH有三个结构域CH1、CH2、CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区(CL)。CL由一个结构域组成。VH和VL可进一步细分为高变区，称为互补决定区(CDR)，其间散布有更保守的区域，称为框架区(FR)。每个VH和VL均由三个CDR和四个FR组成，从氨基端到羧基端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区包括与抗原相互作用的结合结构域。抗体的恒定区介导免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的各种细胞(例如，免疫效应细胞)与经典补体系统的第一组分(C1q)。如果抗原结构域以与其它参考抗原(包括多肽或其他物质)结合相比更大亲和力(或称亲合力)结合抗原，则所述抗原结构域与抗原“特异性结合”或与抗原是“免疫反应性的”。
术语“组合物”指两种或更多种物质的混合物。其可以是溶液、悬液、液体、粉末、糊剂、水性、非水性或其任何组合。
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。
术语“核酸”或“多核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物，包括编码目的多肽或其片段的任何核酸分子。所述核酸分子只需要与内源性核酸序列保持基本同一性即可，不需要与内源性核酸序列100％同源性或同一性。与内源性序列具有“基本同一性”的多核苷酸通常能与双链核酸分子的至少一条链杂交。术语“基本同一性”或“基本同源性”，是指与参考氨基酸序列或核酸序列表现出至少约50％同源性或同一性的多肽或核酸分子。
术语“肽”、“多肽”和“蛋白质”可互换使用，是指由通过肽键共价连接的氨基酸残基组成的化合物。
2.外源受体
本申请外源受体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，包括特异性结合CLD18的胞外域、跨膜域和胞内域；包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)、T细胞融合蛋白(TFP)、抗体-TCR嵌合物。在一实例中，本申请提供特异性结合CLD18.2多肽的外源受体。在一实例中，外源受体结合至CLD18.2多肽的胞外结构域。在一实例中，本申请提供特异性结合CLD18.2多肽的CAR。
CLD18多肽(Claudin 18、密蛋白18、CLDN18)指CLD18基因或编码的蛋白的任何变体、衍生物或同种型，包括剪接变体1(Claudin 18.1、CLD18.1、CLDN18.1)：NP_057453、NM016369，以及剪接变体2(Claudin 18.2、CLD18.2、CLDN18.2)：NP_001002026、NM_001002026。在一实例中，所述CLD18.2多肽为人CLD18.2多肽，包括与SEQ ID NO:9所示序列具有至少约80％、85％、90％、95％、96％、97％、98％、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。CLD18.2多肽在若干癌症类型中强烈表达，包括胆囊癌和胆管癌。
2.1 CAR
在一实例中，本申请包括的外源受体是特异性结合CLD18多肽的CAR。在一实例中，CAR特异性结合CLD18.2多肽。在一实例中，CAR特异性结合CLD18.2多肽胞外结构域。在一实例中，CAR多肽的各个结构域处于相同的多肽链中，例如，作为单个多肽链表达。在一些实例中，CAR多肽的各个结构域彼此不邻接，例如，处于不同的多肽链中。
2.1.1抗原结构域
在一实例中，抗原结构域包括抗体。在一实例中，抗原结构域包括scFV。在一实例中，抗原结构域包括抗体重链可变区(VH)和/或轻链可变区(VL)；或者包括交联的Fab；或者包括F(ab) 2。在一实例中，抗原结构域包括抗体VH和VL，形成可变片段(Fv)。
在一实例中，CAR包括与CLD18.2多肽特异性结合的抗体。在一实例中，CAR的抗原结构域包括特异性结合CLD18.2多肽的Fv。在一实例中，CAR的抗原结合域包含：SEQ ID NO：14所示的HCDR1、SEQ ID NO：15所示的HCDR2、SEQ ID NO：16所示的HCDR3、SEQ ID NO：17所示的LCDR1、SEQ ID NO：18所示的LCDR2、SEQ ID NO：19所示的LCDR3。在一实例中，CAR的抗原结合域包含：SEQ ID NO：10所示的VH，SEQ ID NO：12所示的VL。在一实例中，CAR的抗原结合域包含：SEQ ID NO：2、23、24、25、26、27、28、29、30、31、32或33所示的scFv序列。
在一个方面，本申请考虑到产生功能上等同的分子的起始抗体(例如，VH或VL)氨基酸序列的修饰。例如，可修饰CAR包括特异性结合CLD18.2多肽的抗原结构域的VH或VL的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性序列。
在一实例中，可进一步修饰本申请的抗体，使得它们在氨基酸序列上(例如，相对于野生型)有所变化，但在所需活性上没有变化。例如，可对蛋白质进行另外的核苷酸置换，导致“非必需”氨基酸残基处的氨基酸置换。例如，分子中的非必需氨基酸残基可被来自相同侧链家族的另一个氨基酸残基取代。在另一个实施方案中，氨基酸片段可被结构相似但在顺序和/或组成上与侧链家族成员不同的氨基酸片段取代，例如，可进行保守置换，其中氨基酸残基被具有相似侧链的氨基酸残基所取代。
在一个实例中，抗原结构域与跨膜结构域直接连接或通过铰链连接。在一实例中，所述铰链包括CD8铰链，例如，具有SEQ ID NO:3所示序列95-100％同一性的序列。
在一实例中，编码CAR的核酸分子上游包括编码信号肽的多核苷酸。在一个实施方案中，信号肽的序列具有SEQ ID NO:1所示序列。
2.1.2跨膜结构域
跨膜结构域可以将CAR锚定在细胞膜上。本申请CAR分子的跨膜结构域包括选自以下的跨膜结构域:T细胞受体的α、β、或ζ的跨膜区、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D和/或NKG2C的跨膜结构域。
在一实例中，CAR包括CD8跨膜区，具有SEQ ID NO:4所示序列95-100％同一性的序列。在一实例中，CAR包括CD28跨膜区，具有SEQ ID NO:5所示序列90-100％同一性的序列。
2.1.3胞内信号结构域
在一实例中，本申请提供的CAR包括胞内信号结构域。在一实例中，胞内信号结构域包括一级信号结构域。在一实例中，胞内信号结构域包括共刺激信号结构域。在一实例中，胞内信号结构域包括一级信号结构域和共刺激信号结构域。
在一实例中，一级信号结构域包括选自CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(FcεR1b)、CD79a、CD79b、FcγRIIa、DAP10、和DAP12的蛋白质的功能信号结构域。在一实例中，一级信号结构域包括CD3ζ的胞内域，具有SEQ ID NO:8所示序列90-100％同一性的序列。
在一实例中，共刺激信号结构域包括选自如下一种或多种的蛋白质的功能信号结构域:CD27、CD28、CD137、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原-1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、 TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244,2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、或NKG2D。
在一实例中，共刺激信号结构域包括CD137胞内域，具有SEQ ID NO:6所示序列90-100％同一性的序列。
在一实例中，CAR的胞内信号结构域包括人CD3ζ胞内域。在一实例中，CAR的胞内信号结构域包括人CD3ζ胞内域和CD28胞内域。在一实例中，CAR的胞内信号结构域包括人CD3ζ胞内域和CD137胞内域。在一实例中，CAR的胞内信号结构域包括CD3ζ胞内域、CD28胞内域和CD137胞内域。
本申请考虑到整个CAR分子的修饰，例如，CAR分子的各个结构域的一个或多个氨基酸序列的修饰，以便产生功能上等同的分子。可修饰CAR分子保留起始CAR分子的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
