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WO2022214089A1 - 细胞免疫治疗的应用 - Google Patents

细胞免疫治疗的应用 Download PDF

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Publication number
WO2022214089A1
WO2022214089A1 PCT/CN2022/085921 CN2022085921W WO2022214089A1 WO 2022214089 A1 WO2022214089 A1 WO 2022214089A1 CN 2022085921 W CN2022085921 W CN 2022085921W WO 2022214089 A1 WO2022214089 A1 WO 2022214089A1
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cells
seq
day
cell
patient
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French (fr)
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李宗海
汪薇
肖珺
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Crage Medical Co Ltd
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Crage Medical Co Ltd
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Priority to JP2023562228A priority Critical patent/JP2024527180A/ja
Priority to US18/284,961 priority patent/US20240181055A1/en
Priority to CN202280022826.0A priority patent/CN117545771A/zh
Priority to EP22784155.8A priority patent/EP4321533A4/en
Publication of WO2022214089A1 publication Critical patent/WO2022214089A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4261—Proteoglycans, e.g. glypican, brevican or CSPG4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/51—Stomach
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/52—Intestine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention belongs to the field of immunotherapy; in particular, it relates to immune cell therapy that targets and recognizes tumor antigens, triggers activation of immune effector cells and exerts anti-tumor effects.
  • Programmed death-1 is an immunosuppressive protein that negatively regulates TCR signaling.
  • PD-L1 is the ligand of PD-1 and is expressed in many tumor types, such as ovarian cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, gastric cancer, melanoma, glioblastoma, non-small cell lung cancer, etc.
  • the combination of PD-1 and PD-L1 can provide inhibitory signals, induce T cell apoptosis, and inhibit T cell activation and proliferation. Therefore, drugs targeting PD-1 or PD-L1 can exert anti-tumor effects through immune regulation.
  • PD-1 antibodies or PD-L1 antibodies include Nivolumab, Pembrolizumab, Atezolizumab, Avelumab, Durvalumab, etc., but there are still many patients with disease progression after treatment with PD-1 antibody or PD-L1 antibody, and the treatment fails.
  • Cellular immunotherapy is a treatment method that uses the patient's autologous (or allogeneic) immune cells to repair tissues and organs.
  • Such as CAR-T cell therapy even if the patient's autologous (or allogeneic) T cells are modified with chimeric antigen receptors to treat diseases such as cancer.
  • Similar to PD-1 antibody or PD-L1 antibody treatment of cancer both of them carry out tumor killing through the immune system. Since patients who have failed treatment with PD-1 antibody or PD-L1 antibody may be less suitable for tumor immunotherapy, patients who are resistant to PD-1 antibody or PD-L1 antibody treatment may be more resistant to other immunotherapy (such as CAR-T cell therapy) is also not sensitive.
  • the object of the present invention is to provide a composition for cancer treatment, and a method for treating cancer, especially a method for treating cancer and a composition for treating patients who have failed anti-PD-1 antibody and/or anti-PD-L1 antibody treatment.
  • the present invention relates to the following:
  • a cell therapy product in the manufacture of a medicament for the treatment of cancer in a patient who has failed prior treatment for the cancer, the prior treatment comprising treatment with an anti-PD-1 antibody and/or an anti-PD-L1 antibody.
  • said 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 .
  • immune effector cells are autologous/allogeneic T cells, or primary T cells.
  • T cells comprise memory stem cell-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells Sexual T cells (Tregs), effector memory T cells (Tem), ⁇ T cells, ⁇ T cells, or a combination thereof.
  • exogenous receptor is selected from the group consisting of chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP), T cell antigen couplers (TACs) or combinations thereof.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TFP T cell fusion protein
  • TACs T cell antigen couplers
  • tumor antigen is selected from CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Mucl, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4 or a combination thereof; preferably, the tumor antigen is selected from GPC3, CD19, BCMA, Claudin18.2 or a combination thereof.
  • the cancer comprises a solid tumor and/or a hematological tumor; preferably the solid tumor comprises a tumor of the digestive tract; more preferably the tumor of the digestive tract comprises gastric cancer /Esophagogastric junction adenocarcinoma, gallbladder carcinoma, liver cancer, krukenberg tumor or a combination thereof.
  • the cancer is selected from krukenberg tumor, gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) , head and neck squamous cell carcinoma (HNSCC), classic Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, Liver cancer, Merkel cell carcinoma, renal cell carcinoma (RCC), colorectal cancer (mCRC), breast cancer.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • cHL classic Hodgkin lymphoma
  • PMBCL primary mediastinal large B-cell lymphoma
  • UC urothelial carcinoma
  • esophageal cancer cervical cancer
  • RRCC renal cell carcinoma
  • mCRC colorectal cancer
  • the anti-PD-1 antibody/PD-L1 antibody is selected from the group consisting of Nivolumab, Pembrolizumab, Cemiplimab, Camrelizumab, Toripalizumab , sintilimab, tislelizumab, sepalimumab (GLS-010), tislelizumab, or a combination thereof.
  • the prior treatment comprises drug therapy, surgery, radiotherapy or a combination thereof;
  • the drug therapy comprises administration of chemical and/or biological drugs;
  • the prior treatment includes administration of olaparib, lenvatinib, cabozantinib, axitinib, ipilim, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane compound, bevacizumab, regorafenib, rotinib, apatinib, lenvatinib, fruquintinib, regorafenib, or a combination thereof.
  • chimeric antigen receptor comprises:
  • an antigen-binding domain that specifically binds to a tumor antigen, the transmembrane region of CD28 or CD8, CD3 ⁇ ;
  • an antigen binding domain that specifically binds a tumor antigen, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD28, and CD3 ⁇ ;
  • an antigen binding domain that specifically binds a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD137, and CD3 ⁇ ;
  • an antigen binding domain that specifically binds a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, a costimulatory signaling domain of CD137, and CD3 ⁇ .
  • the dose of cells in the cell therapy product administered per cycle is about 1x107 cells to 5x109 cells/patient, about 1x107 cells to 2x109 cells/patient, or about 1x107 cells to 1x109 cells per patient; or the dose of cells in the cell therapy product administered per cycle is about 1x108 cells to 5x109 cells/patient, about 1x108 cells to 2x109 cells/patient, or about 1x108 cells to 1 x 10 9 cells/; or the dose of cells in the cell therapy product administered per cycle is about 2.5 x 10 8 cells to 5 x 10 8 cells/patient.
  • the chemical drug is selected from any one or at least two of the following: cyclophosphamide, fludarabine, tubulin inhibitor, pyrimidine antineoplastic drugs; or
  • the chemical drugs include cyclophosphamide and fludarabine; or the chemical drugs include cyclophosphamide, fludarabine, and a tubulin inhibitor.
  • tubulin inhibitor is a taxane compound; preferably the taxane compound is selected from paclitaxel, nab-paclitaxel, docetaxel; more preferably The taxane compound is albumin-bound paclitaxel.
  • Cyclophosphamide is administered in an amount of about 200-400 mg/ m2 /day, or about 200-300 mg/ m2 /day, or about 250 mg/ m2 /day, or about 300-700 mg/day, or about 300- 550mg/day, or about 300-500mg/day;
  • the taxane is administered in an amount of no more than about 300 mg/day, or no more than about 200 mg/day, or about 90-120 mg/day.
  • HCDR1 shown in SEQ ID NO:16 HCDR2 shown in SEQ ID NO:17
  • HCDR3 shown in SEQ ID NO:18 LCDR1 shown in SEQ ID NO:19
  • LCDR2 shown in SEQ ID NO:20 LCDR3 shown in SEQ ID NO: 21; or
  • HCDR1 shown in SEQ ID NO:27 HCDR2 shown in SEQ ID NO:28
  • HCDR3 shown in SEQ ID NO:29 LCDR1 shown in SEQ ID NO:30
  • LCDR2 shown in SEQ ID NO:31 LCDR3 shown in SEQ ID NO:32;
  • the antigen binding domain has:
  • antigen binding domain has the sequence shown in SEQ ID NO: 14, 24, 35, or 37.
  • 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 .
  • T cells comprise memory stem-like T cells (Tscm cells), central memory T cells (Tcm), effector T cells (Tef), regulatory T cells Sexual T cells (Tregs), effector memory T cells (Tem), ⁇ T cells, ⁇ T cells, or a combination thereof.
  • exogenous receptor is selected from the group consisting of chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP), T cell antigen couplers (TACs) or combinations thereof.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TFP T cell fusion protein
  • TACs T cell antigen couplers
  • the tumor antigen is selected from CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Mucl, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4 or a combination thereof; preferably, the tumor antigen is selected from GPC3, CD19, BCMA, Claudin18.2 or a combination thereof.
  • the cancer comprises a solid tumor and/or a hematological tumor; preferably the solid tumor comprises a tumor of the digestive tract; more preferably the tumor of the digestive tract comprises gastric cancer /Esophagogastric junction adenocarcinoma, gallbladder carcinoma, liver cancer, krukenberg tumor or a combination thereof.
  • the cancer is selected from krukenberg tumor, gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) , head and neck squamous cell carcinoma (HNSCC), classic Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, Liver cancer, Merkel cell carcinoma, renal cell carcinoma (RCC), colorectal cancer (mCRC), breast cancer.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • cHL classic Hodgkin lymphoma
  • PMBCL primary mediastinal large B-cell lymphoma
  • UC urothelial carcinoma
  • esophageal cancer cervical cancer
  • RRCC renal cell carcinoma
  • mCRC colorectal cancer
  • the anti-PD-1 antibody/PD-L1 antibody is selected from the group consisting of Nivolumab, Pembrolizumab, Cemiplimab, Camrelizumab, Toripalizumab , sintilimab, tislelizumab, sepalimumab (GLS-010), tislelizumab, or a combination thereof.
  • the prior treatment comprises drug therapy, surgery, radiotherapy or a combination thereof;
  • the drug therapy comprises administration of chemical and/or biological drugs;
  • the prior treatment includes administration of olaparib, lenvatinib, cabozantinib, axitinib, ipilim, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane compound, bevacizumab, regorafenib, rotinib, apatinib, lenvatinib, fruquintinib, regorafenib, or a combination thereof.
  • an antigen-binding domain that specifically binds to a tumor antigen, the transmembrane region of CD28 or CD8, CD3 ⁇ ;
  • an antigen binding domain that specifically binds a tumor antigen, the transmembrane region of CD28 or CD8, the costimulatory signaling domain of CD28, and CD3 ⁇ ;
  • an antigen binding domain that specifically binds a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD137, and CD3 ⁇ ;
  • an antigen binding domain that specifically binds a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, a costimulatory signaling domain of CD137, and CD3 ⁇ .
  • the dose of cells in the cell therapy product administered per cycle is about 1x10 cells/kg patient body weight to 2x107 cells/kg patient body weight, or about 1x106 cells/ kg patient body weight to 2x107 cells/kg patient body weight; or
  • the dose of cells in the cell therapy product administered per cycle is about 1x107 cells to 5x109 cells/patient, about 1x107 cells to 2x109 cells/patient, or about 1x107 cells to 1x109 cells per patient; or the dose of cells in the cell therapy product administered per cycle is about 1x108 cells to 5x109 cells/patient, about 1x108 cells to 2x109 cells/patient, or about 1x108 cells to 1 x 10 9 cells/; or the dose of cells in the cell therapy product administered per cycle is about 2.5 x 10 8 cells to 5 x 10 8 cells/patient.
  • the chemical drug is selected from any one or at least two of the following: cyclophosphamide, fludarabine, tubulin inhibitor, pyrimidine antineoplastic drugs; or
  • the chemical drugs include cyclophosphamide and fludarabine; or the chemical drugs include cyclophosphamide, fludarabine, and a tubulin inhibitor.
  • tubulin inhibitor is a taxane compound; preferably the taxane compound is selected from paclitaxel, nab-paclitaxel, docetaxel; more preferably The taxane compound is albumin-bound paclitaxel.
  • fludarabine is administered in an amount of about 10-50 mg/ m2 /day, or about 15-40 mg/ m2 /day, or about 15-30 mg/ m2 /day, or about 20-30 mg/ m2 /day, or about 25 mg/ m2 /day, or about 30-60 mg/day, or about 30-50 mg/day, or about 35-45 mg/day;
  • Cyclophosphamide is administered in an amount of about 200-400 mg/ m2 /day, or about 200-300 mg/ m2 /day, or about 250 mg/ m2 /day, or about 300-700 mg/day, or about 300- 550mg/day, or about 300-500mg/day;
  • the taxane is administered in an amount of no more than about 300 mg/day, or no more than about 200 mg/day, or about 90-120 mg/day.
  • HCDR1 shown in SEQ ID NO:16 HCDR2 shown in SEQ ID NO:17
  • HCDR3 shown in SEQ ID NO:18 LCDR1 shown in SEQ ID NO:19
  • LCDR2 shown in SEQ ID NO:20 LCDR3 shown in SEQ ID NO: 21; or
  • HCDR1 shown in SEQ ID NO:27 HCDR2 shown in SEQ ID NO:28
  • HCDR3 shown in SEQ ID NO:29 LCDR1 shown in SEQ ID NO:30
  • LCDR2 shown in SEQ ID NO:31 LCDR3 shown in SEQ ID NO:32;
  • the antigen binding domain has:
  • antigen binding domain has the sequence shown in SEQ ID NO: 14, 24, 35, or 37.
  • a cell therapy product for the treatment of cancer that has failed prior treatment with an anti-PD-1 antibody or an anti-PD-L1 antibody.
  • a portion of patients who have failed prior therapy with anti-PD-1 antibody or anti-PD-L1 antibody respond to the cell therapy product.
  • At least 40% of patients who have failed prior therapy with an anti-PD-1 antibody or an anti-PD-L1 antibody respond to the cell therapy product.
  • At least 50% of patients who have failed prior therapy with an anti-PD-1 antibody or an anti-PD-L1 antibody respond to the cell therapy product.
  • the response refers to an ORR greater than 40%; preferably, greater than 50%.
  • chemical drugs or biological drugs are also used in the prior treatment; preferably, the chemical drugs or biological drugs are olaparib, lenvatinib, cabozantinib, azithromycin Cetinib, ipilimumab, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxanes, bevacizumab, regorafenib.
  • the anti-PD-1 antibody is selected from the group consisting of Nivolumab, Pembrolizumab, Cemiplimab, camrelizumab, toripalizumab, sintilimab, tislelizumab, Sepalizumab (GLS-010), tislelizumab; preferably, the anti-PD-1 antibody is selected from Nivolumab, pembrolizumab, camrelizumab, toripalizumab, Sindhi Lizumab, Tislelizumab.
  • the anti-PD-L1 antibody is selected from Atezolizumab, Durvalumab, Avelumab, and sugalimumab (CS1001).
  • the cell therapy product contains immune effector cells that express exogenous receptors, preferably, the immune effector cells are selected from T cells, NK cells, NKT cells, mast cells, macrophages, Dendritic cells, CIK cells, or stem cell-derived immune effector cells.
  • the immune effector cells are selected from T cells, NK cells, NKT cells, mast cells, macrophages, Dendritic cells, CIK cells, or stem cell-derived immune effector cells.
  • the immune effector cells are selected from T cells, and the exogenous receptor is selected from chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP) and T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TFP T cell fusion protein
  • TAC T cell antigen coupler
  • the exogenous receptor is selected from a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the immune effector cells are selected from T cells, NK cells, and NKT cells.
  • the cell therapy product specifically recognizes a tumor antigen
  • the tumor antigen is selected from CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Muc1, claudin18.2 , GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4; more preferably, the tumor antigen is selected from CD19, BCMA, Claudin18.2.
  • the cancer is selected from gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classic Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC) ), colorectal cancer (mCRC), breast cancer.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • cHL classic Hodgkin lymphoma
  • PMBCL primary mediastinal large B-cell lymphoma
  • UC urothelial carcinoma
  • esophageal cancer cervical cancer
  • hepatocellular carcinoma Merkel cell carcinoma
  • RRCC renal cell carcinoma
  • the cancer is selected from gastric cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), urothelial carcinoma (UC), liver cancer, melanoma.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • UC urothelial carcinoma
  • liver cancer melanoma
  • the patient is given at least one cycle of cell therapy product for treatment; preferably, the patient is given 1-3 cycles of cell therapy product for treatment.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x109 cells/kg subject body weight, or the dose of cells in the cell therapy product does not exceed about 1x1011 cells/person.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x108 cells/kg subject body weight, or the dose of cells in the cell therapy product does not exceed about 1x1010 cells/person.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x107 cells/kg subject body weight, or the dose of cells in the cell therapy product does not exceed about 1x109 cells/person.
  • the dose of cells in the cell therapy product administered per cycle is about 1x105 cells/kg subject body weight to 2x107 cells/kg subject body weight, or about 1x106 cells/kg Subject body weight to 2 x 107 cells/kg subject body weight.
  • the cell dose of the cell therapy product administered in each cycle is 1x107 cells to 1x109 cells, preferably 1x108 cells to 1x109 cells, more preferably 2.5x108 cells cells to 5x108 cells.
  • subsequent cycles of treatment are administered after the cell product of the previous cycle is undetectable in vivo after treatment.
  • the cell therapy product of the subsequent cycle is administered 30 days after the completion of administration of the cell product of the previous cycle.
  • the dose of the cellular product administered in the subsequent cycle is an amount of cells sufficient to stabilize or reduce tumor burden in the subject.
  • the doses of the cell product administered in the different cycles are different or the same.
  • each cycle of the cell therapy product is divided into 1-5 administrations, preferably, divided into 1-3 administrations.
  • each cycle of the cell therapy product is fully administered within 5 days, more preferably within 3 days.
  • each cell therapy product is infused within 1 hour, preferably within 30 minutes, more preferably within 4-30 minutes.
  • the subject has any of the following characteristics when administered a later cycle of treatment:
  • 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%, and more Excellent, reduce by at least 5%.
  • the CRS level is comparable to the CRS level prior to administration of the cell therapy product of the previous cycle.
  • each cycle administration of the cell therapy product is preceded by a pretreatment comprising administration of chemotherapy and/or radiotherapy to the subject.
  • the radiotherapy includes whole body radiation therapy or local radiation therapy.
  • the pretreatment is performed 1-8 days before the administration of the cell therapy product; preferably, the pretreatment is performed 2-6 days before the administration of the cell therapy product.
  • the chemical drug is selected from any one or at least two of the following: cyclophosphamide, fludarabine, tubulin inhibitor, and pyrimidine antitumor drugs.
  • the chemical drugs are 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, and docetaxel; more preferably, the The taxane compound is albumin-bound paclitaxel.
  • the pyrimidine antitumor drug is selected from the group consisting of 5-fluorouracil, difururacil, carmofur, deoxyfluridine, and capecitabine.
  • the amount of fludarabine administered 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/ m2 /day; or about 25 mg/ m2 /day.
  • the dosage of fludarabine is about 30-60 mg/day, or about 30-50 mg/m 2 /day.
  • the dosage of cyclophosphamide is 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 cyclophosphamide is about 300-700 mg/day, or about 300-550 mg/day, or about 300-500 mg/day.
  • the administration amount of the taxane compound is not higher than about 300 mg/day, or not higher than about 200 mg/day; preferably, the administration amount of the taxane compound is about 90-120 mg/day .
  • 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.
  • the exogenous receptor includes an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises an antibody that specifically recognizes a tumor antigen or the same. fragment, wherein the antibody or fragment thereof has:
  • HCDR1 shown in SEQ ID NO: 1 HCDR2 shown in SEQ ID NO: 2
  • HCDR3 shown in SEQ ID NO: 3 LCDR1 shown in SEQ ID NO: 4
  • LCDR2 shown in SEQ ID NO: 5 LCDR3 shown in SEQ ID NO: 6; or
  • HCDR1 shown in SEQ ID NO: 16 HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, LCDR3 shown in SEQ ID NO: 21; or
  • the antibody or fragment thereof has:
  • the extracellular antigen-binding domain of the exogenous receptor has the sequence shown in SEQ ID NO: 14, 24, 35, or 37.
  • the chimeric antigen receptor has the amino acid sequence shown in any of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, and 52.
  • the subject has not received cell therapy prior to administration of the cell therapy product.
  • the subject has also received drug therapy, surgical therapy, radiation therapy, or any combination thereof, prior to administration of the cell therapy product.
  • the drug therapy comprises chemotherapeutic or biological therapy.
  • the chemotherapeutic treatment comprises anlotinib, apatinib, lenvatinib, fruquintinib, regorafenib.
  • a cytokine indicative of CRS a cytokine indicative of neurotoxicity, an indicator of tumor burden, and/or an indicator of host anti- Serum levels of factors of the CAR immune response were evaluated.
  • the index indicating tumor load is: the total number of tumor cells in the subject, or the total number of tumor cells in the subject's organ, or the total number of tumor cells in the subject's tissue.
  • the dose of the cell therapy product to be administered is determined.
  • the subject exhibits no severe CRS, or no neurotoxicity greater than grade 3 following treatment with the cell therapy product.
  • the cell therapy product is propagated in the subject.
  • a method for treating tumors with exogenous receptor-modified immune effector cells comprising administering a pretreatment drug before administering the immune effector cells to a tumor patient, the pretreatment Including cyclophosphamide, fludarabine and tubulin inhibitors.
  • the fludarabine is administered in an amount of 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/ m2 /day; or about 25 mg/ m2 /day.
  • fludarabine is administered in an amount of about 30-60 mg/day, or about 30-50 mg/ m2 /day.
  • the cyclophosphamide is administered in an amount of about 200-400 mg/ m2 /day, or about 200-300 mg/ m2 /day, or about 250 mg/ m2 /day.
  • cyclophosphamide is administered in an amount of about 300-700 mg/day, or about 300-550 mg/day, or about 300-500 mg/day.
  • the administration amount of the taxane compound is not higher than about 300 mg/day, or not higher than about 200 mg/day; preferably, the administration amount of the taxane compound is about 90-120 mg/day sky.
  • each pretreatment drug is used for no more than 4 consecutive days.
  • the cyclophosphamide is administered 2-3 times;
  • the fludarabine is administered 1-2 times.
  • the tubulin inhibitor is administered once.
  • the tubulin inhibitor is a taxane compound; preferably, the taxane compound is selected from paclitaxel, nab-paclitaxel, and docetaxel; more preferably, The taxane compound is albumin-bound paclitaxel.
  • the exogenous receptor comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the extracellular antigen binding domain comprising an antibody that specifically recognizes a tumor antigen or Fragments thereof, wherein the antibody or fragment thereof has:
  • HCDR1 shown in SEQ ID NO: 1 HCDR2 shown in SEQ ID NO: 2
  • HCDR3 shown in SEQ ID NO: 3 LCDR1 shown in SEQ ID NO: 4
  • LCDR2 shown in SEQ ID NO: 5 LCDR3 shown in SEQ ID NO: 6; or
  • HCDR1 shown in SEQ ID NO: 16 HCDR2 shown in SEQ ID NO: 17, HCDR3 shown in SEQ ID NO: 18, LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, LCDR3 shown in SEQ ID NO: 21; or
  • the antibody or fragment thereof has:
  • the extracellular antigen binding domain of the exogenous receptor has the sequence shown in SEQ ID NO: 14, 24, 35, or 37.
  • the immune effector cells are selected from T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells, or stem cell-derived immune effector cells.
  • the immune effector cells are selected from T cells, and the exogenous receptor is selected from chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP) and T cell antigen coupler (TAC).
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TFP T cell fusion protein
  • TAC T cell antigen coupler
  • the exogenous receptor is selected from a chimeric antigen receptor (CAR); preferably, the chimeric antigen receptor has SEQ ID NOs: 11, 12, 13, 25, 26, The amino acid sequence shown in any of 36, 38, and 52.
  • CAR chimeric antigen receptor
  • the immune effector cells are selected from T cells, NK cells, and NKT cells.
  • the exogenous receptor-modified immune effector cells specifically recognize tumor antigens
  • the tumor antigens are selected from CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Mucl, claudin18.2, GPC3, IL13RA2, SLAMF7, GPRC5D, LILRB4; more preferably, the tumor antigen is selected from CD19, BCMA, and Claudin18.2.
  • the tumor is selected from gastric cancer, pancreatic cancer, gallbladder cancer, melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma (UC), esophageal cancer, cervical cancer, hepatocellular carcinoma, Merkel cell carcinoma, renal cell carcinoma (RCC) ), colorectal cancer (mCRC), breast cancer.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • HNSCC head and neck squamous cell carcinoma
  • cHL classical Hodgkin lymphoma
  • PMBCL primary mediastinal large B-cell lymphoma
  • UC urothelial carcinoma
  • esophageal cancer cervical cancer
  • hepatocellular carcinoma Merkel cell carcinoma
  • RRCC renal cell carcinoma
  • the tumor is selected from gastric cancer, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), urothelial carcinoma (UC), liver cancer, and melanoma.
  • NSCLC non-small cell lung cancer
  • SCLC small cell lung cancer
  • UC urothelial carcinoma
  • liver cancer liver cancer
  • melanoma melanoma
  • the subject has not received cell therapy prior to administration of the immune effector cells.
  • the subject has also received drug therapy, surgical therapy, radiation therapy, or any combination thereof, prior to administration of the immune effector cells.
  • the drug treatment includes chemotherapy or biological treatment; preferably, the chemotherapy includes anlotinib, apatinib, lenvatinib, fruquintinib, Geffini.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x109 cells/kg subject body weight, or the dose of cells in the cell therapy product does not exceed about 1x1011 cells/person.
  • the dose of immune effector cells administered per cycle does not exceed about 2x108 cells/kg subject body weight, or the dose of cells in a cell therapy product does not exceed about 1x1010 cells/person, more preferably, each The dose of immune effector cells administered periodically does not exceed about 2 x 10 7 cells/kg subject body weight, or the dose of cells in a cell therapy product does not exceed about 1 x 10 9 cells/person.
  • the dose of immune effector cells administered per cycle is about 1x105 cells/kg subject body weight to 2x107 cells/kg subject body weight, or about 1x106 cells/kg subject body weight.
  • Subject body weight to 2x10 7 cells/kg subject body weight; or the dose of immune effector cells administered per cycle is 1 x 10 7 cells to 1 x 10 9 cells, preferably 1 x 10 8 cells to 1x 10 9 cells, More preferably, it is 2.5x108 cells to 5x108 cells.
  • Figure 1 is a waterfall plot of the best change in tumor burden from baseline
  • an anti-PD-1 antibody and/or an anti-PD-L1 antibody was used in prior therapy of cancer patients and the therapy failed (progressive disease PD according to RECIST 1.1) , does not affect the efficacy of cell therapy products, and at least 40% of patients still respond to cell therapy products.
  • patients with gastric/esophagogastric junction adenocarcinoma, krukenberg tumor, liver cancer, and gallbladder cancer in solid tumors are treated with cell therapy products (e.g., CAR- T cells) respond.
  • the disease control rate after CAR-T cell therapy was about 79.2%.
  • the disease control rate after administration of CAR-T cell therapy was approximately 54.5%.
  • amino acid or nucleic acid sequence having a specific sequence means that the amino acid or nucleic acid sequence has more than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% of the amino acid or nucleic acid sequence and the specific sequence. %, 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, BLAST, BESTFIT, GAP, PILEUP/PRETTYBOX, etc. programs).
  • 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, where a probability score between e-3 and e-100 indicates closely related sequences.
  • “Failure of prior therapy with anti-PD-1 antibody and/or anti-PD-L1 antibody” as used herein means that at the time of prior therapy, the patient received PD-1 and/or PD-L1 antibody therapy, or Have received treatment with anti-PD-1 and/or PD-L1-containing therapy, and prior treatment failed to control disease progression (eg, disease progression to progressive disease (PD) according to RECIST1.1 Response Evaluation Criteria for Solid Tumors) ).
  • disease progression to progressive disease (PD) according to RECIST1.1 Response Evaluation Criteria for Solid Tumors)
  • the term "therapy comprising antibodies against PD-1 and/or PD-L1" as mentioned herein refers to the use of other drugs in addition to antibodies against PD-1 and/or PD-L1 according to therapeutic needs. or therapy.
  • radiotherapy, stem cell transplantation, etc. are also used in the treatment; or in addition to the use of antibodies against PD-1 and/or PD-L1, other treatments are also used.
  • Chemical or biological drugs such as olaparib, lenvatinib, cabozantinib, axitinib, ipilimumab, platinum-based chemotherapy drugs, pemetrexed, etoposide, taxane alkanes, bevacizumab, etc., or combinations thereof.
  • first-line treatment generally refers to the first round of systemic chemical and/or biological treatment for patients with tumors that cannot be surgically removed after tumor diagnosis; plus adjuvant therapy) for the first round of systemic chemotherapy and/or biologic therapy for relapse.
  • Second-line treatment generally refers to the need to change to a different treatment plan after the failure of first-line treatment (including the recurrence of tumor progression after first-line treatment is effective).
  • Third-line therapy generally refers to the failure of second-line therapy, and then switch to other regimens of therapy. When most tumors reach the third line, there are fewer and fewer drugs to choose from and effective treatment options.
  • PD1 and/or PD-L1 antibody therapy has been approved for different lines of therapy in different tumors. Now clinically, PD1 and/or PD-L1 antibody therapy is given to patients with PD-L1 positive or negative tumors. There are clinical applications of PD1 and/or PD-L1 antibody therapy alone, or PD1 and/or PD-L1 antibody therapy combined with other drugs.
  • the present invention is directed to the administration of a cell therapy product (exemplarily, CAR-T cells) in second-line, third-line or later-line treatment of patients with gastric/esophagogastric junction adenocarcinoma who have failed PD-1 and/or PD-L1 antibody therapy, It shows a higher remission rate of about 50%, disease control rate of about 79.2%, and longer progression-free survival and overall survival, enabling patients to obtain significant clinical benefits: among the 12 patients who achieved PR after CAR-T treatment , the subject's mDOR was 6.3 months; among them, 6 patients had an overall survival of more than about 11 months, and 4 of them were still in the survival follow-up period.
  • a cell therapy product exemplarily, CAR-T cells
  • the present invention also observes that in patients with gastric cancer/esophagogastric junction adenocarcinoma who have failed PD-1 and/or PD-L1 antibody therapy, the curative effect is assessed as complete remission (CR) after administration of CAR-T cell therapy, and the patient is in continuous clinical under observation.
  • CR complete remission
  • Krukenberg tumor is a metastatic malignant ovarian cancer mainly derived from the stomach and intestines. It has a high degree of malignancy and lacks specific clinical features. It is often found with systemic metastases, often associated with ascites and diffuse peritoneal metastases, and is insensitive to chemotherapy. The treatment effect is poor and the prognosis is poor.
  • the present invention is directed to Krukenberg tumor patients who have failed PD-1 and/or PD-L1 antibody therapy, and the administration of cell therapy products (exemplary, CAR-T cells) shows longer progression-free survival and overall survival, allowing patients to Obtained significant clinical benefits: PR achieved after CAR-T cell therapy, disease progression-free survival has exceeded 4 months, and is still in remission.
  • the results of published clinical studies show that the progression-free survival of gallbladder cancer patients who have received first-line standard therapy in the past is less than about 6 months (1.8-5.6 months).
  • the present invention is directed to patients with gallbladder cancer who have failed PD-1 and/or PD-L1 antibody therapy, and the administration of cell therapy products (exemplary, CAR-T cells) shows longer progression-free survival and overall survival, allowing patients to Obtained significant clinical benefits: CAR-T cell therapy has reached SD, and the disease progression-free survival has reached 7 and a half months, and is still in a stable period.
  • Primary liver cancer is the fourth most common malignant tumor and the second leading cause of cancer death in my country. Because most patients are in advanced stage of disease, the tumor has a high degree of deterioration, and the treatment effect is very unsatisfactory.
  • the present invention is aimed at liver cancer patients who have failed PD-1 and/or PD-L1 antibody treatment, and administers a cell therapy product (exemplary, CAR-T cells), which shows longer progression-free survival and overall survival, allowing patients to obtain Significant clinical benefit:
  • the disease control rate after CAR-T cell therapy was about 54.5%; the mean disease progression-free survival of the 6 patients who achieved disease control was about 4.4 months, and 4 patients had an overall survival of more than 1 year. 3 cases are still in the survival follow-up period.
  • the "cell therapy product” mentioned herein refers to a cell product with medical therapeutic effects, such as an immune cell therapy product, which uses immune cells derived from the human body or from a donor, and undergoes in vitro operations, including but not limited to isolation, purification, Culture, expansion, induction of differentiation, activation, genetic manipulation, establishment of cells (lines), cryopreservation and recovery, etc., and then input (or implanted) into patients to treat diseases by inducing, enhancing or inhibiting the body's immune function. product. Including adoptive cell therapy (ACT) products, therapeutic vaccines, etc.
  • ACT adoptive cell therapy
  • the types of immune cell therapy products mainly include but are not limited to Cytokine-Induced Killer (CIK), Tumor Infiltrating Lymphocyte (TIL), Chimeric Antigen Receptor T cells (Chimeric Antigen Recptor T-cells) cell, CAR-T), T Cell Receptor-engineering T-cell (TCR-T), Natural Killer (Natural Killer, NK) cell, Dendritic cell (DC) ), macrophages, etc.
  • CIK Cytokine-Induced Killer
  • TIL Tumor Infiltrating Lymphocyte
  • TIL Chimeric Antigen Receptor T cells
  • CAR-T Chimeric Antigen Recptor T-cells
  • TCR-T T Cell Receptor-engineering T-cell
  • Natural Killer Natural Killer
  • NK Natural Killer
  • DC Dendritic cell
  • macrophages etc.