示例性的外源受体，如包括CAR的序列具有SEQ ID NO：2、23、24、25、26、27、28、29、30、31、32或33所示的scFv序列分别与SEQ ID NO：34、35或36顺序连接的序列。示例性，如SEQ ID NO：2所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：2所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：2所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：23所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：23所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：23所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：24所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：24所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：24所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：25所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：25所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：25所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：26所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：26所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：26所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：27所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：27所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：27所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：28所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：28所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：28所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：29所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：29 所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：29所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：30所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：30所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：30所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：31所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：31所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：31所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：32所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：32所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：32所示序列与SEQ ID NO：36所示序列顺序连接；如SEQ ID NO：33所示序列与SEQ ID NO：34所示序列顺序连接，或SEQ ID NO：33所示序列与SEQ ID NO：35所示序列顺序连接，SEQ ID NO：33所示序列与SEQ ID NO：36所示序列顺序连接。或所述的嵌合抗原受体具有SEQ ID NO：37、38或39所示的核苷酸序列。
2.2修饰TCR受体
本申请提供修饰TCR受体特异性结合CLD18.2多肽，包括但不限于：嵌合T细胞受体、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、抗体-TCR嵌合物。在一实例中，修饰TCR受体的抗原结合域，具有SEQ ID NO：2、23、24、25、26、27、28、29、30、31、32或33所示序列的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性。
3.宿主细胞
本申请提供的宿主细胞是包括表达特异性结合CLD18.2多肽的外源受体的细胞。外源受体与CLD18.2多肽结合后能激活所述细胞。在一实例中，宿主细胞包括干细胞。干细胞包括人多能干细胞(包括人诱导多能干细胞(iPSC)和人胚胎干细胞)。在一实例中，宿主细胞包括免疫效应细胞(也称为免疫细胞)。在一实例中，宿主细胞为原代细胞。本申请提供包含所述宿主细胞的细胞治疗产品。宿主细胞可以指人或非人的、来源于动物的细胞。
本申请的免疫效应细胞包括淋巴谱系的细胞。包括B、T和自然杀伤(NK)细胞的淋巴谱系提供抗体的产生、细胞免疫系统的调节、血液中外源试剂的检测、宿主外源细胞的检测等。淋巴谱系的免疫效应细胞的非限制性实例包括T细胞、自然杀伤T(NKT)细胞及其前体，包括胚胎干细胞和多能干细胞(例如，分化成淋巴样细胞的干细胞或多能干细胞)。T细胞包括在胸腺中成熟的淋巴细胞，主要负责细胞介导的免疫。T细胞参与适应性免疫系统。T细胞包括任何类型的T细胞，包括但不限于辅助T细胞、细胞毒性T细胞、记忆T细胞(包括中央记忆T细胞、干细胞样记忆T细胞(或干样记忆T细胞)和两种效应记忆T细胞：例如TEM细胞和TEMRA细胞)、调节性T细胞(也称为抑制性T细胞)、自然杀伤T细胞、粘膜相关性不变T细胞、γδT细胞或αβT细胞。细胞毒性T细胞(CTL或杀伤性T细胞)是能够诱导被感染的体细胞或肿瘤细胞死亡的T淋巴细胞。在一实例中，本 申请的细胞选自：T细胞、NK细胞、细胞毒性T细胞、NKT细胞、巨噬细胞、CIK细胞、以及干细胞衍生的免疫效应细胞或其组合。在一实例中，免疫效应细胞是T细胞。在一实例中，T细胞包括CD4+T细胞和/或CD8+T细胞。在一实例中，免疫效应细胞包括CD3+T细胞。在一实例中，本申请组合物中的细胞包括由PBMC细胞经CD3磁珠刺激后收集的细胞群。在一实例中，免疫效应细胞选自T细胞、NK细胞、NKT细胞、肥大细胞、巨噬细胞、树突细胞、CIK细胞、干细胞衍生的免疫效应细胞或其组合。在一实例中，免疫效应细胞来源于天然的T细胞和/或经多能干细胞诱导产生的T细胞。在一实例中，免疫效应细胞为自体或同种异体T细胞、或原代T细胞。在一实例中，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、αβT细胞或其组合。
本申请的细胞(例如，T细胞)可以是自体的、非自体的(例如，同种异体的)、或者是体外从工程化的祖细胞或干细胞衍生而来。可从许多来源获得，包括外周血单个核细胞(PBMC)、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。
在本申请的某些方面，可使用本领域技术人员已知的任意数量的技术如Ficoll TM分离技术从收集自受试者的血液样品中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体的循环血液的细胞。单采血液成分术产物通常含有淋巴细胞，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。在一个方面，可洗涤通过单采血液成分术收集的细胞以去除血浆部分并将细胞置于适当的缓冲液或培养基中以供后续处理步骤。在本申请的背景下还可使用多轮选择。在某些方面，可能需要进行选择程序并在激活和扩充过程中使用“未选择的”细胞。“未选择的”细胞也可以经受其他轮选择。
本申请的细胞能够调节肿瘤微环境。
未纯化的CTL来源可以是本领域已知的任何来源，例如骨髓、胎儿、新生儿或成年或其它造血细胞来源，例如胎儿肝、外周血或脐带血。可以采用各种技术来分离细胞。例如，阴性选择法可以最初去除非CTL。mAb对于鉴定与特定细胞谱系和/或阳性和阴性选择的分化阶段相关的标志物特别有用。
最初可以通过相对粗略的分离除去大部分末端分化的细胞。例如，最初可以使用磁珠分离来去除大量不相关的细胞。在某些实施方式中，在分离细胞之前将去除总造血细胞的至少约80％，通常至少约70％。
分离的程序包括但不限于密度梯度离心；重沉(resetting)；偶联至改变细胞密度的颗粒；用抗体包被的磁珠进行磁分离；亲和色谱；与mAb结合或结合使用的细胞毒性剂，包括但不限于补体和细胞毒素；并用附着在固体基质(例如板、芯片、淘析)上的抗体淘选或任何其它方便的技术。
分离和分析的技术包括但不限于流式细胞术，其可以具有不同的复杂程度，例如多个 颜色通道、低角度和钝角光散射检测通道、阻抗通道。
通过使用与死细胞相关的染料，例如碘化丙啶(PI)，可以针对死细胞选择细胞。在某些实施方式中，将细胞收集在包括2％胎牛血清(FCS)或0.2％牛血清白蛋白(BSA)的培养基或任何其它合适的例如无菌等渗培养基中。
4.载体
本文中载体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，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。载体包括自主复制的质粒或病毒。载体还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。
对本申请所述宿主细胞的遗传修饰可以通过用重组核酸分子转导基本上均质的细胞群来完成。在一实例中，病毒载体(AAV、逆转录病毒或慢病毒)用于将核酸分子引入细胞。例如，可以将编码外源受体(例如CAR)的多核苷酸克隆到逆转录病毒载体。也可以使用非病毒载体。转导可以使用任何合适的病毒载体或非病毒递送系统。可以在单个多顺反子表达盒、单个载体的多个表达盒或多个载体中用辅助分子(例如细胞因子)构建CAR。产生多顺反子表达盒的元件的实例包括但不限于各种病毒和非病毒内部核糖体进入位点(IRES，例如，FGF-1IRES、FGF-2IRES、VEGF IRES、IGF-II IRES、NF-κB IRES、RUNX1IRES、p53IRES、甲型肝炎IRES、丙型肝炎IRES、瘟病毒IRES、无杆状病毒IRES、小核糖核酸病毒IRES、脊髓灰质炎病毒IRES和脑心肌炎病毒IRES)和可切割的接头(例如2A肽，例如P2A、T2A、E2A和F2A肽)。
可以使用的其它病毒载体包括，例如，腺病毒、慢病毒和与腺相关的病毒载体、牛痘病毒、牛乳头瘤病毒或疱疹病毒，例如爱泼斯坦-巴尔病毒。
非病毒方法也可以用于免疫效应细胞的遗传修饰。例如，可以通过在脂质转染，脱唾液酸血清类粘蛋白-聚赖氨酸偶联，或手术条件下的微注射将核酸分子引入免疫效应细胞中。其它非病毒的基因转移方法包括使用脂质体、磷酸钙、DEAE葡聚糖、电穿孔和原生质体融合的体外转染。也可以先将核酸分子转移到可离体培养的细胞类型(例如，自体或同种异体原代细胞或其后代)中，再将经所述核酸分子修饰后的细胞(或其后代)注射到受试者目标组织中或全身注射。
5.给药
本申请组合物包括所述宿主细胞或所述细胞治疗产品。在一实例中，组合物系统地或直接提供给受试者，以诱导和/或增强对CLD18.2多肽的免疫应答和/或治疗和/或预防胆道肿瘤。在一实例中，将组合物直接注射到目的器官(例如，受肿瘤影响的器官)中。或者，例如通过向循环系统(例如，静脉、肿瘤脉管系统)给药，将组合物间接地提供给目的器官。可以在施用之前、同时或之后提供扩增和分化剂，以增加体外或体内T细胞、NKT细胞或CTL细胞的扩增。
本申请的组合物中的免疫效应细胞可以包括纯化的细胞群。本领域技术人员可以使用 各种众所周知的方法，例如荧光激活细胞分选(FACS)，容易地确定群体中本申请的免疫效应细胞的百分比。在包括本申请的免疫效应细胞的群体中，纯度的合适范围是约50％至约55％、约5％至约60％、以及约65％至约70％。在一实例中，纯度为约70％至约75％、约75％至约80％或约80％至约85％。在一实例中，纯度为约85％至约90％，约90％至约95％以及约95％至约100％。可以通过注射、导管等引入细胞。
本申请的组合物可以是包括本申请的免疫效应细胞或其祖细胞和药学上可接受的载体的药物组合物。给药可以是自体的或异体的。例如，可以从一个受试者获得免疫效应细胞或祖细胞，并将其施用于相同受试者或不同的相容受试者。外周血来源的免疫效应细胞或其后代(例如，体内、离体或体外来源)可通过局部注射施用，包括导管给药、全身注射、局部注射、静脉内注射或肠胃外给药。当施用本申请的组合物时，可以将其配制成单位剂量可注射形式(溶液剂、悬浮剂、乳剂等)。
6.剂型
包括本申请的组合物以无菌液体制剂的形式提供，例如等渗水溶液剂、悬浮液、乳剂、分散剂或粘性组合物，其可以缓冲至选定的pH。液体制剂通常比凝胶、其它粘性组合物和固体组合物更容易制备。另外，液体组合物在某种程度上更方便施用，尤其是通过注射。另一方面，可以在适当的粘度范围内配制粘性组合物以提供与特定组织的更长的接触时间。液体或粘性组合物可以包括载体，所述载体可以是溶剂或分散介质，其包括例如水、盐水、磷酸盐缓冲盐水、多元醇(例如甘油、丙二醇、液体聚乙二醇等)及其合适的混合物。
可以通过将本申请组合物中的免疫效应细胞掺入所需量的适当溶剂中，并根据需要掺入不同量的其它成分来制备无菌注射溶液。这样的组合物可以与合适的载体、稀释剂或赋形剂例如无菌水、生理盐水、葡萄糖、右旋糖等混合。组合物也可以冻干。所述组合物可包括辅助物质，例如润湿剂、分散剂或乳化剂(例如，甲基纤维素)、pH缓冲剂、胶凝剂或增粘剂、防腐剂、矫味剂、颜料等，这取决于给药途径和所需制剂。
可以添加增强组合物的稳定性和无菌性的各种添加剂，包括抗微生物防腐剂、抗氧化剂、螯合剂和缓冲剂。可以通过各种抗细菌和抗真菌剂，例如对羟基苯甲酸酯、三氯叔丁醇、苯酚、山梨酸等来确保防止微生物的作用。可通过使用延迟吸收的试剂例如单硬脂酸铝和明胶来延长可注射药物形式的吸收。然而，所使用的任何媒介物、稀释剂或添加剂将必须与遗传修饰的免疫效应细胞或其祖细胞相容。
该组合物可以是等渗的，即它们可以具有与血液和/或泪液相同的渗透压。组合物的所需等渗性可以使用氯化钠或其它药学上可接受的试剂例如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。氯化钠可以特别适用于含有钠离子的缓冲剂。
如果需要，可使用药学上可接受的增稠剂将组合物的粘度保持在选定水平。例如，甲基纤维素容易且经济地获得并且易于使用。其它合适的增稠剂包括，例如，黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的浓度可以取决于选择的试剂。重要的是要使用能够达到所选粘度的用量。显然，合适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(例如，是否将组合物配制成溶液剂、悬浮液、凝胶剂或其它液体形式，例如定时释放形式或液体填充形式)。
对于所治疗的受试者，要施用的组合物中的细胞数量将有所不同。可以更少的数量施用更有效的细胞。可以根据每个受试者的个体因素，包括其大小、年龄、性别、体重和受试者的状况，来确定有效剂量的精确确定。本领域技术人员从本申请和本领域知识中可以容易地确定剂量。
本领域技术人员可以容易地确定组合物中和在方法中施用的细胞和任选的添加剂、媒介物和/或载体的量。通常，任何添加剂(除一种或多种活性细胞和/或一种或多种试剂外)在磷酸盐缓冲盐水中的存在量为0.001％至50％(重量)溶液，并且活性成分按微克至毫克的顺序存在，例如约0.0001wt％至约5wt％、约0.0001wt％至约1wt％、约0.0001wt％至约0.05wt％或约0.001wt％至约20wt％、约0.01wt％至约10wt％或约0.05wt％至约5wt％。对于要施用于动物或人的任何组合物，可以确定以下结果：毒性，例如通过在合适的动物模型例如啮齿类动物如小鼠中确定致死剂量(LD)和LD50；组合物的剂量，其中的组分浓度和施用组合物的时间，引起合适的反应。
7.治疗方法
本申请提供一种治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的方法。胆道肿瘤包括胆囊癌和胆管癌。在一实例中，本方法治疗胆道肿瘤细胞的CLD18.2多肽呈“阳性”的受试者，如通过免疫学检测(例如流式细胞术、免疫组化等)胆道肿瘤细胞的CLD18.2多肽的表达水平显著高于采用同种型匹配的对照，和/或基本相似于已知CLD18.2多肽呈阳性的细胞的水平，和/或显著高于已知CLD18.2多肽呈阴性的细胞的水平；所述受试者在给予本方法治疗后能获得临床受益。在一实例中，本方法治疗胆道肿瘤细胞的CLD18.2多肽呈“阴性”的受试者，例如，当通过用特异性地结合CLD18.2多肽的抗体染色，所述染色可被免疫学检测以一定水平检测到，所述水平显著低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CLD18.2多肽呈阳性的细胞的水平，和/或基本相似于已知对CLD18.2多肽呈阴性的细胞的水平；所述受试者在给予本方法治疗后能获得临床受益，则该受试者为怀疑患有CLD18.2阳性的胆道肿瘤患者。