  • anti-PD-1 antibody refers to an antibody capable of recognizing or binding to PD-1, such as an antibody capable of recognizing or binding human PD-1 (extracellular segment SEQ ID NO: 39).
  • the "antibody that recognizes or binds to human PD-1” may specifically bind to human PD1 antigen with a binding affinity of KD value of 1.0x10-8 mol/l or lower.
  • the anti-PD-1 antibody may be Nivolumab, Pembrolizumab, Cemiplimab, Camrelizumab, Toripalizumab, Sintilimab, Tislelizumab, or a combination thereof.
  • anti-PD-L1 antibody refers to an antibody capable of recognizing or binding to PD-L1, such as an antibody capable of recognizing or binding human PD-L1 (extracellular segment SEQ ID NO: 40).
  • the "antibody that recognizes or binds to human PD-L1” may specifically bind to human PD-L1 antigen with a binding affinity of KD value of 1.0x10-8 mol/l or lower.
  • the anti-PD-L1 antibody may be Atezolizumab, Durvalumab, Avelumab, sugelimab (CS1001), or a combination thereof.
  • binding refers to selectively binding to a target.
  • Tumor antigens are recognized by immune effector cells expressing receptors that bind to tumor antigens.
  • Specifically binds 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.
  • a biomolecule of interest eg, a polypeptide
  • Binding affinity can be determined using standard binding assays, such as surface plasmon resonance technology (GE-Healthcare, Uppsala, Sweden).
  • a "dose” may be a dose calculated on a weight basis, a dose calculated on a body surface area (BSA) basis, or a dose calculated on an individual person basis.
  • the dose calculated on a weight basis is the dose administered to a patient calculated on the basis of the patient's body weight, such as mg/kg, number of cells/kg, and the like.
  • the dose calculated on the basis of BSA is the dose administered to the patient calculated on the basis of the surface area of the patient, eg, mg/m 2 , number of cells/m 2 , and the like.
  • the dose calculated on a single person basis refers to the dose per patient per cycle or administered each time, such as mg/person, number of cells/person.
  • number of administrations refers to the frequency with which the cell therapy product or each pretreatment drug is administered within each cycle.
  • fludarabine can be administered as follows: once daily for 4 consecutive days, once daily for 3 consecutive days, once daily for 2 consecutive days, or once daily.
  • Cyclophosphamide may be administered as follows: once daily for 4 consecutive days, once daily for 3 consecutive days, once daily for 2 consecutive days, or once daily.
  • the nab-paclitaxel can be administered as follows: once daily for 4 consecutive days, once daily for 3 consecutive days, once daily for 2 consecutive days, or once daily.
  • the cell therapy product administered in each cycle can be administered at one time; or in 2, 3, 4, 5, 6, 7, 8, 9, 10 administrations, which can be administered on consecutive days, or at intervals of 1, 2 , 3, 4, 5, 6 days to give.
  • responder refers to a complete response (CR) (all target lesions disappear, and all pathological lymph nodes must be reduced to ⁇ 10 mm in short diameter) according to the Response Evaluation Criteria for Solid Tumors Version 1.1 (RECIST 1.1); or a partial response PR: At least 30% reduction in the sum of target lesion diameters from baseline; or stable disease (SD): Target lesions did not decrease to the extent of PR, nor did they increase to the extent of PD, in between, at the time of study The minimum value of the sum of diameters can be used as a reference.
  • PD Disease progression
  • RECIST 1.1 Response Evaluation Criteria for Solid Tumors Version 1.1 (RECIST 1.1): a relative increase of at least 20% ( If the baseline measurement value is the smallest, the baseline value is used as a reference); in addition, the absolute value of the sum of the diameters must be increased by at least 5 mm (the appearance of one or more new lesions is also regarded as PD).
  • Objective response rate (ORR) was defined as the percentage of subjects with a confirmed CR or PR for the best response in the population analyzed
  • DCR disease control rate
  • SD The number of cases with stable disease
  • PFS Progression Free Survival
  • having a response includes CR or PR following treatment by the patented therapeutic method.
  • having a response includes CR, PR or SD after treatment with the patented treatment method; in some embodiments, having a response means that subjects who receive the patented treatment method can obtain significant clinical benefit, PFS And/or OS is significantly prolonged, such as 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer.
  • responding means achieving ORR of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or more, or 100%.
  • responding refers to achieving a DCR of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95% or more, or 100%.
  • 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.
  • a cell or population of cells described herein that is "positive" for a marker means that the presence of a marker (usually a surface marker) of a cell can be confirmed by detection, such as by flow cytometry
  • the presence of surface expression is detected by cytometry, for example, by staining with an antibody that specifically binds to the marker, and detecting the antibody, wherein the staining is detectable at a level by flow cytometry, the a level that is significantly higher than the level of staining detected when the same procedure was performed with an isotype-matched control, other things being equal, and/or is substantially similar to the level of cells known to be positive for the marker, and /or significantly higher than the level of cells known to be negative for the marker.
  • the markers include but are not limited to tumor antigens.
  • tumor antigens include but are not limited to tumor antigens.
  • a cell or population of cells described herein that is "negative" for a marker means that the cell does not have a marker or that it does have it but is not detectable, eg, the marker is not detectable on the surface by flow cytometry Expression, or although surface expression is detected, for example, when surface expression is detected by staining with an antibody that specifically binds to the marker, and detecting the antibody, the staining can be determined by flow cytometry. Detected levels that are significantly lower than the level of staining detected using an isotype-matched control performed with the same steps, other things being equal, and/or significantly lower than those known to be positive for the marker and/or substantially similar to the levels of cells known to be negative for the marker.
  • patients who are positive or negative for the marker PD-L1 in tumor cells can be treated with CAR-T cells after the failure of using anti-PD-1 antibody and/or anti-PD-L1 antibody in the previous treatment .
  • vector is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • Non-plasmid and non-viral compounds that facilitate nucleic acid transfer into cells may also be included, such as polylysine compounds, liposomes, and the like.
  • cell may refer to human or non-human cells of animal origin.
  • Subject and “patient” are used interchangeably herein to refer to an individual, such as a human or other animal, and usually a human, who is being treated with a cell product. Subjects have been treated with PD-1 antibody and/or PD-L1 antibody, and are either responsive or non-responsive to PD-1 antibody or PD-L1 antibody, prior to being administered into the cell therapy study.
  • immune effector cell refers to a cell involved in an immune response that produces an immune effector, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, CIK cells, macrophages , mast cells, or stem cell-derived immune effector cells, etc.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cells may be naive T cells and/or T cells induced by pluripotent stem cells.
  • the T cells can be autologous T cells, xenogeneic T cells, allogeneic T cells.
  • the NK cells can be autologous NK cells or allogeneic NK cells.
  • artificially engineered cells with immune effector cell function refers to cells that do not have immune effector cells, or cells that have acquired immune effector cell function after being artificially engineered or stimulated by stimuli.
  • 293T cells are artificially modified to have the function of immune effector cells; for example, stem cells are induced in vitro to differentiate into immune effector cells.
  • T cells may be pluripotent stem cells from the bone marrow that differentiate and mature within the thymus into immunocompetent mature T cells.
  • a "T cell” can be a population of cells with specific phenotypic characteristics, or a mixed population of cells with different phenotypic characteristics, eg, a "T cell” can be a cell comprising at least one subset of T cells: memory Stem cell-like memory T cells (stem cell-like memory T cells, Tscm cells), central memory T cells (Tcm), effector T cells (Tef, Teff), regulatory T cells (tregs) and/or effector memory T cells ( Tem).
  • a "T cell” may be a particular subtype of T cell, such as ⁇ T or ⁇ T cells.
  • T cells can be obtained from a number of sources including, but not limited to, PBMC (peripheral blood mononuclear cells), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMC peripheral blood mononuclear cells
  • T cells can be obtained from blood collected from an individual using any number of techniques known to those of skill in the art, such as FicollTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove plasma molecules and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from a healthy donor or from a patient diagnosed with cancer.
  • T cells can be of any type and of any developmental stage, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells such as Th1 and Th2 cells, CD8+ T cells (e.g. cytotoxic T cells) , tumor infiltrating cells, memory T cells, naive T cells, etc.
  • exogenous refers to a nucleic acid molecule or polypeptide, cell, tissue, etc. that is not endogenously expressed in the organism itself, or that the level of expression is insufficient to achieve the function it would have when overexpressed.
  • endogenous refers to a nucleic acid molecule or polypeptide or the like that is derived from the organism itself.
  • chimeric receptor refers to a fusion molecule formed by linking DNA fragments or corresponding cDNA or polypeptide fragments of proteins from different sources by gene recombination technology, including extracellular domain, transmembrane domain and intracellular domain.
  • Chimeric receptors include, but are not limited to: Chimeric Antigen Receptor (CAR), Chimeric T Cell Receptor (TCR), T Cell Antigen Coupler (TAC).
  • chimeric antigen receptor includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the extracellular binding domain of the CAR comprises a scFv.
  • Intracellular signaling domains include functional signaling domains of stimulatory and/or costimulatory molecules; or entire intracellular portions, or entire native intracellular signaling structures including stimulatory and/or costimulatory molecules domain, or a functional fragment or derivative thereof.
  • the stimulatory molecule comprises a CD3 ⁇ chain bound to a T cell receptor complex; in one aspect, the intracellular signaling domain further comprises a functional signaling domain of one or more costimulatory molecules, such as 4-1BB (ie CD137), CD27 and/or CD28. In certain embodiments, groups of polypeptides are linked to each other.
  • the extracellular antigen-binding domain of the CAR includes those having SEQ ID NOs: 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71 , 72, 73, 74, 75, 76, 77, 78 or 79.
  • 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 is composed of two peptide chains, ⁇ and ⁇ . Each peptide chain can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region. Its antigen-specific Sex exists in the V region, and the V region (V ⁇ , V ⁇ ) has three hypervariable regions, CDR1, CDR2, and CDR3.
  • the modified TCR extracellular antigen binding domain comprises an antibody that recognizes a tumor antigen.
  • the TCRs include those having SEQ ID NOs: 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, Sequence shown at 75, 76, 77, 78 or 79.
  • TCR fusion protein or "TFP” includes recombinant proteins derived from various proteins of TCR, comprising antigen binding domains, generally capable of: i) binding to surface antigens on target cells; ii) when localized to T cells Interacts with other polypeptide components of the intact TCR complex.
  • the T cells are CD4+ T cells, CD8+ T cells, or CD4+/CD8+ T cells.
  • the TFP T cells are NKT cells.
  • the TFP T cells are ⁇ T or ⁇ T cells.
  • the antigen binding domain of the TFP includes an antibody that recognizes a tumor antigen.
  • the TFP comprises SEQ ID NOs 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, Sequences shown in 76, 77, 78 or 79.
  • T cell antigen coupler includes three functional domains: 1. Antigen binding domain, including single-chain antibody, designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) or other targeting groups; 2. the extracellular domain, a single-chain antibody that binds to CD3, thereby making the TAC receptor close to the TCR receptor; 3. the transmembrane region and the intracellular region of the CD4 co-receptor, wherein , the intracellular domain-linked protein kinase LCK, catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • the TAC antigen binding domain comprises an antibody that recognizes a tumor antigen.
  • the TAC includes 14, 24, 35, 37, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, 74, 75, 76, Sequences shown in 77, 78 or 79.
  • antigen-binding domain refers to an immunoglobulin molecule (used interchangeably with “antibody”) or an immunologically active portion of an immunological molecule, ie, that contains an antigen-binding site that specifically binds ("immunoreactively") an antigen.
  • Molecules which can contain antibodies, such as fully human antibodies, chimeric antibodies, humanized antibodies, murine antibodies, can also be ligands that recognize antigens.
  • antibody is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the indicated desired antigen-binding activity.
  • antibody refers to an antigen binding protein of the immune system.
  • An “antibody” includes an entire full-length antibody having an antigen-binding region and any fragment thereof in which the "antigen-binding portion" or “antigen-binding region” remains, or a single chain thereof, such as a single-chain variable fragment (scFv).
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • VL light chain variable region
  • variable region or variable domain refers to a domain of an antibody heavy or light chain that is involved in antibody antigen binding.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • Antibody fragments include, but are not limited to: (i) Fab fragments composed of VL, VH, CL and CH1 domains, including Fab' and Fab'-SH, (ii) Fd fragments composed of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546); (v) F(ab')2 fragments , a bivalent fragment comprising two linked Fab fragments; (vi) an antigen-binding site for a single-chain Fv molecule (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc.Natl.Acad.Sci.
  • the antigen binding domain may contain scFv, Fv, Fab, Fab', Fab'-SH, F(ab')2, single domain fragments, or natural ligands that bind antigen, and any of these derivative.
  • the extracellular antigen binding domain eg, scFv
  • the antibody or fragment thereof contained in the antigen binding domain may have the following CDR sequences: HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR2 shown in SEQ ID NO: 3 HCDR3 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, LCDR3 shown in SEQ ID NO: 6; or HCDR1 shown in SEQ ID NO: 16, LCDR1 shown in SEQ ID NO: 17 HCDR2 shown in SEQ ID NO: 18, LCDR1 shown in SEQ ID NO: 19, LCDR2 shown in SEQ ID NO: 20, LCDR3 shown in SEQ ID NO: 21; or SEQ ID NO: 27 HCDR1 shown in SEQ ID NO: 28, HCDR3 shown in SEQ ID NO: 29, LCDR1 shown in SEQ ID NO: 30, LCDR2 shown in SEQ ID NO: 31, SEQ ID NO: 32 LCDR3 shown.
  • the antibody or fragment thereof comprised by the antigen binding domain may have the following heavy chain variable region and light chain variable region sequences: SEQ ID NO: 7 for the heavy chain variable region and SEQ ID NO: 7 : the light chain variable region shown in 9; or the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 23; or the heavy chain variable region shown in SEQ ID NO: 33 chain variable region and the light chain variable region shown in SEQ ID NO:34.
  • the antibody or fragment thereof comprised by the antigen binding domain may have SEQ ID NOs: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 70, 71, 72, 73, scFv sequences shown in 74, 75, 76, 77, 78 or 79.
  • the extracellular region of the CAR includes an antigen-binding domain and a linker fragment (also known as a hinge, spacer) that recognizes a tumor antigen.
  • Linked fragments can be considered as part of a CAR used to provide flexibility to the extracellular antigen binding domain.
  • Linked fragments can be of any length.
  • the CAR includes a hinge domain, exemplarily, the CD8 ⁇ hinge, as set forth in SEQ ID NO:41.
  • transmembrane domain can anchor a foreign receptor to the plasma membrane of a cell.
  • CD28 the transmembrane domain of CD8, having the sequence shown in SEQ ID NO: 42 or 43
  • signaling domain refers to a functional portion of a protein that functions by transmitting information within a cell, for regulation via a defined signaling pathway, either by producing a second messenger or by acting as an effector in response to such a messenger cell activity.
  • the intracellular signaling domain may include the entire intracellular portion of the molecule, or the entire native intracellular signaling domain, or functional fragments or derivatives thereof. Exemplarily, the intracellular signaling domain has the sequence set forth in SEQ ID NO: 44, 45, or 46.
  • the functional signaling domain of the stimulatory molecule modulates the initial activation of the CAR or TCR complex in a stimulatory manner.
  • the primary signaling domain is initiated by, for example, binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, thereby mediating T cell responses (including, but not limited to, proliferation, activation, differentiation, etc.).
  • Primary signaling domains that act in a stimulatory manner may contain immunoreceptor tyrosine activation motifs or signaling motifs of ITAMs.
  • ITAM-containing primary signaling domains examples include, but are not limited to, those derived from CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS" ) and sequences of CD66d.
  • costimulatory molecule refers to binding to a cell stimulatory signal molecule, such as TCR/CD3, in combination with a signal that results in T cell proliferation and/or up- or down-regulation of key molecules, having a sequence as set forth in SEQ ID NO: 44 or 45. is an cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, including but not limited to cell proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137 ).
  • intracellular signaling domains can be designed to contain several possible stimulatory molecules and/or costimulatory signaling domains: Exemplarily, first-generation CARs contain a single stimulatory domain, such as the CD3 ⁇ chain; The second generation CAR contains one costimulatory domain, such as CD3 ⁇ chain and CD28, or CD3 ⁇ chain and 4-1BB; the third generation CAR contains two costimulatory domains (third generation CAR), such as CD3 ⁇ chain and CD28 and OX40, or comprising CD3 ⁇ chain with CD28 and 4-1BB.
  • first-generation CARs contain a single stimulatory domain, such as the CD3 ⁇ chain
  • the second generation CAR contains one costimulatory domain, such as CD3 ⁇ chain and CD28, or CD3 ⁇ chain and 4-1BB
  • the third generation CAR contains two costimulatory domains (third generation CAR), such as CD3 ⁇ chain and CD28 and OX40, or comprising CD3 ⁇ chain with CD28 and 4-1BB.
  • the CAR includes an optional leader sequence.
  • the CAR further comprises a leader sequence N-terminal to the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane.