临床受益是指受试者对本方法有应答或有治疗效果。本文中“有应答”是指根据实体肿瘤的疗效评价标准1.1版(RECIST1.1)评估为完全缓解CR(Complete Response，所有靶病灶消失，全部病理淋巴结短直径必须减少至<10mm)；或者部分缓解PR：靶病灶直径之和比基线水平减少至少30％；或者疾病稳定(SD):靶病灶减小的程度没达到PR，增加的程度也没达到PD水平，介于两者之间，研究时可以直径之和的最小值作为参考。根据实体肿瘤的疗效评价标准1.1版(RECIST1.1)评估为疾病进展(PD，progressive disease)：以整个实验研究过程中所有测量的靶病灶直径之和的最小值为参照，直径和相对增加至少20％(如果基线测量值最小就以基线值为参照)；除此之外，必须满足直径和的绝对值增加 至少5mm(出现一个或多个新病灶也视为PD)。无进展生存期(PFS)被定义为从本申请治疗方法的第一剂量到疾病进展的第一次出现或由于任何原因的死亡的时间。总生存期(OS)被测量为从本申请治疗方法的第一剂量到由于任何原因引起的死亡的时间，并且将被以与针对PFS描述的方式类似的方式分析。
在一实例中，有应答包括本申请治疗方法处理后出现CR或PR。在一实例中，有应答包括本申请治疗方法处理后出现CR、PR或SD；在一实例中，有应答是指接受本申请治疗方法的受试者能获得明显的临床获益，包括PFS和/或OS明显延长，比如延长2周、3周、4周、1个月、2个月、3个月、4个月、5个月、6个月或更长。
在一实例中，给予本申请治疗方法治疗后，疾病负荷约减少1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或100％。
在一实例中，通过本申请提供的方法进行的治疗，与其它方法相比，复发的概率降低。例如，在一实例中，治疗后复发或进展的概率小于约80％，小于约70％，小于约60％，小于约50％，小于约40％，小于约30％，小于约20％，小于约10％，小于约9％，小于约8％，小于约7％，小于约6％。
本文中“受试者”与“患者”可以交换使用，是指接受细胞产品治疗的个体，如人或其它动物，通常是人。
本文中“治疗”指在试图改变疾病过程的干预措施，完全或部分减轻或减少疾病或与其相关的症状。所述的治疗效果包括但不限于，预防胆道肿瘤的发生或复发、减轻症状、减少胆道肿瘤的任何直接或间接病理学结果、防止转移、减缓胆道肿瘤进展速率、改善或减轻胆道肿瘤状态，和减轻或改善预后、或延迟胆道肿瘤的发展。所述“延迟胆道肿瘤的发展"表示推迟、阻碍、减缓、减慢、稳定、遏制和/或拖延胆道肿瘤的发展。该延迟可具有不同的时间长度，这取决于疾病史和/或待治疗的个体。本领域技术人员应理解，充分或显著的延迟可涵盖预防(对于未发展胆道肿瘤的个体而言)。
本方法包括给予受试者包含本申请所述宿主细胞、细胞治疗产品或组合物。在一实例中，给予受试者有效量或治疗有效量的包含本申请所述宿主细胞、细胞治疗产品或组合物。
本文中“有效量”或“治疗有效量”是指足以预防或治疗个体胆道肿瘤的剂量。治疗性或预防性使用的有效剂量取决于所治疗胆道肿瘤的阶段和严重程度、受试者的年龄、体重和一般健康状况以及处方医生的判断。剂量的大小还取决于所选择的活性物质、给药方法、给药时间和频率、可能伴随特定活性物给药的不良副作用的存在、性质和程度以及所需的生理效应。根据处方医生或本领域技术人员判断可能需要一个周期轮或多个周期给予本申请所述宿主细胞、细胞治疗产品或组合物。
本文中“肿瘤负荷”，包括肿瘤体积大小或分化程度，或转移类型、阶段，和/或中晚期癌症常见的并发症的出现与消失，例如癌性胸腹水，和/或肿瘤标志物出现或表达水平的变化，和/或受试者发生毒性结果的可能性或发生率，例如CRS，巨噬细胞活化综合征， 肿瘤溶解综合征，神经毒性和/或针对给予的细胞和/或嵌合抗原受体的宿主免疫应答。在一实例中，肿瘤的大小通过PET(正电子发射型计算机断层显像)和CT(计算机X线断层扫描)自带的标尺进行测量。肿瘤标志物，是指特征性存在于恶性肿瘤细胞，或由恶性肿瘤细胞异常产生的物质，或是宿主对肿瘤的刺激反应而产生的物质，并能反映肿瘤发生、发展，监测肿瘤对治疗反应的一类物质。肿瘤标志物存在于肿瘤患者的组织、体液和排泄物中，能够用免疫学、生物学及化学的方法检测到，示例性的，包括甲胎蛋白(AFP)、CA125、CA15-3、鳞状细胞癌抗原(SCC)、细胞角蛋白19的可溶性片段(CYFRA21-1)、癌胚抗原(CEA)、CA199、CA724等。
本文中“周期”指每一次细胞治疗从接受治疗前的基线期(即评估阶段)开始，到给下一次接受治疗前的基线期前这段时间；如从给予预处理药物开始，直至下一周期开始预处理药物治疗前。在一些实施方式中，每个周期给予细胞治疗产品之前都需要进行预处理。
本文中“预处理”是指在给予所述宿主细胞、细胞治疗产品或组合物之前，先给予受试者其他的药物或治疗，使得受试者的身体状态更适合输注所述宿主细胞、细胞治疗产品或组合物。在一实例中，预处理包括给予受试者化学药物、生物药物、放疗或其组合。优选的，给予化学药物进行预处理，更优选，所述的化学药物为化疗药。在一实例中，受试者在接受预处理之后的淋巴细胞相比预处理之前减少约50％、55％、60％、65％或者70％。
本文中“化学药物”是指通过化学合成的方法制备的抗癌药物，包括传统的化疗药，例如烷化剂、抗代谢药、抗癌抗生素等，也包括靶向药，如阿法替尼、爱乐替尼等。在一实例中，化学药物包括但不限于烷化剂、抗代谢药、抗肿瘤抗生素、植物类抗癌药、激素、免疫制剂等；例如，二萜生物碱类化合物(如紫杉烷)、环磷酰胺、氟达拉滨、环孢菌素、雷帕霉素、美法仑、苯达莫司汀、天冬酰胺酶，白消安，卡铂，顺铂，柔红霉素，多柔比星，氟尿嘧啶，羟基脲，甲氨蝶呤，利妥昔单抗，长春碱和/或长春新碱等。在一实例中，抗代谢药包括但不限于卡莫氟、替加氟、喷司他丁、去氧氟尿苷、曲美沙特、氟达拉滨、吉美嘧啶、奥替拉西钾、双呋氟尿嘧啶、卡莫氟、卡培他滨、加洛他滨、阿糖胞苷十八烷基磷酸钠、fosteabine钠水合物、雷替曲塞、paltitrexid、乙嘧替氟、噻唑呋林(tiazofurin)、诺拉曲塞、培美曲塞、奈拉滨(nelzarabine)、2’-脱氧-2’-亚甲基胞苷、2’-氟亚甲基-2’-脱氧胞苷、N-[5-(2,3-二氢-苯并呋喃基)磺酰基]-N’-(3,4-二氯苯基)脲、N6-[4-脱氧-4-[N2-[2(E),4(E)-十四碳二烯酰基]甘氨酰基氨基]-L-甘油-B-L-甘露糖-吡喃庚糖基]腺嘌呤、aplidine、海鞘素、4-[2-氨基-4-氧代-4,6,7,8-四氢-3H-嘧啶并[5,4-b]噻嗪-6-基-(S)-乙基]-2,5-噻吩酰基(thienoyl)-L-谷氨酸、氨基蝶呤、5-氟尿嘧啶、阿拉诺新、11-乙酰基-8-(氨基甲酰氧基甲基)-4-甲酰基-6-甲氧基-14-氧杂-1,11-二氮杂四环(7.4.1.0.0)-十四碳-2,4,6-三烯-9-基醋酸酯、苦马豆碱、洛美曲索、右雷佐生、蛋氨酸酶、2’-氰基-2’-脱氧-N4-棕榈酰基-1-B-D-阿糖呋喃糖基(arabinofuranosyl)胞嘧啶和3-氨基吡啶-2-醛缩氨基硫脲等。在一实例中，烷化剂包括但不限于达卡巴嗪、苯丙氨酸氮芥、环磷酰胺、替莫唑胺、苯丁酸氮芥、 白消安、氮芥和亚硝基脲等。本文中“生物药物(即其他生物药物)”是指通过分子生物学或基因工程的手段制备的药物，如抗体药物、ADC等。本文中“微管蛋白抑制剂”包括微管蛋白聚合促进剂和微管蛋白聚合抑制剂。微管蛋白抑制剂包括但不限于例如紫杉烷类、埃坡霉素类、海绵内酯类以及Laulimalide等。本文中“紫杉烷类(taxanes)药物”指主要成分含有紫杉烷类化合物的药物，所述的紫杉烷类化合物具有与紫杉烷类似的桥亚甲基苯并环癸烯母核结构。在一实例中，紫杉烷类化合物的所述桥亚甲基苯并环癸烯母核结构中含有不饱和键、或不含不饱和键。在一实例中，紫杉烷类的所述桥亚甲基苯并环癸烯母核结构上的碳原子被选自N、O、S、P的杂原子取代。在一实例中，紫杉烷类化合物的给药方式为注射给药。
在一实例中，预处理包括给予受试者环磷酰胺、氟达拉滨、微管蛋白抑制剂、嘧啶类抗肿瘤药物中的任意一种或至少两种或其组合。
在一实例中，每个周期给予所述宿主细胞、细胞治疗产品或组合物之前，先对受试者进行预处理，所述预处理包括给予受试者化疗药物、生物药物和/或放疗。在一实例中，所述放疗是全身辐射治疗，或是局部辐射治疗。
本文中“剂量”，是以重量为基础计算的剂量，或是以体表面积(BSA)为基础计算的剂量，或是以单个人为基础计算的剂量。以重量为基础计算的剂量是以受试者体重为基础所计算出的剂量，例如mg/kg受试者或患者体重，细胞个数/kg受试者或患者体重等。以BSA为基础计算的剂量是以受试者的表面积为基础所计算出的剂量，例如mg/m 2，细胞个数/m 2等。以单个人为基础计算的剂量是指每个受试者每个周期或每次给予的剂量，例如mg/受试者，细胞个数/受试者。
本文中“给药次数”是指每个周期内给予所述宿主细胞、细胞治疗产品或组合物或每个预处理药物的频率。例如，氟达拉滨给药次数：给予1次；或每日给予1次，连续给予4、3或2日。环磷酰胺给药次数：给予1次；或每日给予1次，连续给予4、3或2日。白蛋白结合型紫杉醇给药次数：给予1次；或每日给予1次，连续给予4、3或2日。例如，每个周期内给予的所述宿主细胞、细胞治疗产品或组合物一次性给予；或分2、3、4、5、6、7、8、9、10次给予，可以是连续日给予，或间隔1、2、3、4、5、6天给予。
在一实例中，给予受试者至少一个周期的所述宿主细胞、细胞治疗产品或组合物进行治疗。在一实例中，每个周期的剂量可以是相同的，也可以是不同的。在一实例中，一个周期内的剂量一次或分多次给予受试者，每次给药的分剂量可以是相同或不同。如，一个周期内的所述宿主细胞、细胞治疗产品或组合物1次给予受试者，或分2次、3次、4次、5次，或更多次给予受试者。在一实例中，当一个周期内的剂量分多次给予受试者时，每次给予的所述宿主细胞、细胞治疗产品或组合物或预处理药物的剂量由医生根据受试者的具体情况确定。受试者的具体情况可以是受试者的整体健康情况、疾病的严重程度、对同周期前次给药量的反应、对先前周期的反应、受试者联合用药情况、毒性反应程度或可能性、并发症、癌症转移情况、以及医生所认为的会影响到受试者接受细胞治疗或预处理 药物的剂量的任何其他因素。在一实例中，每次给予的剂量呈递增趋势。在一实例中，每次给予的剂量呈递减趋势。在一实例中，每次给予的剂量呈先递增后递减的趋势。在一实例中，每次给予的剂量呈先递减后递增的趋势。
给予多周期的所述宿主细胞、细胞治疗产品或组合物是指具有多个时间段，在每个时间段内给予一定总量的所述宿主细胞、细胞治疗产品或组合物。在一实例中，每个时间段的间隔时间是一致的。在一实例中，每个时间段的间隔时间是不一致的。在一实例中，给予至少2个周期的所述宿主细胞、细胞治疗产品或组合物，如给予2、3、4、5、6、7、8、9或10个周期。在一实例中，每个周期给予的剂量及给予次数，不同周期之间的间隔时间的设计，是以改善一种或多种结果为目标来评估，例如，结果可以是降低受试者的副作用的程度或发生副作用的可能性，也可以是改善疗效。
在一实例中，每个周期给予特异性结合CLD18.2多肽的外源受体的细胞(例如CLD18.2-CAR-T细胞)的剂量不超过约2ⅹ10 9个细胞/kg受试者体重，或不超过约1ⅹ10 11个细胞/人。优选的，每个周期给予的剂量不超过约2ⅹ10 8个细胞/kg受试者体重，或不超过约1ⅹ10 10个细胞/人。更优选地，每个周期给予的剂量不超过约2ⅹ10 7个细胞/kg受试者体重，或不超过约5ⅹ10 9个细胞/人、或2ⅹ10 9个细胞/人、或1ⅹ10 9个细胞/人、或5ⅹ10 8个细胞/人。在一实例中，每个周期给予的剂量为约1ⅹ10 5个细胞/kg受试者体重至2ⅹ10 7个细胞/kg受试者体重，或约1ⅹ10 6个细胞/kg受试者体重至2ⅹ10 7个细胞/kg受试者体重。在一实例中，每个周期给予的剂量是约1ⅹ10 7个细胞至1ⅹ10 9个细胞、1ⅹ10 7个细胞至2ⅹ10 9个细胞、1ⅹ10 7个细胞至5ⅹ10 9个细胞、或1ⅹ10 7个细胞至1ⅹ10 10个细胞，优选的，为1ⅹ10 8个细胞至1ⅹ10 9个细胞、1ⅹ10 8个细胞至2ⅹ10 9个细胞、1ⅹ10 8个细胞至5ⅹ10 9个细胞，更优选的，为1ⅹ10 8个细胞至5ⅹ10 8个细胞、或2.5ⅹ10 8个细胞至5ⅹ10 8个细胞、3.75ⅹ10 8个细胞至5ⅹ10 8个细胞。
在一实例中，预处理包括给予受试者环磷酰胺和氟达拉滨；或者环磷酰胺、氟达拉滨以及微管蛋白抑制剂。在一实例中，微管蛋白抑制剂是紫杉烷类化合物，优选的，所述紫杉烷类化合物选自紫杉醇、白蛋白结合型紫杉醇、多西他赛，更优选的，所述紫杉烷类化合物是白蛋白结合型紫杉醇。
在一实例中，可在同日或不同日给予氟达拉滨、环磷酰胺和白蛋白结合型紫杉醇。如果在同日给予氟达拉滨、环磷酰胺和白蛋白结合型紫杉醇，则可在氟达拉滨前或后给予环磷酰胺和/或白蛋白结合型紫杉醇；或在环磷酰胺前或后给予氟达拉滨和/或白蛋白结合型紫杉醇；或在白蛋白结合型紫杉醇前或后给予环磷酰胺和/或氟达拉滨。在一实例中，可同时给予或依次给予氟达拉滨、环磷酰胺、白蛋白结合型紫杉醇。在一实例中，在给予氟达拉滨前给予环磷酰胺。在一实例中，在给予氟达拉滨后给予环磷酰胺。在一实例中，在给予氟达拉滨前给予白蛋白结合型紫杉醇。在一实例中，在给予氟达拉滨后给予白蛋白结合型紫杉醇。在一实例中，在给予环磷酰胺前给予白蛋白结合型紫杉醇。在一实例中，在给予环磷酰胺后给予白蛋白结合型紫杉醇。
在一实例中，氟达拉滨的给予量约为20-60mg/天、或约30-55mg/天、或约30-50mg/天。在一实例中，氟达拉滨的给予量约为10-50mg/m 2/天、或约15-40mg/m 2/天、或约15-30mg/m 2/天、或约20-30mg/m 2/天；或约25mg/m 2/天。在一实例中，环磷酰胺的给予量约为200-1000mg/天、或约300-600mg/天、或约300-560mg/天、或约300-550mg/天、或约300-500mg/天。在一实例中，环磷酰胺的给予量约为200-400mg/m 2/天、或约200-300mg/m 2/天、或约250mg/m 2/天。在一实例中，紫杉烷类化合物给予量为不高于约300mg/天、或不高于约200mg/天，优选的，所述紫杉烷类化合物给予量约为90-200mg/天，更优选的，所述紫杉烷类化合物给予量约为90-120mg/天。在一实例中，每种预处理药物连续使用不超过4天。在一实例中，环磷酰胺给予2-3次。在一实例中，氟达拉滨给予1-2次。在一实例中，紫杉烷类化合物给予一次。
将对受试者每个周期给予CAR-T细胞疗法之日指定为第0日。在一实例中，在第-6和-5日给予氟达拉滨。在一实例中，在第-5和-4日给予氟达拉滨。在一实例中，在第-4和-3日给予氟达拉滨。在一实例中，在第-6、-5、-4和-3日给予氟达拉滨。在一实例中，在第-7、-6、-5和-4日给予氟达拉滨。在一实例中，在第-6、-5、-4和-3日给予环磷酰胺。在一实例中，在第-6、-5和-4日给予环磷酰胺。在一实例中，在第-5、-4和-2日给予环磷酰胺。在一实例中，在第-5、-4和-3日给予环磷酰胺。在一实例中，在第-4、-3和-2日给予环磷酰胺。在一实例中，在第-4和-3日给予环磷酰胺。在一实例中，在第-6和-5日给予环磷酰胺。在一实例中，在第-5和-4日给予环磷酰胺。在一实例中，在第-2日、第-3日、第-4日、第-5日、或第-6日，给予紫杉烷类化合物(如白蛋白结合型紫杉醇)。
在一实例中，本文所述的用途或方法还包括监控受试者对所述宿主细胞、细胞治疗产品或组合物的毒性反应程度或者风险，并根据所述毒性程度或者风险确定后续分次给药或者在后周期的给药剂量和间隔时间。在一实例中，所述毒性反应程度或者风险包括但不限于例如CRS、神经毒性、巨噬细胞活化综合征和肿瘤溶解综合征等。
在一实例中，通常在给予所述宿主细胞、细胞治疗产品或组合物之后，当毒性反应或其症状或生物化学指标(例如CRS或神经毒性、巨噬细胞活化综合征或肿瘤溶解综合征)的风险等于或低于可接受水平的情况下，给予在后周期的治疗。在一实例中，所述毒性反应或其症状或生物化学指标包括发热、低血压、缺氧、神经障碍、炎性细胞因子、C反应性蛋白(CRP)的血清水平中的一种或多种。在一实例中，所述毒性反应或其症状或生物化学指标的风险的可接受的水平是指在给予在先周期的治疗之后的峰值水平的95％、90％、85％、80％、75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、15％、10％、或者5％。在一实例中，在基于在先周期的治疗之后，在受试者中指示细胞因子-释放综合征(CRS)的因子的血清水平不超过该受试者在给予在先周期之前的血清水平的5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30倍的情况下给予在后周期的治疗。在一实例中，经在先周期的治疗，在肿瘤负荷呈现稳定或减少后，适应性宿主免疫应答(即，机体对CAR-T细胞产生 的免疫排斥反应)产生之前，给予一个或多个在后周期的治疗。在这种条件下，在后周期可以安全有效地提供免疫监测、清除或预防残留肿瘤细胞的增殖或转移。因此，在一实例中，在后周期是疾病巩固剂量。
在一实例中，在给予在先周期的细胞治疗产品后至少28、29、30、31、32、33、34、35天之后，给予在后周期的治疗。
给予细胞治疗产品的方法是已知的。在一实例中，所述宿主细胞、细胞治疗产品或组合物通过自体回输的方式给予受试者。在一实例中，特异性结合CLD18.2多肽的外源受体的细胞(例如CLD18.2-CAR-T细胞)源自需要本方法治疗的受试者，并在体外进行基因修饰后回输。在一实例中，特异性结合CLD18.2多肽的外源受体的细胞(例如CLD18.2-CAR-T细胞)通过同种异体回输的方式给予受试者。在一实例中，特异性结合CLD18.2多肽的外源受体的细胞(例如CLD18.2-CAR-T细胞)源自健康供者，供者和回输细胞的受试者不同。