  • the leader sequence comprises the sequence shown in SEQ ID NO:53.
  • the antigen binding domain targeting GPC3-CAR of the present invention comprises a sequence shown in SEQ ID NO: 70, 71, 72, 73, 74, 75, 76, 77, 78 or 79, a transmembrane domain Including the sequence shown in SEQ ID NO: 42 or 43, the intracellular signaling domain includes the sequence shown in SEQ ID NO: 44 and 46, or SEQ ID NO: 45 and 46, or SEQ ID NO: 45, 44 and 46. the sequence shown.
  • administration of the GPC3-CAR-T cells to liver cancer patients who failed PD1 and/or PD-L1 antibody therapy showed longer progression-free survival and overall survival, and the patients obtained significant clinical benefits.
  • the GPC3-CAR-T cells and transcription factor RUX3 polypeptide are administered to liver cancer patients who have failed PD1 and/or PD-L1 antibody therapy, and some patients are evaluated as PR after treatment, showing significant clinical benefit .
  • the GPC3-CAR-T expresses a RUNX3 polypeptide.
  • RUNX3 (Runt-related transcription factor 3)
  • osteogenesis-related transcription factor 3 is a member of the runt domain family of transcription factors, the amino acid sequence of GenBank Accession No.NP_004341.1. It plays an important role in cell growth, development, apoptosis and signal transduction and its biological effects.
  • activation and “activation” are used interchangeably and can refer to the process by which cells change from a resting state to an active state. This process may include responses to phenotypic or genetic changes in antigenic, migratory and/or functionally active states.
  • activation can refer to the process of stepwise activation of T cells. The activation process is co-regulated by the first stimulatory signal and the co-stimulatory signal.
  • T cell activation or “T cell activation” refers to the state of T cells that are stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function. Using CD3/CD28 magnetic beads, antigen stimulation in vitro or in vivo will affect the degree and duration of T cell activation.
  • the engineered T cells are co-incubated with tumor cells containing a specific target antigen or activated after viral infection.
  • treatment refers to interventions that attempt to alter the course of a disease, fully or partially alleviate or reduce the disease or symptoms associated therewith.
  • the therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving the prognosis.
  • “delaying the development of a tumor” means delaying, hindering, slowing, slowing, stabilizing, arresting and/or delaying the development of the tumor. This delay can be of varying lengths, depending on the history of the disease and/or the individual being treated. As will be understood by those of skill in the art, a sufficient or significant delay may encompass prophylaxis (for individuals who have not developed the tumor). For example, the development of advanced cancers, such as metastases, can be delayed.
  • provided cell therapy products are used to delay tumor development or slow tumor progression.
  • inhibitor refers to a reduction in function or activity when compared to the same disorder in other contexts or compared to another disorder.
  • an “effective amount” or “therapeutically effective amount” refers to a dose sufficient to prevent or treat a disease (cancer) in a subject. Effective doses for therapeutic or prophylactic use depend on the stage and severity of the disease 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 selected, the method of administration, the timing and frequency of administration, the presence, nature and extent of adverse side effects that may accompany administration of the particular active substance, and the desired physiological effect. One or more rounds, or multiple administrations of the cell therapy products described herein may be required according to the judgment of the prescribing physician or those skilled in the art.
  • tumor antigen refers to an antigen that is new or overexpressed during the development, progression of a hyperproliferative disease.
  • the hyperproliferative disorders of the present invention refer to cancer (including tumors).
  • the tumor antigens of the present invention can be solid tumor antigens or hematological tumor antigens.
  • cycle refers to the period from the baseline period before receiving the treatment (ie the evaluation period) to the baseline period before the next treatment with each cell therapy; such as starting from the administration of the pretreatment drug, until the next cycle Before starting preconditioning medication.
  • pretreatment refers to the administration of other drugs or treatments to patients prior to administration of a product (eg, a cell therapy product) to make the patient's physical condition more suitable for product treatment.
  • a product eg, a cell therapy product
  • chemical drugs, biological drugs, radiotherapy or a combination thereof are administered for pretreatment.
  • a chemical drug is administered for pretreatment first, and more preferably, the chemical drug is a chemotherapeutic drug.
  • preconditioning is required prior to each cycle administration of the cell therapy product.
  • the patient's lymphocytes are reduced by about 50%, 55%, 60%, 65%, or 70% compared to before pretreatment.
  • chemical drug refers to anticancer drugs prepared by chemical synthesis, which can be traditional chemotherapeutic drugs, such as alkylating agents, antimetabolites, anticancer antibiotics, etc., or targeted drugs. , such as afatinib, alectinib, etc.
  • biological drugs or “biological drugs” refers to drugs prepared by means of molecular biology or genetic engineering, such as antibody drugs, ADCs, and the like.
  • chemotherapeutic drug refers to a drug that treats tumors.
  • Chemotherapy drugs can kill tumor cells, or inhibit tumor cell growth, migration or invasion. These drugs can act on different aspects of tumor cell growth and reproduction, inhibiting or killing tumor cells. Chemotherapy is one of the main methods of cancer treatment at present.
  • chemotherapeutic drugs described herein refer to drugs used in chemotherapy, and refer to chemical drugs that have preventive and therapeutic effects on malignant tumors.
  • Chemotherapeutic drugs include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immune agents, and the like.
  • diterpene alkaloids eg, taxanes
  • cyclophosphamide fludarabine
  • cyclosporine e.g., rapamycin
  • melphalan e.g., asparaginase
  • bendamustine asparaginase
  • white Zulfan carboplatin
  • cisplatin daunorubicin
  • doxorubicin fluorouracil
  • hydroxyurea methotrexate
  • rituximab rituximab
  • vinblastine and/or vincristine etc.
  • the antimetabolites include, but are not limited to, antimetabolites such as carmofur, tegafur, pentostatin, deoxyfluridine, trimexat, fludarabine, cape citabine, galotabine, cytarabine sodium octadecyl phosphate, fosteabine sodium hydrate, raltitrexed, paltitrexid, pyritrifluoride, tiazofurin, noradrexed, pemetrexed Troxet, nelzarabine, 2'-deoxy-2'-methylenecytidine, 2'-fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dioxycytidine Hydro-benzofuranyl)sulfonyl]-N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-ten Tetracarbamate, c
  • the alkylating agents include, but are not limited to, dacarbazine, chlorambucil, cyclophosphamide, temozolomide, chlorambucil, busulfan, chlorambucil, and nitrosoureas, for example, but not limited to Wait.
  • the present invention provides the use or method of a cell therapy product for the treatment of cancer (used interchangeably with "tumor”) that has failed prior therapy with anti-PD-1 antibodies and/or anti-PD-L1 antibodies. .
  • the patient's ORR is greater than 40%; preferably, greater than 50%.
  • dose refers to the amount of drug product administered, eg, the amount of cell therapy product administered in a cycle, or the amount of cell therapy product administered per cycle in a cycle, or the amount of cell therapy product administered in a cycle , the amount of pretreatment drug administered each time.
  • the patient is administered at least one cycle of the cell therapy product for treatment.
  • the dose for each cycle can be the same or different.
  • a dose is administered to a subject in one or more divided doses within a cycle, and the divided doses for each administration may be the same or different.
  • the cell therapy product is administered to the subject once in a cycle, or administered to the subject in 2, 3, 4, 5, or more divided doses.
  • the dose of the cell therapy product or pretreatment drug administered each time is determined by the physician according to the specific condition of the subject.
  • the specific condition of the subject can be the subject's overall health, the severity of the disease, the response to the previous dose in the same cycle, the response to the previous cycle, the subject's combination medication, the degree of toxicity or the possibility of sex, complications, cancer metastasis, and any other factor deemed by the physician to affect the dose of cell therapy or preconditioning drug administered to the subject.
  • the dose per administration is escalating.
  • the dose per administration is in a decreasing trend.
  • the dose for each administration is escalating and then decreasing.
  • the dose for each administration has a trend of decreasing first and then increasing.
  • the administration of a multi-cycle cell therapy product means that there are multiple time periods, and a certain total amount of the cell therapy product is administered in each time period.
  • the interval time of each time period is consistent. In some embodiments, the interval time of each time period is not consistent.
  • the cell therapy product is administered for at least 2 cycles, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles of the cell therapy product.
  • the dose and number of doses of cells administered per cycle, and the time interval between cycles are designed to be assessed with the goal of improving one or more outcomes, eg, the outcome may be a reduction in the subject's
  • the degree of side effects, or the likelihood of side effects, can also be an improvement in treatment outcomes.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x109 cells/kg subject body weight, or the dose of cells in the cell therapy product does not exceed about 1x1011 cells/person.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x108 cells/kg subject body weight, or the dose of cells in the cell therapy product does not exceed about 1x1010 cells/person.
  • the dose of cells in the cell therapy product administered per cycle does not exceed about 2x107 cells/kg subject body weight, or the dose of cells in the cell therapy product does not exceed about 5x109 cells /human, or 2x109 cells/human, or 1x109 cells/human, or 5x108 cells/human.
  • the dose of cells in the cell therapy product administered per cycle is about 1x105 cells/kg subject body weight to 2x107 cells/kg subject body weight, or about 1x106 cells/kg Subject body weight to 2 x 107 cells/kg subject body weight.
  • the dose of cells in the cell therapy product administered per cycle is about 1x107 cells to 1x109 cells, 1x107 cells to 2x109 cells, 1x107 cells to 5x109 cells, or 1x107 cells to 1x1010 cells, preferably 1x108 cells to 1x109 cells, 1x108 cells to 2x109 cells, 1x108 cells to 5x109 cells, more preferably 1x10 8 cells to 5x108 cells, or 2.5x108 cells to 5x108 cells, 3.75x108 cells to 5x108 cells.
  • the patient is pretreated prior to each cycle of administration of the cell therapy product, the pretreatment comprising administering to the subject a chemotherapeutic agent and/or radiation therapy.
  • the radiation therapy may be systemic radiation therapy or local radiation therapy.
  • the pretreated chemotherapeutic agent may be any one or at least two of cyclophosphamide, fludarabine, tubulin inhibitor, pyrimidine antineoplastic drugs.
  • Tubulin inhibitors described herein include tubulin polymerization promoters and tubulin polymerization inhibitors.
  • Tubulin inhibitors include, but are not limited to, for example, taxanes, epothilones, spongolides, and Laulimalide, among others.
  • taxanes refers to drugs whose main components contain taxanes, which have a metamethylenebenzocyclodecene core structure similar to those of taxanes .
  • the metamethylenebenzocyclodecene core structure of the taxane compound contains an unsaturated bond.
  • the metamethylenebenzocyclodecene core structure of the taxane compound does not contain unsaturated bonds.
  • the carbon atoms on the metamethylenebenzocyclodecene core structure of the taxane are substituted with heteroatoms selected from N, O, S, P.
  • the taxane compound is administered by injection.
  • the pretreatment chemotherapeutic agent may be 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 The taxane compound is albumin-bound paclitaxel.
  • the pyrimidine antitumor drug is selected from the group consisting of 5-fluorouracil, gimeracil, oteracil potassium, difurfluorouracil, carmofur, deoxyfluridine, and capecitabine.
  • fludarabine is administered in an amount of about 20-60 mg/day, or about 30-55 mg/day, or about 30-50 mg/day.
  • fludarabine is administered in an amount of about 10-50 mg/ m2 /day, or about 15-40 mg/ m2 /day, or about 15-30 mg/ m2 /day, or about 20- 30 mg/ m2 /day; or about 25 mg/ m2 /day.
  • cyclophosphamide is administered in an amount of about 200-700 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 sky. .
  • cyclophosphamide is administered in an amount of about 200-400 mg/ m2 /day, or about 200-300 mg/ m2 /day, or about 250 mg/ m2 /day.
  • the taxanes are administered in an amount of no more than about 300 mg/day, or no more than about 200 mg/day, preferably, the taxanes are administered in an amount of about 90-200 mg/day , more preferably, the administration amount of the taxane compound is about 90-120 mg/day.
  • each chemotherapeutic agent is used for no more than 4 consecutive days.
  • cyclophosphamide is administered 2-3 times.
  • fludarabine is administered 1-2 times.
  • the taxane is administered once.
  • the methods or uses provided herein are based on applicants' observations of a subject's response and therapeutic outcome to an administered cell therapy product. For example, subjects with CLDN18A2-positive cancer who have failed prior therapy with anti-PD-1 antibodies and/or anti-PD-L1 antibodies are given different doses, or different number of cycles of CLDN18A2-targeting CAR-T cells, CAR -T cells can still show better therapeutic results. For example, subjects with GPC3-positive cancer who have failed prior therapy with anti-PD-1 antibodies and/or anti-PD-L1 antibodies are given different doses, or different number of cycles of GPC3-targeting CAR-T cells, CAR-T cells. -T cells can still show better therapeutic results.
  • GPC3-CAR-T cells expressing RUNX3 at different doses, or in different cycles are administered to subjects with GPC3-positive cancer who have failed prior therapy with anti-PD-1 antibodies and/or anti-PD-L1 antibodies, Still able to show better treatment results.
  • these treatment outcomes include patient survival, remission, or stabilization of the disease.
  • the uses or methods described herein further comprise monitoring the subject for the degree or risk of toxicity to the cell therapy product, and determining subsequent fractionation or subsequent cycles of administration based on the degree or risk of toxicity or risk Dosage and interval.
  • the degree or risk of toxicity includes, but is not limited to, for example, CRS, neurotoxicity, macrophage activation syndrome, tumor lysis syndrome, and the like.
  • toxicity or symptoms or biochemical markers eg, CRS or neurotoxicity, macrophage activation syndrome, or tumor lysis syndrome
  • the toxicity or symptoms or biochemical indicators thereof comprise one or more of fever, hypotension, hypoxia, neurological disorders, inflammatory cytokines, serum levels of C-reactive protein (CRP) kind.
  • CRP C-reactive protein
  • the acceptable level of risk of toxicity or symptoms or biochemical indicators refers to 95%, 90%, 85%, 80%, 80%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%.
  • the serum level of a factor indicative of cytokine-release syndrome (CRS) in a subject after a prior cycle based cell therapy product does not exceed the subject's level prior to administration of the prior cycle Serum levels of 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, and 30 times the treatment in the later cycle.
  • CRS cytokine-release syndrome
  • the timing of administration of a subsequent cycle is selected to avoid the potential induction of a host immune response generated by a prior cycle that has been administered such that the efficacy of the subsequent cycle is reduced.
  • administration occurs in the post-cycle prior to the development of a host immune response, eg, fitness or specificity, eg, a humoral or cell-mediated immune response, against the administered cells and/or their expressed chimeric antigen receptors.
  • the subsequent period is administered before such a response can be detected, eg, by one or more specialized detection methods.
  • One or more subsequent cycles are typically administered when the host adaptive immune response against the cells has not been detected, has not yet been established, and/or has not yet reached a certain level or extent or stage.
  • the post-cycle is a disease consolidation dose.
  • the treatment of the subsequent cycle is administered at least 28, 29, 30, 31, 32, 33, 34, 35 days after the administration of the cell therapy product of the prior cycle.
  • tumor burden includes tumor size or degree of differentiation, or the type, stage, and/or appearance and disappearance of complications common to intermediate and advanced cancers, such as cancerous pleural and ascites, and/or appearance or expression of tumor markers Changes in 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 chimeric antigens The host immune response of the recipient.
  • the size of the tumor is measured by scales that come with PET (positron emission tomography) and CT (computed tomography).
  • the tumor markers also known as tumor markers, refer to substances characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or substances produced by the host's stimulatory response to tumors, and can reflect tumor occurrence, A class of substances developed to monitor tumor response to therapy.
  • Tumor markers are present in the tissues, body fluids and excretions of tumor patients and can be detected by immunological, biological and chemical methods, exemplary, including alpha-fetoprotein (AFP), CA125, CA15-3, squamous Cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.
  • AFP alpha-fetoprotein
  • SCC squamous Cell carcinoma antigen
  • CYFRA21-1 soluble fragment of cytokeratin 19
  • CEA carcinoembryonic antigen
  • the uses or methods provided by the present invention include administering at least one cycle of cell therapy products, such as cells expressing CAR, TCR, TFP, or immune effector cells of TAC or a combination thereof.
  • cell therapy products such as cells expressing CAR, TCR, TFP, or immune effector cells of TAC or a combination thereof.
  • Tumor antigens include, but are not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); ); interleukin 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDG)
  • the tumor antigen may be at least one of CD19, CD20, CD22, CD30, Mesothelin, BCMA, EGFR, EGFRvIII, PSMA, Mucl, claudin18.2, GPC3, IL13RA2.
  • the tumor antigen may be at least one of CD19, BCMA, Claudin18.2, GPC3.
  • the tumor or cancer can be colon cancer, gallbladder cancer, krukenberg tumor, rectal cancer, renal cell cancer, liver cancer, lung cancer, small cell lung cancer, small bowel cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer , skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer , Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureter cancer, Renal pelvis carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brainstem
  • CNS central
  • Claudin 18 (Claudin 18, CLD18) molecules (Genbank accession numbers: splice variant 1 (Claudin 18.1, CLD18A1): NP_057453, NM016369, and splice variant 2 (Claudin 18.2, CLD18A2): NM_001002026, NP_001002026) are approximately MW 27,9/27,72kD intrinsic transmembrane protein.
  • Claudin is an intrinsic membrane protein located in the tight junctions of the epithelium and endothelium.
  • CLD18A2 comprises the sequence shown in SEQ ID NO:56.
  • CLD18A2 is strongly expressed in several cancer types, including gastric/esophagogastric junction adenocarcinoma, esophagus, gallbladder, pancreatic, lung, krukenberg tumor. Expression was predominantly in adenocarcinoma subtypes of these indications.
  • GPC3 or “Glypican 3” as used herein is a member of the Glypican family, which plays an important role in regulating cell growth and differentiation.
  • GPC3 comprises the sequence shown in SEQ ID NO:68.
  • the abnormal expression of GPC3 is closely related to the occurrence and development of various tumors, such as abnormal expression in liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, etc.
  • the cell therapy product (eg, CAR-T cells) can be administered to a patient by autologous infusion.
  • the cell therapy product is derived from a subject in need of the cell therapy product and administered to the same subject after genetic modification in vitro.
  • the cell therapy product (eg, CAR-T cells) can be administered to a patient by allogeneic infusion.
  • the cell therapy product is derived from a healthy donor, and the donor and the subject to whom the cells are reinfused are different.
  • the cell therapy product is administered to a subject with high genetic histocompatibility, eg, the donor and the subject are genetically identical or similar.
  • the donor and subject have the same HLA class or supertype.
  • the cells can be administered by any suitable means, eg, by injection, eg, intravenous or subcutaneous injection, intraperitoneal injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, retroseptal injection, subscleral injection, Intrachoroidal injection, anterior chamber injection, subconjectval injection, subconjuntival injection, episcleral injection, retrobulbar injection, periocular injection or peribulbar delivery.
  • they are administered parenterally, intrapulmonary, and intranasally, as well as intralesional if local treatment is desired.
  • Extraperitoneal infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.
  • the appropriate dose may depend on the type of disease being treated, the type of chimeric antigen receptor or cell, the severity and course of the disease, whether the cell is administered for prophylactic or therapeutic purposes, prior treatment, receiving The subject's clinical history and response to the cells, and the judgment of the attending physician.
  • the compositions and cells are suitable for administration to a subject at one time point or in a series of treatments.
  • Pretreatment prior to administration of cell therapy products may improve the efficacy of immune effector cell therapy.
  • the day of the first infusion of immune effector cells (e.g., CAR-T cells) in each cycle was set as day 0, and pretreatment was given before the infusion of immune effector cells.
  • pretreatment is performed at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to administration of adoptive cells or immune effector cells.
  • the pretreatment is performed 2-8 days prior to administration of the cell therapy product; preferably, the pretreatment is performed 3-6 days prior to administration of the cell therapy product.
  • the day on which each cycle of CAR-T cell therapy will be administered to patients will be designated as Day 0.
  • pretreatment can be administered at any time prior to administration of CAR-T cell therapy.
  • the efficacy of the cell therapy product can be maximized by adjusting the timing or number of administrations of chemotherapeutic agents or treatments in the pretreatment.
  • the cyclophosphamide is administered 2-3 times; or the fludarabine is administered 1-2 times; or the nab-paclitaxel is administered once.
  • each chemotherapeutic agent is used for no more than 4 consecutive days.
  • fludarabine is administered in an amount of about 20-60 mg/day, or about 30-60 mg/day, or about 30-55 mg/day, or about 30-50 mg/day.
  • fludarabine is administered in an amount of about 10-50 mg/ m2 /day, or about 15-40 mg/ m2 /day, or about 15-30 mg/ m2 /day, or about 20- 30 mg/ m2 /day; or about 25 mg/ m2 /day.
  • cyclophosphamide is administered in an amount of about 200-700 mg/day, or about 300-700 mg/day, or about 300-600 mg/day, or about 300-560 mg/day, or about 300-550 mg/day day, or about 300-500 mg/day.
  • cyclophosphamide is administered in an amount of about 200-400 mg/ m2 /day, or about 200-300 mg/ m2 /day, or about 250 mg/ m2 /day.
  • the taxanes are administered in an amount of no more than about 300 mg/day, or no more than about 200 mg/day; preferably, the taxanes are administered in an amount of about 90-200 mg/day . More preferably, the administration amount of the taxane compound is about 90-120 mg/day.
  • fludarabine is administered to the patient on days -6 and -5. In some embodiments, fludarabine is administered to the patient on days -5 and -4. In some embodiments, fludarabine is administered to the patient on days -4 and -3. In some embodiments, fludarabine is administered to the patient on days -6, -5, -4, and -3. In some embodiments, fludarabine is administered to the patient on days -7, -6, -5, and -4. In some embodiments, cyclophosphamide is administered to the patient on days -6, -5, -4, and -3.
  • cyclophosphamide is administered to the patient on days -6, -5, and -4. In some embodiments, cyclophosphamide is administered to the patient on days -5, -4, and -2. In some embodiments, cyclophosphamide is administered to the patient on days -5, -4, and -3. In some embodiments, cyclophosphamide is administered to the patient on days -4, -3, and -2. In some embodiments, the patient is administered cyclophosphamide on days -4 and -3. In some embodiments, cyclophosphamide is administered to the patient on days -6 and -5.
  • cyclophosphamide is administered to the patient on days -5 and -4.
  • the patient is administered a taxane (eg, nab-paclitaxel) on Day -2, Day -3, Day -4, Day -5, or Day -6.
  • a taxane eg, nab-paclitaxel
  • Fludarabine, cyclophosphamide, and nab-paclitaxel may 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 may be given before or after fludarabine; or before or after cyclophosphamide Administer fludarabine and/or nab-paclitaxel; or administer cyclophosphamide and/or fludarabine before or after nab-paclitaxel.
  • fludarabine, cyclophosphamide, and nab-paclitaxel may be administered concurrently or sequentially.
  • cyclophosphamide is administered to the patient prior to administration of fludarabine.
  • cyclophosphamide is administered to the patient after fludarabine is administered.
  • the patient is administered nab-paclitaxel prior to administration of fludarabine.
  • the patient is administered nab-paclitaxel before and after administration of fludarabine.
  • the patient is administered nab-paclitaxel prior to administration of cyclophosphamide.
  • the patient is administered nab-paclitaxel after administration of cyclophosphamide.
  • Fludarabine, cyclophosphamide, and nab-paclitaxel can be administered by any route, including intravenous (IV) injection.
  • IV intravenous
  • cyclophosphamide and fludarabine may cause adverse time in patients after administration. It is within the scope of the present invention to administer compositions to patients such that certain of these adverse events are reduced.
  • the method comprises administering saline to the patient. Physiological saline may be administered to the patient before or after administration of cyclophosphamide and/or fludarabine, or before and after administration of cyclophosphamide and/or fludarabine.
  • saline is administered to the patient prior to administration of cyclophosphamide and/or fludarabine on each infusion day, and after administration of cyclophosphamide and/or fludarabine.
  • adjuvants and excipients may also be administered to the patient.
  • mesna sodium 2-mercaptoethanesulfonate
  • exogenous cytokines may also be administered to the patient.
  • the biological activity of the engineered immune effector cell population can be measured by any of a variety of known methods after administration of the cell therapy product to the subject. Parameters to be assessed include: specific binding of cells to antigen, either in vivo (eg, by imaging) or ex vivo (eg, by ELISA or flow cytometry). In certain embodiments, the ability of engineered immune effector cells to destroy target cells can be detected using any suitable method known in the art, such as cytotoxicity assays described in, eg, Kochenderfer et al., J. Immunotherapy , 32(7):689-702 (2009) and Herman et al. J. Immunological Methods, 285(1):25-40 (2004).
  • the biological activity of immune effector cells can also be measured by measuring the expression and/or secretion of certain cytokines, such as CD107a, IFNy, IL-2 and TNF.
  • biological activity is assessed by assessing clinical outcomes, such as tumor burden or reduction in burden.
  • the reduction in tumor markers is assessed.
  • the cells are assessed for toxicity outcome, persistence and/or proliferation, and/or the presence or absence of a host immune response.
  • a "preceding cycle" relative to a “later cycle” is a dosing cycle prior to dosing in a subsequent cycle, eg, a second cycle of dosing is administered after completion of the first cycle of dosing And the administration of the third cycle, the second cycle is relative to the first cycle, the first cycle is the previous cycle, the second cycle is the subsequent cycle, the third cycle is relative to the second cycle, the second cycle is The preceding period, the third period is the succeeding period.
  • the dosage of the cell therapy product refers to the number of cells contained in the cell therapy product.
  • the amount or size in the later cycle is sufficient to reduce tumor burden or an indicator thereof, and/or one or more symptoms of a disease or disorder.
  • the dose is of a magnitude effective to improve survival of the subject, eg, to improve survival of the subject, eg, to induce survival of the subject, relapse-free survival, or event-free survival for at least 1 month or at least 1, 2 , 3, 4 or 5 years.
  • tumor burden including tumor size, tumor volume, and/or tumor mass
  • the number of immune effector cells administered in a later cycle is lower than the number of immune effector cells administered in a prior cycle.
  • whether to administer a subsequent cycle of treatment is determined based on one or more criteria, eg, based on the subject's response to a prior cycle of treatment, eg, chemotherapy, the subject's tumor burden, eg, tumor volume, size or Degree, extent, or type of metastasis, stage, and/or complications common to intermediate and advanced cancers, such as cancerous pleural ascites and/or likelihood or incidence of toxic outcomes in the subject, such as CRS, macrophage activation syndrome , tumor lysis syndrome, neurotoxicity and/or host immune responses against administered cells and/or chimeric antigen receptors.
  • the subject's tumor burden eg, tumor volume, size or Degree, extent, or type of metastasis, stage, and/or complications common to intermediate and advanced cancers, such as cancerous pleural ascites and/or likelihood or incidence of toxic outcomes in the subject, such as CRS, macrophage activation syndrome , tumor lysis syndrome, neurotoxicity and/or host immune responses against administered cells and/or chimeric antigen receptor
  • the doses specifically disclosed in the present invention are safe and effective doses obtained by the inventor through research.
  • Factors such as tumor burden and the patient's own physical condition determine the dose of administration in each cycle; if further administration is required in subsequent cycles, those skilled in the art can determine the dose in subsequent cycles based on, for example, changes in tumor burden after administration of immune effector cells. dose.
  • the dose of the cell therapy product administered in the subsequent cycle is determined based on the tumor burden prior to administration of the treatment in the prior cycle. In some embodiments, eg, where the prior cycle results in a reduction or reduction in tumor burden, or has fallen below a certain threshold amount or level, eg, above which indicates an increased risk of toxic outcome, the dose of the later cycle is increased.
  • the dose of the cell therapy product in the later cycle can be increased even if the subject's tumor burden does not decrease after receiving the prior cycle of treatment.
  • T cells can be administered by any route, including intravenous (IV) injection.
  • IV intravenous
  • the CAR-T cells are administered IV for 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.
  • biochemical markers or indicators indicative of side effects eg, cytokine release syndrome (CRS), macrophage activation syndrome, or tumor lysis syndrome, or neurotoxicity
  • CRS cytokine release syndrome
  • macrophage activation syndrome or tumor lysis syndrome, or neurotoxicity
  • whether to administer the treatment in the later cycle, when to administer the treatment in the later cycle, and/or the dosage of the cell therapy product administered in the later cycle is based on the cells in the subject for the previous cycle or its The presence, absence or extent of an immune response or detectable immune response to the expressed chimeric antigen receptor is determined.
  • the time of administration in the later cycle is from the completion of the cell therapy product of the prior cycle (the date of completion of the total dose infusion of the prior cycle is designated as day 0).
  • the serum level of the factor indicative of CRS in the subject does not exceed about 10-fold, 25-fold, 50-fold or 100-fold the serum level of the indicator in the subject administered the time prior to the cycle , administer the treatment in the later cycle.
  • the results associated with CRS e.g., serum factors associated with or indicative of CRS
  • clinical signs or symptoms thereof such as fever, hypoxia, hypotension, or neurological disorders
  • the treatment of the subsequent cycle is given when the decline begins after the prior cycle is given.
  • subsequent cycles of treatment are administered when the host adaptive immune response has not been detected, has not been established, or has not yet reached a certain level, extent or stage. In some aspects, subsequent cycles of treatment are administered prior to the development of a memory immune response in the subject.
  • tumor burden is monitored between a prior cycle and a subsequent cycle.
  • a detectable immune response refers to an amount detectable by any of a number of known methods for assessing specific immune responses to specific antigens and cells. For example, in some embodiments, specific binding and/or neutralization is detected on cells by performing ELISPOT, ELISA, or cell-based antibody detection methods (eg, by flow cytometry) on the serum of the subject. The presence of an antigen, such as an antibody that binds an epitope of a chimeric antigen receptor, such as a CAR, is used to detect a specific type of immune response. In some such assays, the isotype of the antibody detected is determined and can indicate the type of response and/or whether the response is a memory response.
  • the number and magnitude of the immune response is detected or measured.
  • disease burden is measured or detected prior to administration of a prior cycle of treatment, or between administration of a prior cycle and a subsequent cycle of treatment, or after administration of a subsequent cycle of treatment.
  • disease burden is reduced by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% following administration of a prior cycle of treatment %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%.
  • administration following treatment in a later cycle further reduces disease burden, eg, a reduction of about 1%, 2%, 3%, 4%, 5%, 6% after treatment compared to disease burden before and after treatment in a later cycle %, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%.
  • progression-free survival or overall survival of a subject is improved by this method as compared to other methods.
  • the subject has a progression-free survival rate of greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, Greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%.
  • the overall survival rate is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90% % or greater than about 95%.
  • the subject exhibits progression-free survival, relapse-free survival, or overall survival of at least about 1 month, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
  • the duration of progression-free survival eg, greater than or about 1 month, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10 years.
  • treatment by this method results in a reduced probability of relapse compared to other methods.
  • the probability of recurrence or progression 1 month after the preceding cycle 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%.
  • Cells can be exogenously modified by known biological techniques to express exogenous receptors, such as exogenous expression of TCR receptor, chimeric antigen receptor (CAR) and T cell fusion protein (TFP) and T cell antigen coupler (TAC).
  • exogenous receptors such as exogenous expression of TCR receptor, chimeric antigen receptor (CAR) and T cell fusion protein (TFP) and T cell antigen coupler (TAC).
  • Exemplary exogenous receptors such as sequences comprising a CAR, may have the amino acid sequence set forth in any of SEQ ID NOs: 11, 12, 13, 25, 26, 36, 38, 52.
  • the CAR of the present application includes a CAR having the sequence shown in SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, 65, 66 or 67 and the sequence shown in SEQ ID NO: 80, 81 or 82, respectively Sequence sequential concatenation combination.
  • sequence shown in SEQ ID NO: 58 is sequentially linked with the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 58 is sequentially linked with the sequence shown in SEQ ID NO: 81, SEQ ID NO: 58
  • the sequence shown is sequentially connected with the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 59 is sequentially connected with the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 59 is connected with the sequence shown in SEQ ID NO:
  • the sequence shown in 81 is connected in sequence
  • the sequence shown in SEQ ID NO: 59 is sequentially connected with the sequence shown in SEQ ID NO: 82;
  • sequence shown in SEQ ID NO: 60 is connected in sequence with the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 80.
  • the sequence shown in ID NO: 60 is sequentially connected with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 60 is sequentially connected with the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 61 is connected with the sequence shown in SEQ ID NO: 61
  • the sequence shown in ID NO: 80 is sequentially connected, or the sequence shown in SEQ ID NO: 61 is sequentially connected with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 61 is connected in sequence with the sequence shown in SEQ ID NO: 82 ;
  • the sequence shown in SEQ ID NO: 62 is sequentially connected with the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 62 is sequentially connected with the sequence shown in SEQ ID NO: 81, and shown in SEQ ID NO: 62
  • the sequence is sequentially connected with the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 63 is sequential
  • sequence shown in SEQ ID NO: 63 is sequentially connected with the sequence shown in SEQ ID NO: 82; if the sequence shown in SEQ ID NO: 64 is connected in sequence with the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 80 :
  • the sequence shown in 64 is sequentially connected with the sequence shown in SEQ ID NO: 81, the sequence shown in SEQ ID NO: 64 is sequentially connected with the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 65 is connected with the sequence shown in SEQ ID NO: 82.