所述宿主细胞、细胞治疗产品或组合物可通过任何合适的方式给予，例如，通过注射，例如，静脉内或皮下注射、腹腔注射、眼内注射、眼底注射、视网膜下注射、玻璃体内注射、反间隔注射、巩膜下注射、脉络膜内注射、前房注射、结膜下注射、结膜下注射、巩膜上腔注射、球后注射、眼周注射或球周递送。在一实例中，通过胃肠外、肺内的和鼻内，以及病灶内给予。腹膜外输注包括肌内、静脉内、动脉内、腹膜内或皮下给予。在一实例中，可利用任何途径(包括静脉注射(IV))给予氟达拉滨、环磷酰胺和白蛋白结合型紫杉醇。
可利用任何途径(包括静脉注射(IV))给予所述宿主细胞、细胞治疗产品或组合物(例如CAR-T细胞)。在一些实施方式中，在下列时间内利用IV给予CAR-T细胞：约3分钟、约4分钟、约5分钟、约6分钟、约7分钟、约8分钟、约9分钟、约10分钟、约11分钟、约12分钟、约13分钟、约14分钟、约15分钟、约16分钟、约17分钟、约18分钟、约19分钟、约20分钟、约21分钟、约22分钟、约23分钟、约24分钟、约25分钟、约26分钟、约27分钟、约28分钟、约29分钟、约30分钟、约40分钟、约50分钟、约60分钟。
本申请所述方法中可包括各种其他干预。例如，环磷酰胺与氟达拉滨在给予后可能引起受试者的不良事件。本申请的范围包括对受试者给予组成物以使这些不良事件中的某些事件减少。在一实例中，包含对受试者给予生理盐水。在一实例中，包含给予佐剂与赋形剂。例如，美司钠(2-巯基乙烷磺酸钠)。在一实例中，包含给予外源性细胞激素。
给予受试者所述宿主细胞、细胞治疗产品或组合物后，可用多种已知方法中的任一种来测量给予的细胞群的生物活性。评估的参数包括：细胞与抗原的特异性结合，体内方式(例如，通过成像)或离体方式(例如，通过ELISA或流式细胞术)。在一实例中，通过测定细胞因子，例如CD107a，IFNγ，IL-2和TNF的表达和/或分泌，来测量细胞的生物活性。在一实例中，生物活性通过评估临床结果，例如肿瘤负荷的减少。在一实例中，评估 肿瘤标志物的降低。在一实例中，评估细胞的毒性结果、持久性和/或增殖，和/或存在或不存在宿主免疫应答。
在一实例式中，如果指示副作用的生物化学指标或指示物(如：细胞因子释放综合征(CRS)、巨噬细胞活化综合征、或肿瘤溶解综合征、或神经毒性)显示的临床风险不存在或已过去，在需要的情况下，可以给予在后周期的治疗。在一实例中，是否给予在后周期的治疗，何时给予在后周期的治疗，和/或在后周期中给予的细胞治疗产品的剂量基于受试者中针对在先周期的细胞或其表达的嵌合抗原受体的免疫应答或可检测免疫应答的存在、不存在或程度确定。在一实例中，在受试者中指示CRS的因子的血清水平不超过给予在先周期前时刻的受试者中该指示物的血清水平的约10倍、25倍、50倍或100倍时，给予在后周期的治疗。在一实例中，在受试者中与CRS相关的结果(例如与CRS相关或指示CRS的血清因子)或其临床迹象或症状如发热、缺氧、低血压或神经障碍已经达到峰值水平，并且在给予在先周期后开始下降之后时给予在后周期的治疗。在一实例中，在宿主适应性免疫应答未被检测到、尚未建立、或尚未达到一定水平、程度或阶段时给予在后周期的治疗。在一些方面，在受试者的记忆免疫应答发展之前给予在后周期的治疗。
可检测的免疫应答是指通过用于评估对特定抗原和细胞的特异性免疫应答的许多已知方法中的任何一种可检测到的量。在一实例中，通过在受试者的血清上进行ELISPOT、ELISA或基于细胞的抗体检测方法(例如，通过流式细胞术)以检测特异性结合和/或中和存在于细胞上的抗原，例如结合嵌合抗原受体的表位，例如CAR的抗体的存在来检测特定类型的免疫应答。在一实例中，确定检测到的抗体的同种型并且可以指示应答的类型和/或应答是否是记忆应答。
本申请实施例中，通过基于“去单个核细胞分离术”从患有癌症的人类受试者的外周血单个核细胞(PBMC)制备T细胞，并且用编码嵌合抗原受体(CAR)的病毒载体培养和转导所述T细胞，所述嵌合抗原受体(CAR)特异性结合受试者中由肿瘤细胞表达的抗原，其是肿瘤相关抗原或肿瘤特异性抗原。细胞在单独的柔性冷冻袋中的输注介质中冷冻保存。每个包含单个单位剂量的细胞，其为约1×10 6个细胞至5×10 7个细胞。每个受试者输注的在先周期不高于约1×10 12个细胞，优选地，不高于约1×10 11个细胞，更优选地，不高于约1×10 10个细胞或约5ⅹ10 9个细胞或约2ⅹ10 9个细胞。在输注之前，将细胞保持在约低于-130℃或约低于-175℃的温度下。
在开始细胞治疗之前，从受试者获得血液，并且任选地通过ELISA和/或MSD和/或CBA的方法评估血清中指示细胞因子释放综合征(CRS)的一种或多种血清因子的水平，例如肿瘤坏死因子α(TNFα)、干扰素γ(IFNγ)、IL-10或IL-6。在治疗开始之前，可以通过例如通过PET或CT扫描测量实体肿瘤的大小或质量来任选地评估肿瘤负荷。
通过升温至约38℃进行复苏，并且受试者通过多次输注给予在先周期的细胞。每次输注为约3-30分钟时间内连续输注静脉内(IV)给予。
在给予在先周期后，受试者接受身体检查，并监测任何毒性或毒性结果的症状，例 如发热、低血压、缺氧、神经障碍或炎性细胞因子或C反应蛋白(CRP)的血清水平升高。任选地，在给予在先周期后，在一次或多次的情况下，从受试者获得血液，并通过ELISA和/或MSD和/或CBA的方法评估指示CRS的血清因子的水平。将血清因子的水平与刚好给予在先周期之前获得的血清因子的水平进行比较。如有必要，给予抗IL6或其他CRS治疗以减少CRS的症状。
在给予在先周期后，例如在给药开始后1、2、3和/或4周，任选地检测受试者中抗CAR免疫应答的存在或不存在，例如，通过qPCR、ELISA、ELISPOT、基于细胞的抗体测定和/或混合淋巴细胞反应。
通过在先周期实现的肿瘤负荷减少百分比可任选地在通过扫描(例如PET和CT扫描)在实体瘤患者中给予在先周期后一次或多次进行测量，和/或通过量化在血液或肿瘤部位肿瘤阳性细胞。
从首剂开始并持续长达数年，定期监测受试者。在随访期间，测量肿瘤负荷、和/或通过流式细胞术和定量聚合酶链反应(qPCR)检测CAR-表达细胞，以测量所给予的细胞的体内增殖和持久性、和/或评估抗CAR免疫应答的发展。
每个受试者输注的在先周期不高于约1×10 12个细胞，优选地，不高于约1×10 11个细胞，优选地，不高于约1×10 10个细胞、或不高于约5×10 9个细胞、或不高于2×10 9个细胞。在输注之前，将细胞保持在低于-175℃的温度下。在输注时，升温至38℃左右进行复苏。
在后周期的给药剂量是受试者特异性的，并且基于肿瘤负荷，抗CAR免疫应答的存在和CRS相关结果的水平。在后周期的给药剂量不高于约1×10 12个细胞，优选地，不高于约1×10 11个细胞，更优选地，不高于约1×10 10个细胞,更优选地，不高于约5×10 9个细胞或不高于约2×10 9个细胞。
采用描述性统计方法(如例数、平均数、中位数、标准差[SD]、最小值和最大值)分析。分类变量采用频数表(频数和百分比)分析。所有不良事件(AE)将依据ICH国际医学用语辞典MedDRA最新版本的编码进行分类，并按照不良事件常用标准术语标准(CTCAE v5.0版)分级，采用频率分布、图表或其它描述性指标进行分析，将按系统器官分类、首选术语和组别计算发生治疗后出现的不良事件(TEAE)的受试者例数和百分比。将生成包含不同检测时间点CAR-T细胞拷贝数。对于ORR和DCR采用Clopper-Pearson方法计算95％置信区间。
8.试剂盒
本申请提供用于在受试者中治疗和/或预防胆道肿瘤的试剂盒。在一实例中，试剂盒包括有效量的本申请的所述宿主细胞、细胞治疗产品、组合物组或药物组合物。在一实例中，试剂盒包括无菌容器；包括盒子、安瓿、瓶、小瓶、管、袋、小袋、泡罩包装或本领域已知的其它合适的容器形式。容器包括由塑料、玻璃、层压纸、金属箔或其它适合于容纳药物的材料制成。在一实例中，试剂盒包括编码本申请的CAR的核酸分子，其以可表达的形式识别CLD18.2多肽，可以任选地包括在一种或多种载体中。
在一实例中，将本申请的组合物和/或核酸分子，与将所述组合物或核酸分子施用于患有肿瘤或病原体或免疫疾病或有发展成肿瘤或病原体或免疫疾病的受试者的说明书一起提供。说明书通常包括有关组合物用于治疗和/或预防肿瘤或病原体感染的信息。在一实例中，说明书包括以下至少一项：治疗剂的描述；用于治疗或预防肿瘤、病原体感染或免疫疾病或其症状的剂量表和给药；注意事项；警告；适应症；不适应症；用药信息；不良反应；动物药理学；临床研究；和/或参考。这些说明书可以直接打印在容器上，或者作为粘贴在容器上的标签，或者作为单独的纸页、小册子、卡片或文件夹提供在容器内或与容器一起。
本申请的优点：
1.本申请显著提高利用表达嵌合抗原受体的免疫效应细胞进行胆道肿瘤(包括胆囊癌、胆管癌)治疗的疗效：肿瘤数量减少或体积缩小，和/或转移的数量和/或程度降低，和/或肿瘤标志物降低，和/或中晚期癌症常见的并发症的消失或减弱表示。
2.对实体瘤的抗肿瘤效果显著，提供了一种安全有效，能稳定甚至治愈CLA18.2阳性的中晚期胆道肿瘤(包括胆囊癌、胆管癌)的治疗手段。
3.提供一种提高免疫效应细胞抗胆道肿瘤(包括胆囊癌、胆管癌)治疗的预处理组合物，在给予免疫效应细胞之前给予氟达拉滨、环磷酰胺、白蛋白结合型紫杉醇组合物，能显著促进免疫效应细胞的抗肿瘤疗效，大幅降低受试者的肿瘤负荷、提高受试者的生活质量，以及延长生存期。
下面结合具体实施例，进一步阐述本申请。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
材料与方法：
材料：
本申请采用的各种材料，包括试剂均可自商业渠道购得。
本申请的示例性的抗原受体，包括CAR，以及用于工程改造和将受体导入细胞中的方法，参考例如例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、CN 108884459A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN 109908176A、CN109880803A、CN 110055275A、CN110123837A、CN 110438082A、CN 110468105A国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、 WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018/219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029中公开的全文内容。
实施例1.CAR-T细胞的制备
采用本领域常规分子生物学方法制备表达特异性识别CLD18.2的CAR-T细胞。以PRRLSIN为载体，分别插入表1所示的嵌合抗原受体序列构建了表达CLD18.2抗体的嵌合抗原受体的慢病毒载体，分别感染活化后的T细胞并培养扩增至需要的数量，得到CLD18.2-BBZ CAR-T细胞、CLD18.2-28Z CAR-T细胞、CLD18.2-28BBZ CAR-T细胞。
表1嵌合抗原受体
Figure PCTCN2022100762-appb-000001
注：CD8α信号肽(SEQ ID NO:1)；CLDN18.2scFV(SEQ ID NO:2)；CD8铰链区(SEQ ID NO:3)；CD8跨膜区(SEQ ID NO:4)；CD28跨膜区(SEQ ID NO:5)；CD137胞内域(SEQ ID NO:6)；CD28胞内域(SEQ ID NO:7)；CD3ζ胞内域(SEQ ID NO:8)。
实施例2.CAR-T细胞的细胞毒性测定
通过乳酸脱氢酶法检测CAR-T细胞在体外杀伤。采用CytoTox
Figure PCTCN2022100762-appb-000002
试剂盒(Promega，G1780)，具体步骤参照试剂盒说明书。
(1)靶细胞：CLD18.2表达阴性的人胆管癌细胞RBE、人胆囊癌细胞GBC-SD；外源性表达人CLD18.2的RBE-CLD18.2细胞、GBC-SD-CLD18.2细胞。
(2)效应细胞：UTD(未感染病毒的T细胞)、CLD18.2-28Z CAR-T细胞。
分别按效靶比3:1、1:1、1:3铺入96孔板中，在37℃、5％CO 2下共培养18小时检测。如图1所示：CLD18.2-28Z CAR-T杀伤CLD18.2表达阳性的细胞；CLD18.2-28Z CAR-T对CLD18.2表达阴性的细胞RBE、GBC-SD基本无杀伤。
实施例3.CAR-T细胞体内杀伤实验
接种3×10 6个GBC-SD-CLD18.2细胞于5～6周龄，雌性NPG小鼠(购自北京维通达生物技术有限公司)右侧腋部皮下，肿瘤接种日记为D0。接种后D15,肿瘤体积长至约150mm 3，随机分为4组，分别为UTD、CLD18.2-28Z CAR-T组，其中CLD18.2-28Z CAR-T组包括CAR-T1(5*10^5细胞/小鼠)、CAR-T2(1*10^6细胞/小鼠)、CAR-T3(3*10^6细胞/小鼠)。如图2所示，肿瘤细胞接种后D36天时，与UTD相比，各组抑瘤率分别为：CAR-T1：42.43％，CAR-T2：80％；CAR-T3：100％(5只小鼠肿瘤 均消退)。
实施例4.CAR-T细胞治疗胆道肿瘤
对CLD18.2阳性的胆道肿瘤受试者进行靶向CLD18A2的CAR-T细胞治疗，采用既往手术或活检的肿瘤组织切片进行CLD 18.2表达检测。在给予CAR-T细胞之前，受试者接受“去单个核细胞分离术”的单采分离技术，并进行预处理治疗。通过单采，从受试者体内获得PBMC，通过编码抗CLD18A2的CAR(示例性CAR采用CLD18.2-28Z CAR)的病毒载体转导和扩增，得到CAR-T细胞。在给予CAR-T之前，给予受试者预处理：氟达拉滨、环磷酰胺和白蛋白结合型紫杉醇。
CAR-T治疗之前，通过例如PET或CT扫描测量实体肿瘤的大小或性状来任选地评估肿瘤负荷，且还可以通过检测肿瘤标志物和/或观察肿瘤并发症发生及严重程度来评估肿瘤负荷。
当受试者需要进行至少一个周期的CAR-T治疗时，在后周期的CAR-T给药，在前一周期的CAR T给药完成后的4周后给予。每个周期的CAR-T给药，可以一次给予，也可以分两次或两次以上静脉(IV)滴注给予，每次给予的细胞在约3-30分钟完成输注，优选5-25分钟时间完成输注。每个周期在CAR-T给药完毕后，受试者会接受身体检查，并监测任何毒性或毒性结果的症状，例如发热、低血压、缺氧、神经障碍或炎性细胞因子或C反应蛋白(CRP)的血清水平升高。检查可以是从受试者获得血液，通过ELISA、和/或MSD、和/或CBA的方法评估指示CRS的细胞因子水平。如有必要，给予抗IL-6治疗，或给予其他CRS治疗，以减少CRS的症状。每个周期在CAR-T给药完毕后，例如在给药开始后1、2、3、和/或4周，可以通过qPCR、ELISA、ELISPOT、抗体测定等评估受试者体内的CAR-T数量。
每个周期的治疗后肿瘤负荷的减少情况可通过扫描(例如PET和CT扫描)，和/或通过量化在血液或肿瘤部位呈现抗原(如claudin18.2)阳性的细胞得出。
示例性，受试者1：男、65岁、44kg，胆囊低分化腺癌，CLD18.2表达呈+++(强阳性染色)、染色的肿瘤细胞百分比为90％。既往接受过姑息性胆囊切除术，氟尿嘧啶等化疗药治疗，现有远处转移。CAR-T治疗前预处理：氟达拉滨为35mg/d第1-2天，环磷酰胺为350mg/d第1-3天，白蛋白结合型紫杉醇为100mg/d第2天。接受2个周期的CAR-T输注，周期1的输注剂量为3.75×10 8个细胞，周期2的输注剂量为2.5×10 8个细胞，均为一次性回输。
受试者2：女、63岁、59kg，胆囊高中分化腺癌，CLD18.2表达呈+++(强阳性染色)、染色的肿瘤细胞百分比为80％。既往接受过胆囊切除术，白蛋白结合型紫杉醇、S-1等化疗药和帕博利珠单抗免疫治疗，现有远处转移。CAR-T治疗前预处理：氟达拉滨为38.75mg/d第1-2天，环磷酰胺为387.5mg/d第1-3天，白蛋白结合型紫杉醇为100mg/d第2天。该受试者接受了1个周期的CAR-T输注，输注剂量为2.5×10 8个细胞，为一次性回输。
受试者3：男、57岁、65kg，肝外胆管中-低分化癌，CLD18.2表达呈+++(强阳性染色)、染色的肿瘤细胞百分比为80％。既往接受过胰十二指肠切除术，吉西他滨、雷替曲塞、顺铂等化疗药治疗以及PD-L1/TGF-β双抗(Bintrafusp alfa)或安慰剂治疗，现有远处转移。CAR-T治疗前预处理：氟达拉滨为42.25mg/d第1-2天，环磷酰胺为422.5mg/d第1-3天，白蛋白结合型紫杉醇为100mg/d第2天。该受试者接受了1个周期的CAR-T细胞输注，输注剂量为2.5×10 8个细胞，为一次性回输。
实施例5：治疗的评估
通过影像学检测靶病灶和非靶病灶对治疗前和治疗后的肿瘤负荷进行评估，并确定靶病灶的数目及大小，疗效评价标准参见实体肿瘤的疗效评价标准1.1版。所有不良事件(AE)将依据ICH国际医学用语辞典MedDRA最新版本的编码进行分类，并按照不良事件常用标准术语标准(CTCAE v5.0版分级)进行分析。其中，细胞因子释放综合征(CRS)和神经毒性按照ASTCT共识进行分级和处理。
3例胆道肿瘤受试者没有发生严重CRS及神经毒性，靶向CLD18.2的CAR-T治疗是安全的。2例胆囊癌受试者CAR-T回输发生1-2级CRS。临床表现以发热、心动过速、外周水肿为主，亦可见低血压、低血氧，经对症支持治疗后可恢复。1例胆管癌受试者CAR-T回输未发生CRS。CAR-T治疗前后，3例受试者体重均保持稳定。