  • the sequence shown in SEQ ID NO: 80 is sequentially connected, or the sequence shown in SEQ ID NO: 65 is sequentially connected with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 65 is sequentially connected with the sequence shown in SEQ ID NO: 82; as The sequence shown in SEQ ID NO:66 is sequentially linked with the sequence shown in SEQ ID NO:80, or the sequence shown in SEQ ID NO:66 and SEQ ID NO: The sequence shown in 81 is sequentially connected, and the sequence shown in SEQ ID NO: 66 is sequentially connected with the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 67 is connected in sequence with the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 80. The sequence shown in ID NO: 67 is sequentially connected with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 67 is sequentially connected with the sequence shown in SEQ ID NO: 82.
  • the CAR of the present application includes a sequence with the sequence shown in SEQ ID NO: 70 and the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 70 and the sequence shown in SEQ ID NO: 81.
  • the sequence shown in SEQ ID NO:70 is sequentially connected with the sequence shown in SEQ ID NO:82
  • the sequence shown in SEQ ID NO:71 is sequentially connected with the sequence shown in SEQ ID NO:80, or the sequence shown in SEQ ID NO:71
  • the sequence is sequentially connected with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 71 is sequentially connected with the sequence shown in SEQ ID NO: 82
  • the sequence shown in SEQ ID NO: 72 is shown in SEQ ID NO: 80
  • the sequences are connected sequentially, or the sequence shown in SEQ ID NO: 72 is connected in sequence with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 72 is connected in sequence with the sequence shown in SEQ
  • sequence shown in SEQ ID NO: 81 is connected in sequence, and the sequence shown in SEQ ID NO: 75 is connected in sequence with the sequence shown in SEQ ID NO: 82; the sequence shown in SEQ ID NO: 76 is connected with the sequence shown in SEQ ID NO: 80.
  • connection or the sequence shown in SEQ ID NO: 76 is sequentially connected with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 76 is sequentially connected with the sequence shown in SEQ ID NO: 82; as shown in SEQ ID NO: 77
  • the sequence shown in SEQ ID NO: 80 is connected in sequence, or the sequence shown in SEQ ID NO: 77 is connected in sequence with the sequence shown in SEQ ID NO: 81, and the sequence shown in SEQ ID NO: 77 is connected with the sequence shown in SEQ ID NO: 82.
  • sequence shown in SEQ ID NO: 78 is sequentially connected with the sequence shown in SEQ ID NO: 80, or the sequence shown in SEQ ID NO: 78
  • sequence shown in SEQ ID NO: 81 is connected in sequence
  • sequence shown in SEQ ID NO: 78 is connected in sequence with the sequence shown in SEQ ID NO: 82
  • sequence shown in SEQ ID NO: 79 is connected with the sequence shown in SEQ ID NO: 80
  • Sequential connection or the sequence shown in SEQ ID NO: 79 is sequentially connected with the sequence shown in SEQ ID NO: 81
  • sequence shown in SEQ ID NO: 79 is sequentially connected with the sequence shown in SEQ ID NO: 82.
  • sequences shown in SEQ ID NO: 83, 84 or 85 are examples of the sequence shown in SEQ ID NO: 83, 84 or 85.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • polypeptides that include the provided receptors and other polypeptides (eg, linkers or peptides) can include amino acid residues, including natural and/or unnatural amino acid residues.
  • the term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation and phosphorylation.
  • the polypeptide may comprise modifications to the original or native sequence, so long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by host mutation to produce the protein or errors due to PCR amplification.
  • cells expressing the exogenous receptor are generally eukaryotic cells, such as mammalian cells, and usually human cells.
  • the cells are derived from blood, bone marrow, lymph, or lymphoid organs, and are cells of the immune system, such as cells of innate or adaptive immunity, eg, myeloid or lymphoid cells, including lymphocytes, typically T cells, NKT cells and/or NK cells.
  • Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells are generally primary cells, such as cells isolated directly from the subject and/or isolated from the subject and frozen.
  • the cells comprise one or more subsets of T cells or other cell types, such as total T cell populations, CD4+ T cells, CD8+ T cells, and subsets thereof, as determined by function, activation state, maturation , differentiation potential, expansion, recycling, localization, and/or persistence capacity, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation definition of T cells.
  • the cells can be allogeneic and/or autologous. Methods of making such exogenous receptor-expressing cells include off-the-shelf methods.
  • the method comprises isolating cells from a subject, preparing, processing, culturing and/or engineering them, and reintroducing them into the same subject before or after cryopreservation.
  • T cells and/or CD4+ T cells and/or CD8+ T cells include: naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T cells (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells , cytotoxic T cells, mucosa-associated non-variant T (MAIT) cells, naturally occurring and adoptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells or combinations thereof.
  • TN naive T
  • TSCM stem cell memory T cells
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor
  • the cells are natural killer (NK) cells.
  • the cells are monocytes or granulocytes, eg, myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, or its combination.
  • the cells comprise one or more nucleic acids introduced by genetic engineering, and thereby express recombinant or genetically engineered products of the nucleic acids.
  • the nucleic acid is heterologous, typically not present in a cell or a sample obtained from the cell, eg, a sample obtained from another organism or cell, or eg, a cell that is not typically engineered and/or or such cell-derived organisms.
  • the nucleic acid is not naturally occurring, such as the nucleic acid is not found in nature, including chimeric combinations comprising nucleic acids encoding various domains from a variety of different cell types, such as CARs, chimeric cytokine receptors .
  • the present invention also provides methods, compositions and kits for generating genetically engineered cells expressing chimeric antigen receptors.
  • Genetic engineering generally involves introducing a nucleic acid encoding the recombinant or engineered portion into a cell, eg, by viral (eg, lentivirus, retrovirus, AAV) transduction, transfection, or transformation.
  • viral eg, lentivirus, retrovirus, AAV
  • the term "engineering” refers to the application of the principles and methods of cell biology and molecular biology, through some engineering means, at the overall level of cells or at the level of organelles, to change the genetic material in cells or obtain cells according to people's wishes. A comprehensive science and technology of products.
  • the engineering refers to one or more alterations of nucleic acids, such as nucleic acids within the genome of an organism.
  • the engineering refers to changes, additions and/or deletions of genes.
  • the engineered cell or the engineered cell may also refer to a cell with added, deleted and/or altered genes.
  • gene transfer is performed by first, stimulating the cell, eg, by combining it with a stimulator that induces a response, eg, proliferation, survival, and/or activation, eg, by cytokines or The activated cells are then transduced and expanded in culture to numbers sufficient for clinical use.
  • a stimulator that induces a response, eg, proliferation, survival, and/or activation, eg, by cytokines or
  • the activated cells are then transduced and expanded in culture to numbers sufficient for clinical use.
  • the cells are also engineered to promote the expression of cytokines or other factors.
  • recombinant infectious virus particles are used to transfer recombinant nucleic acids into cells, eg, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV).
  • recombinant lentiviral or retroviral vectors such as gamma-retroviral vectors, are used to transfer recombinant nucleic acids into T cells.
  • the recombinant nucleic acid is transferred into T cells by electroporation. In some embodiments, the recombinant nucleic acid is transferred into T cells by translocation (see, e.g., Manuri et al, (2010) Hum Gene Ther 21(4):427-437; Sharma et al, (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (eg, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY).
  • the preparation of engineered cells includes one or more culturing and/or one or more preparation steps.
  • Cells for introduction of nucleic acid encoding a transgenic receptor can be isolated from a sample (eg, a biological sample, eg, a sample obtained or derived from a subject).
  • the subject from which the cells are isolated is a subject suffering from a disease or disorder or in need of or to be administered cell therapy.
  • the subject is a human in need of a particular therapeutic intervention, eg, a human in need of adoptive cell therapy or immune effector cell therapy for which cells are isolated, processed and/or engineered .
  • the cells are primary cells, eg, primary human cells.
  • the samples include tissues, body fluids, and other samples taken directly from a subject, and obtained from one or more processing steps, such as isolation, centrifugation, genetic engineering (eg, transduction with viral vectors), washing, and/or Incubation samples.
  • the biological sample may be a sample obtained directly from a biological source or a processed sample.
  • Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood-derived sample or, is or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, PBMC, leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemia, lymphoma, lymph nodes, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver , lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organs, and/or cells derived therefrom.
  • samples include samples from autologous and allogeneic sources.
  • the cells are derived from a cell line, eg, a T cell line.
  • the cells are obtained from a xenogeneic source, eg, from a mouse, rat, non-human primate, or pig.
  • the isolation of cells includes one or more preparative and/or non-affinity-based cell isolation steps.
  • cells are washed, centrifuged and/or incubated in the presence of one or more substances, eg, to remove unwanted components, enrich for desired components, lyse or remove specific substances sensitive cells.
  • cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity and/or resistance to particular components.
  • cells from the subject's circulating blood are obtained, eg, by apheresis or leukapheresis.
  • the sample comprises lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated blood leukocytes, red blood cells, and/or platelets and in some aspects with red blood cells and platelets different cells.
  • the method comprises a density-based cell separation method, eg, by lysing red blood cells or not by lysing red blood cells and by centrifuging the peripheral blood through a Percoll or Ficoll gradient or by a single sample or leukapheresis sample preparation to obtain peripheral blood single cells nuclear cells (PBMC).
  • a density-based cell separation method eg, by lysing red blood cells or not by lysing red blood cells and by centrifuging the peripheral blood through a Percoll or Ficoll gradient or by a single sample or leukapheresis sample preparation to obtain peripheral blood single cells nuclear cells (PBMC).
  • PBMC peripheral blood single cells nuclear cells
  • the method of separation comprises separating different cell types based on the expression or presence of one or more specific molecules, eg, surface markers, eg, surface proteins, intracellular markers, or nucleic acids, in the cells. In some embodiments, any known method for isolation based on such markers can be employed. In some embodiments, the separation is an affinity-based or immunoaffinity-based separation.
  • the isolating comprises isolating cells and cell populations based on the expression or level of expression of one or more markers (usually cell surface markers) of the cells, eg, by specifically binding thereto
  • the marker-like antibody or binding partner is incubated, usually followed by a washing step, and separation of cells that have bound the antibody or binding partner from those cells that have not yet bound to the antibody or binding partner.
  • the isolation need not result in a 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • a specific subset of T cells eg, one or more surface marker positive cells or cells expressing high levels of one or more surface markers, eg, CD3+, CD28+, CD62L+, CCR7+ , CD27+, CD127+, CD4+, CD8+, CD45RA+ and/or CD45RO+ T cells, isolated by positive or negative selection techniques.
  • one or more surface marker positive cells or cells expressing high levels of one or more surface markers eg, CD3+, CD28+, CD62L+, CCR7+ , CD27+, CD127+, CD4+, CD8+, CD45RA+ and/or CD45RO+ T cells, isolated by positive or negative selection techniques.
  • CD3+, CD28+ T cells can be positively selected using CD3/CD28 linked magnetic beads (eg, DYNA beads M-450 CD3/CD28 T cell expander).
  • CD3/CD28 linked magnetic beads eg, DYNA beads M-450 CD3/CD28 T cell expander
  • T cells are isolated from a PBMC sample by negative selection for markers (eg, CD14) expressed on non-T cells (eg, B cells, monocytes, or other blood leukocytes).
  • markers eg, CD14
  • non-T cells eg, B cells, monocytes, or other blood leukocytes.
  • CD4+ or CD8+ selection steps are used to isolate helper CD4+ and CD8+ cytotoxic T cells.
  • Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed on or to a relatively high degree on one or more naive, memory and/or effector T cell subsets group.
  • CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and/or central memory stem cells, eg, by positive or negative selection based on surface antigens associated with corresponding subpopulations.
  • enrichment for central memory T (TCM) cells is performed to increase efficacy, eg, to improve long-term survival, expansion, and/or engraftment after administration, which in some aspects is in such subgroups Especially strong in the group.
  • the method of preparation includes a freezing step, eg, before or after isolation, incubation and/or engineering, freezing the cells.
  • the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes in the cell population.
  • the cells are suspended in a freezing solution.
  • any of a variety of known freezing solutions and parameters can be employed.
  • the cells are generally frozen to -80°C or -90°C according to a predetermined procedure or principle such as a rate of 1°/min with a programmed cooling device, and stored in the vapor phase of a liquid nitrogen storage tank.
  • the cells are incubated and/or cultured prior to or with genetic engineering. Incubation steps can include culturing, cultivating, stimulating, activating, and/or proliferating. In some embodiments, the cells or compositions are incubated in the presence of stimulating conditions or stimulating agents. Such conditions include those designed to induce proliferation, reproduction, activation, and/or survival of cells in a population to mimic antigen contact, and/or to prime cells for genetic engineering, such as those used to introduce recombinant antigen receptors.
  • the stimulating condition or agent includes one or more substances, eg, ligands, that are capable of activating the intracellular signaling domain of the TCR complex.
  • the agent turns on or initiates the TCR/CD3 intracellular signaling cascade in T cells.
  • substances may include antibodies, eg, those specific for TCR components and/or costimulatory receptors, eg, anti-CD3, anti-CD28, eg, which are bound to a solid support, eg, beads, and/or a or multiple cytokines.
  • the expansion method may further comprise the step of adding anti-CD3 and/or anti-CD28 antibodies to the culture medium (eg, at a concentration of at least about 0.5 ng/ml).
  • the stimulating agent comprises IL-2 and/or IL-15 and/or IL-7 and/or IL-21, eg, IL-2 at a concentration of at least about 10 units/mL.
  • antigen-specific T cells such as antigen-specific CD4+ and/or CD8+ T cells
  • antigen-specific T cell lines or clones can be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.
  • T cells were prepared from peripheral blood mononuclear cells (PBMCs) of human subjects with cancer by "demonocyte isolation"-based, cultured and transduced with viral vectors encoding chimeric antigen receptors (CARs).
  • the T cell, the chimeric antigen receptor (CAR) specifically binds an antigen expressed by a tumor cell in the subject, which is either a tumor-associated antigen or a tumor-specific antigen.
  • Cells are cryopreserved in infusion medium in separate 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 10 12 cells per subject infused prior cycle preferably, no more than about 1 x 10 11 cells, more preferably, no more than about 1 x 10 10 cells or about 5x109 cells or about 2x109 cells.
  • the cells are maintained at a temperature below about -130°C or below about -175°C prior to infusion.
  • tumor necrosis factor alpha TNF ⁇
  • IFN ⁇ interferon gamma
  • IL-10 IL-6
  • Tumor burden can optionally be assessed by measuring the size or mass of solid tumors, eg, by PET or CT scans, prior to initiation of treatment.
  • Resuscitation was performed by warming to about 38°C, and subjects were given cells from previous cycles by multiple infusions. Each infusion is administered intravenously (IV) as a continuous infusion over a period of approximately 3-30 minutes.
  • IV intravenously
  • subjects undergo a physical examination and are monitored for symptoms of any toxicity or toxicity outcomes, such as fever, hypotension, hypoxia, neurological disturbances, or serum levels of inflammatory cytokines or C-reactive protein (CRP) rise.
  • any toxicity or toxicity outcomes such as fever, hypotension, hypoxia, neurological disturbances, or serum levels of inflammatory cytokines or C-reactive protein (CRP) rise.
  • blood is obtained from the patient and the levels of serum factors indicative of CRS are assessed by means of ELISA and/or MSD and/or CBA. Serum factor levels were compared to serum factor levels obtained just prior to administration of the previous cycle. If necessary, administer anti-IL6 or other CRS therapy to reduce symptoms of CRS.
  • the subject is optionally tested for the presence or absence of an anti-CAR immune response, eg, by qPCR, ELISA, ELISPOT , cell-based antibody assays and/or mixed lymphocyte reactions.
  • an anti-CAR immune response eg, by qPCR, ELISA, ELISPOT , cell-based antibody assays and/or mixed lymphocyte reactions.
  • the percent reduction in tumor burden achieved by the prior cycle can optionally be measured one or more times after administration of the prior cycle by scans (eg, PET and CT scans) in solid tumor patients, and/or by quantification in blood or tumor Site tumor positive cells.
  • scans eg, PET and CT scans
  • Subjects are monitored regularly, starting with the first dose and continuing 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 anti-CAR Development of the immune response.
  • qPCR quantitative polymerase chain reaction
  • No more than about 1 x 10 12 cells preferably no more than about 1 x 10 11 cells, preferably no more than about 1 x 10 10 cells, Either no more than about 5 x 109 cells, or no more than 2 x 109 cells.
  • Cells were kept below -175°C prior to infusion. During the infusion, the temperature was raised to about 38°C for resuscitation.
  • Dosing in later cycles is patient-specific and based on tumor burden, presence of anti-CAR immune responses, and levels of CRS-related outcomes.
  • the dose administered in the later cycle is not higher than about 1 x 1012 cells, preferably, not higher than about 1 x 1011 cells, more preferably, not higher than about 1 x 1010 cells, more preferably , no more than about 5 x 109 cells or no more than about 2 x 109 cells.