受试者1经过治疗后处于PR，PFS为4.2个月，OS约10个月。受试者2和3经过治疗后达到SD，无疾病进展生存期分别为约9.3个月、7.5个月，并且均仍在持续稳定期。
检测CAR-T细胞在体内持续存活期，即CAR-T细胞“植入”体内持续存活的期间。从初次输注(为第0天)结束后起每个访视点采用Q-PCR的方法(所用探针(SEQ ID NO:20)，上游引物(SEQ ID NO:21)，下游引物(SEQ ID NO:22))检测外周血中含有CAR-CLD18DNA的拷贝数。结果显示，CAR-T输注后，受试者1体内扩增拷贝数D5达峰值6713拷贝/μg gDNA，持续存在至少62天；受试者2于D7达峰值8237拷贝/μg gDNA，持续存在至少85天；受试者3于D14达峰值，5698拷贝/μg gDNA，持续存在至少28天。
在本申请提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本申请的上述讲授内容之后，本领域技术人员可以对本申请作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
以下表格中列出了本申请中使用的序列：
Figure PCTCN2022100762-appb-000003
Figure PCTCN2022100762-appb-000004
Figure PCTCN2022100762-appb-000005
Figure PCTCN2022100762-appb-000006

Claims (22)

  1. 一种治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的方法，其特征在于，包括向所述受试者给予靶向CLD18的外源受体的细胞。
  2. 如权利要求1所述的方法，其特征在于，所述CLD18为CLD18.2。
  3. 如权利要求1所述的方法，其特征在于，所述胆道肿瘤包括胆囊癌和胆管癌。
  4. 如权利要求1所述的方法，其特征在于，所述细胞为免疫效应细胞，优选所述免疫效应细胞选自T细胞、NK细胞、NKT细胞、肥大细胞、巨噬细胞、树突细胞、CIK细胞、干细胞衍生的免疫效应细胞或其组合，进一步优选免疫效应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。
  5. 如权利要求4所述的方法，其特征在于，所述免疫效应细胞为自体或同种异体T细胞、或原代T细胞。
  6. 如权利要求1-5任一所述的方法，其特征在于，所述外源受体选自嵌合抗原受体(CAR)、T细胞受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)、抗体-TCR嵌合物或其组合；所述外源受体的抗原结合域特异性结合CLD18.2。
  7. 如权利要求6所述的方法，其特征在于，所述CAR包括：
    (i)特异性结合CLD18.2的抗体、CD8或CD28的跨膜区和CD3ζ胞内域；
    (ii)特异性结合CLD18.2的抗体、CD8或CD28的跨膜区、CD137胞内域和CD3ζ胞内域；
    (iii)特异性结合CLD18.2的抗体、CD28或CD8的跨膜区、CD28胞内域和CD3ζ胞内域；或
    (iv)特异性结合CLD18.2的抗体、CD8或CD28的跨膜区、CD28胞内域、CD137胞内域和CD3ζ胞内域。
  8. 如权利要求1-7任一所述的方法，其特征在于，给予所述受试者至少一个周期的所述细胞进行治疗；优选的，给予受试者1-3个周期的所述细胞进行治疗。
  9. 如权利要求8所述的所述的方法，其特征在于，
    每个周期给予的细胞治疗产品中细胞的剂量不超过约2ⅹ10 9个细胞/kg受试者体重、2ⅹ10 8个细胞/kg受试者体重、或2ⅹ10 7个细胞/kg受试者体重；或
    细胞治疗产品中细胞剂量不超过约1ⅹ10 11个细胞/受试者、1ⅹ10 10个细胞/受试者、5ⅹ10 9个细胞/受试者、2ⅹ10 9个细胞/受试者或1ⅹ10 9个细胞/受试者。
  10. 如权利要求9所述的方法，其特征在于，
    每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 5个细胞/kg受试者体重至2ⅹ10 7个细胞/kg受试者体重，或约1ⅹ10 6个细胞/kg受试者体重至2ⅹ10 7个细胞/kg受试者体重；或者
    每个周期给予的细胞治疗产品中细胞剂量为约1ⅹ10 7个细胞至5ⅹ10 9个细胞/受试者、约 1ⅹ10 7个细胞至2ⅹ10 9个细胞/受试者、或约1ⅹ10 7个细胞至1ⅹ10 9个细胞/受试者；或者
    每个周期给予的细胞治疗产品中细胞剂量为约1ⅹ10 8个细胞至5ⅹ10 9个细胞/受试者、约1ⅹ10 8个细胞至2ⅹ10 9个细胞/受试者、或约1ⅹ10 8个细胞至1ⅹ10 9个细胞/受试者；或者
    每个周期给予的细胞治疗产品中细胞剂量为约2.5ⅹ10 8个细胞至5ⅹ10 8个细胞/受试者。
  11. 如权利要求8-10任一项所述的方法，其特征在于，每个周期给予所述细胞治疗产品之前进行预处理，所述预处理包括给予所述受试者化学药物、生物药物、放疗或其组合。
  12. 如权利要求11所述的方法，其特征在于，所述预处理在给予细胞治疗产品前1-8天实施；优选的，在给予细胞治疗产品前2-6天实施；优选地，每种化学药物、生物药物、放疗或其组合连续使用不超过4天。
  13. 如权利要求11或12所述的方法，其特征在于，所述化学药物选自以下任意一种或至少两种：环磷酰胺、氟达拉滨、微管蛋白抑制剂、嘧啶类抗肿瘤药物；或所述化学药物包括环磷酰胺和氟达拉滨；或者所述化学药物包括环磷酰胺、氟达拉滨以及微管蛋白抑制剂。
  14. 如权利要求13所述的方法，其特征在于，所述微管蛋白抑制剂包括紫杉烷类化合物；或所述微管蛋白抑制剂包括紫杉醇、白蛋白结合型紫杉醇、多西他赛；或所述微管蛋白抑制剂为白蛋白结合型紫杉醇。
  15. 如权利要求13或14所述的方法，其特征在于，
    氟达拉滨的给予量约为10-50mg/m 2/天、或约15-40mg/m 2/天、或约15-30mg/m 2/天、或约20-30mg/m 2/天；或约25mg/m 2/天、或约为30-60mg/天、或约30-50mg/天、或约35-45mg/天；或者
    环磷酰胺的给予量约为200-400mg/m 2/天、或约200-300mg/m 2/天、或约250mg/m 2/天、或约为300-1000mg/天、或约300-550mg/天、或约300-500mg/天；或者
    紫杉烷类化合物给予量为不高于约300mg/天、或不高于约200mg/天，或约为90-120mg/天。
  16. 如权利要求13-15任一所述的方法，其特征在于，所述环磷酰胺给予2-3次；或所述氟达拉滨给予1-2次；或所述紫杉烷类化合物给予一次。
  17. 如权利要求6-16任一所述的方法，其特征在于，所述抗原结合域或所述抗体具有：SEQ ID NO：2、23、24、25、26、27、28、29、30、31、32或33所示的scFv序列；或
    所述抗原结合域或所述抗体具有SEQ ID NO：14所示的HCDR1、SEQ ID NO：15所示的HCDR2、SEQ ID NO：16所示的HCDR3、SEQ ID NO：17所示的LCDR1、SEQ ID NO：18所示的LCDR2、SEQ ID NO：19所示的LCDR3；或
    所述抗原结合域或所述抗体具有SEQ ID NO：10所示的重链可变区，SEQ ID NO：12所示的轻链可变区。
  18. 如权利要求6-17任一所述的方法，其特征在于，所述的嵌合抗原受体具有SEQ ID NO： 2、23、24、25、26、27、28、29、30、31、32或33所示的scFv序列分别与SEQ ID NO：34、35或36顺序连接的序列或所述的嵌合抗原受体具有SEQ ID NO：37、38或39所示的核苷酸序列。
  19. 靶向CLD18的外源受体的细胞用于制备治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的药物的用途。
  20. 表达靶向CLD18的外源受体的细胞和化学药物或其他生物药物或放疗用于制备治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的药物的用途。
  21. 靶向CLD18的外源受体的细胞用于治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的用途。
  22. 表达靶向CLD18的外源受体的细胞和化学药物或其他生物药物或放疗用于治疗患有或怀疑患CLD18阳性胆道肿瘤受试者的用途。
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