  • AEs adverse events
  • CCAE v5.0 Common Criteria for Adverse Events
  • Exemplary antigen receptors of the present invention including CARs, and methods for engineering and introducing receptors into cells, refer to, eg, Chinese Patent Application Publication Nos. ⁇ CN105331585A ⁇ CN106397593A ⁇ CN106467573A ⁇ CN108884459A ⁇ CN108610420A ⁇ CN108341872A ⁇ CN108456250A ⁇ CN109796532A ⁇ CN108866003A ⁇ CN108853144A ⁇ CN109385403A ⁇ CN109385400A ⁇ CN109468279A ⁇ CN109503715A ⁇ CN109880803A ⁇ CN110055275 A ⁇ CN110123837A ⁇ CN 110438082 A ⁇ CN 110468105 A, ⁇ WO2018006882A1 ⁇ WO2015172339A8 ⁇ WO2016086813A1 ⁇ WO2017032293A1 ⁇ WO2017020812A1 ⁇ WO2017080377A1 ⁇ WO2018108106A1 ⁇ WO2018/133877 A1 ⁇ WO2018/149358 A1 ⁇ WO 2018/2192
  • the scFv of the chimeric antigen receptor can recognize the tumor antigen claudin18.2 and has the sequence shown in SEQ ID NO: 14, and the chimeric antigen receptor has the sequence shown in SEQ ID NO: 11.
  • the construction method of CAR-T cells firstly, construct the CAR gene, which includes: CD8 ⁇ signal peptide (nucleotide sequence shown in SEQ ID NO: 47), scFv (nucleotide sequence shown in SEQ ID NO: 47) from the 5' end to the 3' end. ID NO: 15), CD8 hinge region (nucleotide sequence shown in SEQ ID NO: 48), CD28 transmembrane region (nucleotide sequence shown in SEQ ID NO: 49) and intracellular signaling of CD28
  • the conduction domain (nucleotide sequence shown in SEQ ID NO:50) and the intracellular signaling domain CD3 ⁇ of CD3 ⁇ (nucleotide sequence shown in SEQ ID NO:51).
  • the CAR gene was cloned into the plasmid PRRLSIN-cPPT.EF-1 ⁇ to obtain the target plasmid containing the CAR gene.
  • the constructed target plasmid and packaging plasmid were co-transfected into 293T cells to prepare a lentiviral vector, and the lentiviral vector was used to transduce T cells obtained from the peripheral blood of patients to obtain CAR-T cells.
  • the positive intensity of antigen is defined by the staining intensity of immunohistochemistry, which is part or all of the cell membrane or cytoplasm staining.
  • the staining intensity is divided into: 0 (negative, no staining), + (weak positive staining) ), ++ (positive staining, ) and +++ (strong positive staining).
  • the percentage of stained tumor cells refers to the percentage of tumor cells with each staining intensity in the total tumor cells.
  • PBMCs were obtained from subjects, and CAR-T cells were obtained by transduction and amplification with a viral vector encoding anti-CLDN18A2 CAR.
  • the resulting CAR-T cells were cryopreserved in a freezing solution using a freezing bag.
  • tumor burden can optionally be assessed by measuring the size or shape of a solid tumor by, for example, a PET or CT scan, and also by detecting tumor markers and/or observing the occurrence and severity of tumor complications to assess tumor burden.
  • the CAR-T cell administration in the later cycle is administered 4 weeks after the completion of the CAR-T cell administration in the previous cycle.
  • the administration of CAR T cells in each cycle can be administered once or divided into two or more intravenous (IV) infusions. 25 minutes to complete the infusion.
  • IV intravenous
  • CAR-T cell administration After each cycle of CAR-T cell administration, subjects undergo a physical examination and are monitored for symptoms of any toxicity or toxicity outcomes, such as fever, hypotension, hypoxia, neurological disturbances, or inflammatory cytokines or C-reactivity Serum levels of protein (CRP) are elevated.
  • the examination may be to obtain blood from the patient and assess the levels of cytokines indicative of CRS by means of ELISA, and/or MSD, and/or CBA. If necessary, give anti-IL6 therapy, or give other CRS therapy to reduce the symptoms of CRS.
  • the CAR-T cells in the subject can be assessed by qPCR, ELISA, ELISPOT, antibody assays, etc. number of T cells.
  • the reduction in tumor burden after each cycle of treatment can be obtained by scanning (eg, PET and CT scans), and/or by quantifying cells that are positive for antigens (eg, claudin 18.2) in the blood or at the tumor site.
  • scanning eg, PET and CT scans
  • quantifying cells that are positive for antigens eg, claudin 18.2 in the blood or at the tumor site.
  • fludarabine was administered twice, on the 6th, 5th day (D-6, -5), or 5th, 4th day (D-5, -4), or on the 5th and 4th day (D-5, -4) before CAR-T cell infusion, respectively.
  • Cyclophosphamide was administered 2 or 3 times, wherein, when cyclophosphamide was administered twice, on the 6th, 5th day (D-6, -5), or 5th and 4th day before CAR-T cell administration, respectively (D-5,-4), or on days 4 and 3 (D-4,-3), when cyclophosphamide was administered three times, on days 6 and 5 before CAR-T cell administration, respectively (D- 6,-5), or the 5th, 4th day (D-5,-4), or the 4th, 3rd day (D-4,-3) administration, before CAR-T cell administration 6th, 5th, 4 days (D-6, -5, -4), or 5, 4, 3 days (D-5, -4, -3), or 4, 3, 2 days (D-4, -3, -2) Administration, the dose of cyclophosphamide administered is 300-600 mg/day, wherein the amount of cyclophosphamide administered to the patient is 300-400 mg/day or 401-500 mg/day,
  • Nab-paclitaxel was administered once, 3-5 days before administration of CAR-T cells, such as nab-paclitaxel 90-200 mg/day on D-4.
  • the mean daily dose of nab-paclitaxel was 102.2 ( ⁇ 8.60) mg.
  • the treatment evaluation of the 12 subjects involved in Example 1 uses the Kaplan Meier method to perform statistical analysis on PFS, DDC, DOR and OS, and perform descriptive analysis on TTR.
  • the tumor burden before and after treatment was assessed by imaging detection of target lesions and non-target lesions, and the number and size of target lesions were determined.
  • neurotoxicity neurotoxicity complications, including confusional symptoms, aphasia, seizures, convulsions, somnolence, and/or altered mental status
  • neurotoxicity neurotoxicity complications, including confusional symptoms, aphasia, seizures, convulsions, somnolence, and/or altered mental status
  • graded according to severity using a 1-5 scale, e.g., Guido Cavaletti and Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010), where 3 (severe symptoms), 4 (life-threatening symptoms) or 5 (death) Considered severe neurotoxicity.
  • systemic therapy such as antipyretics and antiemetics (eg, fever, nausea, fatigue, headache, myalgia, malaise).
  • monovasopressor eg, norepinephrine ⁇ 20ug/kg/min, dopamine ⁇ 10ug/kg/min, Hypotension with phenylephrine ⁇ 200ug/kg/min, or
  • Grade 4 life-threatening
  • - ventilator support required, or Grade 4 organ toxicity (exclude transaminases).
  • Grade 1 (asymptomatic or mild) - mild or no clinical symptoms.
  • Grade 2 There are symptoms that limit active activities of daily living (ADL), such as cooking, grocery shopping or clothing, using the phone, managing money.
  • ADL active activities of daily living
  • Grade 3 There are symptoms of restrictive self-management of ADL, such as bathing, dressing or undressing, eating, toilet use, taking medication.
  • the second cycle of treatment was performed on some patients, and the start time of the second cycle and the curative effect after treatment are shown in Table 3.
  • the 6 subjects who achieved objective response had a mDOR (median duration of response) of 5.4 months (1.9, NE).
  • 1 subject who received only one treatment continued to remission for 6.4 months; 1 patient had no target lesions detected in the efficacy evaluation after one treatment, and the continuous remission time had exceeded 4 months.
  • Progression-free survival (PFS) and overall survival (OS) The inverse K-M curve method was used to calculate the median follow-up time of 12 subjects since apheresis and after the first CAR-T cell infusion, respectively, about 6.4 months and 5.6 months; mPFS was calculated by K-M curve method (95%CI), and the mPFS of all subjects receiving CAR-T cell therapy was 4.2 months (2.2, NE); mOS was calculated by K-M curve method (95%CI) was 7.4 months (2.6, NE). The results suggest that subjects can obtain more obvious clinical benefits from CAR-T treatment, and PFS and OS are significantly prolonged.
  • CAR-T cell survival in vivo the period during which the CAR-T cells are "engrafted” in vivo, was examined. From the end of the initial infusion (which was day 0) using the method of Q-PCR at each visit point (probe used: FAM-5'-CTGAGCAGCGTGACCGCCGC-3'TAMRA (SEQ ID NO: 49); upstream primer sequence It is: 5'-TGGAGTGGATCGGCTACATC-3' (SEQ ID NO:50); the downstream primer sequence is: 5'-AGTAGTAGATGGCGGTGTCG-3' (SEQ ID NO:51)) to detect the copy number of CAR-CLDN18 DNA in peripheral blood.
  • the results of cell metabolism in 12 subjects showed that after the first CAR-T cell infusion, the median time to peak the number of amplified copies in vivo was about 7 days, and the median peak value of CAR copies (minimum, maximum value) ) were 7196 (3422, 39315) [copy number/ ⁇ g gDNA], respectively.
  • CAR-T cell expansion persisted in vivo for up to 203 days after the first infusion. No apparent dose-dependent relationship was observed.
  • the median time to peak cell expansion was advanced to within 5 days, and the amplitude was significantly lower than that of the first cycle, and the median Cmax was 1074 copies/ ⁇ g gDNA.
  • AEs adverse events
  • CCAE v5.0 Common Criteria for Adverse Events
  • the mDOR of the 12 subjects who achieved PR after CAR-T treatment was 6.3 months (1.9, 9.5). Among them, 6 patients had an overall survival of more than 11 months and were still in the follow-up period.
  • the cell metabolism results of 24 subjects showed that after the first CAR-T cell infusion, the median time to peak the number of amplified copies in vivo was about 7 days, and the median peak value of CAR copies (minimum, maximum value) ) were 4536 (433, 39315) [copy number/ ⁇ g gDNA], respectively.
  • this example exemplifies the progression of 11 patients with tumor antigen GPC3 positive liver cancer after PD-1 and/or PD-L1 antibody treatment (progressive disease PD according to RECIST 1.1).
  • Received GPC3-CAR-T cell therapy Antibodies to PD-1 and/or PD-L1 previously used by patients are: camrelizumab, pembrolizumab, sintilimab, toripalizumab, and some in clinical trials. monoclonal antibody. All 11 patients had received at least 1 line of standard therapy, including 1 1st line, 8 2nd lines, 1 4th line, and 1 6th line.
  • Example 11 According to the RECIST 1.1 standard evaluation, after the patients with liver cancer in Example 11 received about 2.5 ⁇ 10 8 -5.0 ⁇ 10 8 CAR-T cells/cycle treatment for 1 cycle or 2 cycles, 5 patients were in SD, and 1 patient was in SD. PR was achieved, and 5 cases were PD, indicating that CAR-T cells can still produce significant curative effect on liver cancer patients who have used PD-1 or PD-L1 antibody, and the disease control rate is about 54.5% (6/11). The mean progression-free survival of the 6 patients who achieved disease control was approximately 4.4 months, of which 4 patients had an overall survival of more than 1 year, and 3 of these 4 patients were still followed for survival.
  • the method of Q-PCR was used to detect the copy number of CAR-GPC3 DNA contained in the peripheral blood of patients.
  • the results showed that after the first CAR-T cell infusion, the median time to peak copy number in vivo was about 7 days.

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Abstract

一种癌症治疗的组合物，以及癌症治疗方法，提供了针对抗PD-1抗体或抗PD-L1抗体治疗失败的患者的癌症治疗方法和治疗组合物。

Description

细胞免疫治疗的应用 技术领域
本发明属于免疫治疗领域；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
程序性细胞死亡蛋白-1(programmed death-1，PD-1)是负调节TCR信号的免疫抑制蛋白。PD-L1是PD-1的配体，在许多肿瘤类型中表达，如卵巢癌、乳腺癌、宫颈癌、结肠癌、胰腺癌、胃癌、黑色素瘤、胶质母细胞瘤、非小细胞肺癌等(Callahan 2014J Leukoc Biol 94(1)：41-53)，PD-1与PD-L1结合后可提供抑制性信号，诱导T细胞凋亡，抑制T细胞的活化和增殖。因此，PD-1或PD-L1为靶点的药物可以通过免疫调节，发挥抗肿瘤作用。目前已上市的PD-1抗体或PD-L1抗体有Nivolumab、Pembrolizumab、Atezolizumab、Avelumab、Durvalumab等，但是依然有很多患者经有PD-1抗体或PD-L1抗体治疗后疾病进展，治疗失败。
细胞免疫疗法是利用患者自体(或异体)的免疫细胞对组织、器官进行修复的治疗方法。如CAR-T细胞治疗，即使采用嵌合抗原受体修饰患者自体(或异体)的T细胞对癌症等疾病进行治疗。与PD-1抗体或PD-L1抗体治疗癌症相似，二者均是通过免疫系统来进行肿瘤杀伤。由于使用PD-1抗体或PD-L1抗体治疗失败的患者可能更不适合肿瘤免疫治疗，因此，对PD-1抗体或PD-L1抗体治疗抵抗的患者可能对其他的免疫治疗(如CAR-T细胞治疗)也不敏感。
发明内容
本发明的目的是提供了癌症治疗的组合物，以及癌症治疗方法，特别是还提供针对抗PD-1抗体和/或抗PD-L1抗体治疗失败的患者的癌症治疗方法和治疗组合物。
具体地，本发明涉及如下各项：
1.细胞治疗产品在制备用于治疗在先治疗癌症失败的患者中的癌症的药物中的用途，所述在先治疗包括使用抗PD-1抗体和/或抗PD-L1抗体治疗。
2.如项1所述的用途，其中所述细胞治疗产品含有表达有外源受体的免疫效应细胞。
3.如项2所述的用途，其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。
4.如项2所述的用途，其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。
5.如项2-4中任一项所述的用途，其中所述免疫效应细胞为自体/同种异体T细胞、或原代T细胞。
6.如项3-5中任一项所述的用途，其中所述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。
7.如项2-6中任一项所述的用途，其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)或其组合。
8.如项2-7中任一项所述的用途，其中所述外源受体的抗原结合结构域特异性识别肿瘤抗原。
9.如项8所述的用途，其中所述肿瘤抗原选自CD19、CD20、CD22、CD30、Mesothelin、BCMA、EGFR、EGFRvIII、PSMA、Muc1、claudin18.2、GPC3、IL13RA2、SLAMF7、GPRC5D、LILRB4或其组合；优选地，所述肿瘤抗原选自GPC3、CD19、BCMA、Claudin18.2或其组合。
10.如项1-9中任一项所述的用途，其中所述癌症包括实体瘤和/或血液肿瘤；优选地所述实体瘤包括消化道肿瘤；更优选地所述消化道肿瘤包括胃癌/食管胃结合部腺癌、胆囊癌、肝癌、krukenberg瘤或其组合。
11.如项1-10中任一项所述的用途，其中所述癌症选自krukenberg瘤、胃癌、胰腺癌、胆囊癌、黑色素瘤、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、头颈部鳞状细胞癌(HNSCC)、经典霍奇金淋巴瘤(cHL)、原发性纵膈大B细胞淋巴瘤(PMBCL)、尿路上皮癌(UC)、食管癌、宫颈癌、肝癌、Merkel细胞癌、肾细胞癌(RCC)、结直肠癌(mCRC)、乳腺癌。
12.如项1-11中任一项所述的用途，其中所述抗PD-1抗体/PD-L1抗体选自Nivolumab、Pembrolizumab、Cemiplimab、卡瑞利珠单抗、特瑞普利单抗、信迪利单抗、替雷利珠单抗、赛帕利单抗(GLS-010)、替雷利珠单抗或其组合。
13.如项1-12中任一项所述的用途，其中所述在先治疗包括药物治疗、手术治疗、放疗或其组合；所述药物治疗包括给予化学药和/或生物药；优选的，所述在先治疗包括给予奥拉帕利、乐伐替尼、卡博替尼、阿西替尼、伊匹木、铂类的化疗药物、培美曲塞、依托泊苷、紫杉烷类化合物、贝伐珠单抗、瑞戈非尼、罗替尼、阿帕替尼、乐伐替尼、呋喹替尼、瑞格菲尼或其组合。
14.如项1所述的用途，其中给予患有所述癌症的患者至少一个周期的细胞治疗产品进行治疗；优选的，给予患有所述癌症的患者1-3个周期的细胞治疗产品进行治疗。
15.如项7-14中任一项所述的用途，其中所述嵌合抗原受体(CAR)包括：
(i)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD3ζ；
(ii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；
(iii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
(iv)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
16.如项14或15所述的用途，其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、2ⅹ10 8个细胞/kg或2ⅹ10 7个细胞/kg患者体重；或细胞治疗产品中细胞的剂量不超过约1ⅹ10 11个细胞/患者、1ⅹ10 10个细胞/患者、5ⅹ10 9个细胞/患者、2ⅹ10 9个细胞/患者或1ⅹ10 9个细胞/患者。
17.如项16所述的用途，其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患者体重至2ⅹ10 7个细胞/kg患者体重，或约1ⅹ10 6个细胞/kg患者体重至2ⅹ10 7个细胞/kg患者体重；或者
每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 7个细胞至5ⅹ10 9个细胞/患者、约1ⅹ10 7个细胞至2ⅹ10 9个细胞/患者、或约1ⅹ10 7个细胞至1ⅹ10 9个细胞/患者；或者每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 8个细胞至5ⅹ10 9个细胞/患者、约1ⅹ10 8个细胞至2ⅹ10 9个细胞/患者、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的剂量为约2.5ⅹ10 8个细胞至5ⅹ10 8个细胞/患者。
18.如项14-17中任一项所述的用途，其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患者化学药物、生物药物、放疗或其组合。
19.如项18所述的用途，其中所述预处理在给予所述细胞治疗产品前1-8天实施；优选地，在给予所述细胞治疗产品前2-6天实施；优选地，化学药物、生物药物、放疗或其组合连续使用不超过4天。
20.如项18或19所述的用途，其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。
21.如项20所述的用途，其中所述微管蛋白抑制剂是紫杉烷类化合物；优选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；更优选所述紫杉烷类化合物为白蛋白结合型紫杉醇。
22.如项20或21所述的用途，其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300-700mg/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。
23.如项20-22中任一项所述的用途，其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。
24.如项8-15中任一项所述的用途，其中所述抗原结合结构域具有：
SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3、SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2、SEQ ID NO:6所示的LCDR3；或者
SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2、SEQ ID NO:18所示的HCDR3、SEQ ID NO:19所示的LCDR1、SEQ ID NO:20所示的LCDR2、SEQ ID NO:21所示的LCDR3；或者
SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2、SEQ ID NO:29所示的HCDR3、SEQ ID NO:30所示的LCDR1、SEQ ID NO:31所示的LCDR2、SEQ ID NO:32所示的LCDR3；
或所述抗原结合结构域具有：
SEQ ID NO:7所示的重链可变区和SEQ ID NO:9所示的轻链可变区；或者
SEQ ID NO:22所示的重链可变区和SEQ ID NO:23所示的轻链可变区；或者
SEQ ID NO:33所示的重链可变区和SEQ ID NO:34所示的轻链可变区；
或所述抗原结合结构域具有：SEQ ID NO:14、24、35、或37所示的序列。
25.如项7-24中任一项所述的用途，其中所述的嵌合抗原受体具有SEQ ID NO:11、12、13、25、26、36、38、52任一所示氨基酸序列、或具有SEQ ID NO：58、59、60、61、62、63、64、65、66或67任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽、或具有SEQ ID NO：70、71、72、73、74、75、76、77、78或79任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽。
26.如项14-25中任一项所述的用途，其中在每个周期给予所述细胞治疗产品之前，对所述患者的指示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。
27.如项1-26中任一项所述的用途，其中在给予所述细胞治疗产品治疗后，所述患者 没有显示严重CRS，或没有显示超过3级的神经毒性。
28.如项1-27中任一项所述的用途，其中所述使用抗PD-1抗体或抗PD-L1抗体治疗癌症失败的患者中的至少一部分，优选至少40％，更优选至少50％对所述细胞治疗产品有应答。
29.如项28所述的用途，其中所述应答是指ORR大于40％，优选大于50％。
30.一种使用细胞治疗产品治疗在先治疗癌症失败的患者中的癌症的方法，其中所述在先治疗包括使用抗PD-1抗体和/或抗PD-L1抗体治疗。
31.如项30所述的方法，其中所述细胞治疗产品含有表达有外源受体的免疫效应细胞。
32.如项31所述的方法，其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。
33.如项31所述的方法，其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。
34.如项31-33中任一项所述的方法，其中所述免疫效应细胞为自体/同种异体T细胞、或原代T细胞。
35.如项32-34中任一项所述的方法，其中所述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。
36.如项31-35中任一项所述的方法，其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)或其组合。
37.如项31-36中任一项所述的方法，其中所述外源受体的抗原结合结构域特异性识别肿瘤抗原。
38.如项37所述的方法，其中所述肿瘤抗原选自CD19、CD20、CD22、CD30、Mesothelin、BCMA、EGFR、EGFRvIII、PSMA、Muc1、claudin18.2、GPC3、IL13RA2、SLAMF7、GPRC5D、LILRB4或其组合；优选地，所述肿瘤抗原选自GPC3、CD19、BCMA、Claudin18.2或其组合。
39.如项30-38中任一项所述的方法，其中所述癌症包括实体瘤和/或血液肿瘤；优选地所述实体瘤包括消化道肿瘤；更优选地所述消化道肿瘤包括胃癌/食管胃结合部腺癌、胆囊癌、肝癌、krukenberg瘤或其组合。
40.如项30-39中任一项所述的方法，其中所述癌症选自krukenberg瘤、胃癌、胰腺癌、胆囊癌、黑色素瘤、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、头颈部鳞状细胞癌(HNSCC)、经典霍奇金淋巴瘤(cHL)、原发性纵膈大B细胞淋巴瘤(PMBCL)、尿路上皮癌(UC)、食管癌、宫颈癌、肝癌、Merkel细胞癌、肾细胞癌(RCC)、结直肠癌(mCRC)、乳 腺癌。
41.如项30-40中任一项所述的方法，其中所述抗PD-1抗体/PD-L1抗体选自Nivolumab、Pembrolizumab、Cemiplimab、卡瑞利珠单抗、特瑞普利单抗、信迪利单抗、替雷利珠单抗、赛帕利单抗(GLS-010)、替雷利珠单抗或其组合。
42.如项30-41中任一项所述的方法，其中所述在先治疗包括药物治疗、手术治疗、放疗或其组合；所述药物治疗包括给予化学药和/或生物药；优选的，所述在先治疗包括给予奥拉帕利、乐伐替尼、卡博替尼、阿西替尼、伊匹木、铂类的化疗药物、培美曲塞、依托泊苷、紫杉烷类化合物、贝伐珠单抗、瑞戈非尼、罗替尼、阿帕替尼、乐伐替尼、呋喹替尼、瑞格菲尼或其组合。
43.如项30所述的方法，其中给予患有所述癌症的患者至少一个周期的细胞治疗产品进行治疗；优选的，给予患有所述癌症的患者1-3个周期的细胞治疗产品进行治疗。
44.如项36-43中任一项所述的方法，其中所述嵌合抗原受体(CAR)包括：
(i)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD3ζ；
(ii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；
(iii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
(iv)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
45.如项43或44所述的方法，其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、2ⅹ10 8个细胞/kg或2ⅹ10 7个细胞/kg患者体重；或细胞治疗产品中细胞的剂量不超过约1ⅹ10 11个细胞/患者、1ⅹ10 10个细胞/患者、5ⅹ10 9个细胞/患者、2ⅹ10 9个细胞/患者或1ⅹ10 9个细胞/患者。
46.如项45所述的方法，其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患者体重至2ⅹ10 7个细胞/kg患者体重，或约1ⅹ10 6个细胞/kg患者体重至2ⅹ10 7个细胞/kg患者体重；或者
每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 7个细胞至5ⅹ10 9个细胞/患者、约1ⅹ10 7个细胞至2ⅹ10 9个细胞/患者、或约1ⅹ10 7个细胞至1ⅹ10 9个细胞/患者；或者每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 8个细胞至5ⅹ10 9个细胞/患者、约1ⅹ10 8个细胞至2ⅹ10 9个细胞/患者、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的剂量为约2.5ⅹ10 8个细胞至5ⅹ10 8个细胞/患者。
47.如项43-46中任一项所述的方法，其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患者化学药物、生物药物、放疗或其组合。
48.如项47所述的方法，其中所述预处理在给予所述细胞治疗产品前1-8天实施；优选地，在给予所述细胞治疗产品前2-6天实施；优选地，化学药物、生物药物、放疗或其组合连续使用不超过4天。
49.如项47或48所述的方法，其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。
50.如项49所述的方法，其中所述微管蛋白抑制剂是紫杉烷类化合物；优选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；更优选所述紫杉烷类化合物为白蛋白结合型紫杉醇。
51.如项49或50所述的方法，其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300-700mg/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。
52.如项49-51中任一项所述的方法，其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。
53.如项37-44中任一项所述的方法，其中所述抗原结合结构域具有：
SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3、SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2、SEQ ID NO:6所示的LCDR3；或者
SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2、SEQ ID NO:18所示的HCDR3、SEQ ID NO:19所示的LCDR1、SEQ ID NO:20所示的LCDR2、SEQ ID NO:21所示的LCDR3；或者
SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2、SEQ ID NO:29所示的HCDR3、SEQ ID NO:30所示的LCDR1、SEQ ID NO:31所示的LCDR2、SEQ ID NO:32所示的LCDR3；
或所述抗原结合结构域具有：
SEQ ID NO:7所示的重链可变区和SEQ ID NO:9所示的轻链可变区；或者
SEQ ID NO:22所示的重链可变区和SEQ ID NO:23所示的轻链可变区；或者
SEQ ID NO:33所示的重链可变区和SEQ ID NO:34所示的轻链可变区；
或所述抗原结合结构域具有：SEQ ID NO:14、24、35、或37所示的序列。
54.如项36-53中任一项所述的方法，其中所述的嵌合抗原受体具有SEQ ID NO:11、12、13、25、26、36、38、52任一所示氨基酸序列、或具有SEQ ID NO：58、59、60、61、62、63、64、65、66或67任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽、或具有SEQ ID NO：70、71、72、73、74、75、76、77、78或79任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽。
55.如项43-54中任一项所述的方法，其中在每个周期给予所述细胞治疗产品之前，对所述患者的指示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。
56.如项30-55中任一项所述的方法，其中在给予所述细胞治疗产品治疗后，所述患者没有显示严重CRS，或没有显示超过3级的神经毒性。
57.如项30-56中任一项所述的方法，其中所述使用抗PD-1抗体或抗PD-L1抗体治疗癌症失败的患者中的至少一部分，优选至少40％，更优选至少50％对所述细胞治疗产品有应答。
58.如项57所述的方法，其中所述应答是指ORR大于40％，优选大于50％。
为了实现上述目的，本发明提供的技术方案如下：
在本发明的第一方面，提供了细胞治疗产品用于在先治疗中使用抗PD-1抗体或抗PD-L1抗体治疗失败的癌症治疗的用途。
在一优选实施方式中，在先治疗中使用抗PD-1抗体或抗PD-L1抗体治疗失败的部分患者对细胞治疗产品有应答。
在一优选实施方式中，至少40％的在先治疗中使用抗PD-1抗体或抗PD-L1抗体治疗失败的患者对细胞治疗产品有应答。
在一优选实施方式中，至少50％的在先治疗中使用抗PD-1抗体或抗PD-L1抗体治疗失败的患者对细胞治疗产品有应答。
在一优选实施方式中，所述应答是指ORR大于40％；优选的，大于50％。
在一优选实施方式中，所述在先治疗中，还使用到化学药或生物药；优选的，所述化学药或生物药是奥拉帕利、乐伐替尼、卡博替尼、阿西替尼、伊匹木单抗、铂类的化疗药物、培美曲塞、依托泊苷、紫杉烷类化合物、贝伐珠单抗、瑞戈非尼。
在一优选实施方式中，所述的抗PD-1抗体选自Nivolumab、Pembrolizumab、Cemiplimab、卡瑞利珠单抗、特瑞普利单抗、信迪利单抗、替雷利珠单抗、赛帕利单抗(GLS-010)、替雷利珠单抗；优选的，所述抗PD-1抗体选自Nivolumab、pembrolizumab， 卡瑞利珠单抗，特瑞普利单抗，信迪利单抗、替雷利珠单抗。
在一优选实施方式中，所述的抗PD-L1抗体选自Atezolizumab、Durvalumab、Avelumab、舒格利单抗(CS1001)。
在一优选实施方式中，所述细胞治疗产品含有表达有外源受体的免疫效应细胞，优选的，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、或干细胞衍生的免疫效应细胞。
在一优选实施方式中，所述免疫效应细胞是选自T细胞，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)和T细胞抗原耦合器(TAC)。
在一优选实施方式中，所述外源受体选自嵌合抗原受体(CAR)。
在一优选实施方式中，所述免疫效应细胞是选自T细胞、NK细胞、NKT细胞。
在一优选实施方式中，所述细胞治疗产品特异性识别肿瘤抗原，优选的，所述肿瘤抗原选自CD19、CD20、CD22、CD30、Mesothelin、BCMA、EGFR、EGFRvIII、PSMA、Muc1、claudin18.2、GPC3、IL13RA2、SLAMF7、GPRC5D，LILRB4；更优选的，所述肿瘤抗原选自CD19、BCMA、Claudin18.2。
在一优选实施方式中，所述癌症选自胃癌、胰腺癌、胆囊癌、黑色素瘤、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、头颈部鳞状细胞癌(HNSCC)、经典霍奇金淋巴瘤(cHL)、原发性纵膈大B细胞淋巴瘤(PMBCL)、尿路上皮癌(UC)、食管癌、宫颈癌、肝细胞癌、Merkel细胞癌、肾细胞癌(RCC)、结直肠癌(mCRC)、乳腺癌。
在一优选实施方式中，所述癌症选自胃癌、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、尿路上皮癌(UC)、肝癌、黑色素瘤。
在一优选实施方式中，给予所述患者至少一个周期的细胞治疗产品进行治疗；优选的，给予患者1-3个周期的细胞治疗产品进行治疗。
在一优选实施方式中，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，或细胞治疗产品中细胞剂量不超过约1ⅹ10 11个细胞/人。优选的，每个周期给予的细胞治疗产品中细胞的剂量不超过约2ⅹ10 8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 10个细胞/人。更优选地，每个周期给予的细胞治疗产品中细胞的剂量不超过约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 9个细胞/人。
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。
在一优选例中，每个周期给予的细胞治疗产品中细胞剂量是1ⅹ10 7个细胞至1ⅹ10 9个细胞，优选的，为1ⅹ10 8个细胞至1ⅹ10 9个细胞，更优选的，为2.5ⅹ10 8个细胞至5ⅹ10 8个细胞。
在一优选实施方式中，待在先周期的细胞产品治疗后在体内检测不到后，再给予后续周期的治疗。
在一优选实施方式中，在所述在先周期的细胞产品给予完毕后的30天后，再给予后续周期的细胞治疗产品。
在一优选实施方式中，所述的后续周期给予的细胞产品的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。
在一优选实施方式中，所述不同周期给予的细胞产品剂量不同或相同。
在一优选实施方式中，每个周期的细胞治疗产品分为1-5次给予，优选的，分为1-3次给予。
在一优选实施方式中，每个周期的细胞治疗产品在5天内完成全部给药，更优选的，在3天内，完成全部给药。
在一优选实施方式中，每次的细胞治疗产品在1小时内完成输注，优选的，在30min内完成输注，更优选的，在4-30min内完成输注。
在一优选实施方式中，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，上述(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％。
在一优选实施方式中，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。
在一优选实施方式中，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。
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，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。
在一优选例中，氟达拉滨的给予量约为30-60mg/天、或约30-50mg/m 2/天。
在一优选例中，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。
在一优选例中，环磷酰胺的给予量约为300-700mg/天、或约300-550mg/天、或约300-500mg/天。
在一优选例中，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90-120mg/天。
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。
在一优选例中，所述外源受体包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述胞外抗原结合结构域包含特异性识别肿瘤抗原的抗体或其片段，其中，所述抗体或其片段具有：
SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3、SEQ ID NO：4所示的LCDR1、SEQ ID NO：5所示的LCDR2、SEQ ID NO：6所示的LCDR3；或者
SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；或者
SEQ ID NO：27所示的HCDR1、SEQ ID NO：28所示的HCDR2、SEQ ID NO：29所示的HCDR3、SEQ ID NO：30所示的LCDR1、SEQ ID NO：31所示的LCDR2、SEQ ID NO： 32所示的LCDR3。
优选的，所述抗体或其片段具有：
SEQ ID NO：7所示的重链可变区和SEQ ID NO：9所示的轻链可变区；或者
SEQ ID NO：22所示的重链可变区和SEQ ID NO：23所示的轻链可变区；或者
SEQ ID NO：33所示的重链可变区和SEQ ID NO：34所示的轻链可变区。
在一优选例中，所述外源受体的胞外抗原结合结构域具有SEQ ID NO：14、24、35、或37所示的序列。
在一优选例中，所述的嵌合抗原受体具有SEQ ID NO：11、12、13、25、26、36、38、52任一所示氨基酸序列。
在一优选实施方式中，给予所述细胞治疗产品之前，所述受试者没有接受过细胞治疗。
在一优选实施方式中，在给予所述细胞治疗产品之前，所述受试者还接受了药物治疗、手术治疗、放疗、或任意的组合。
在一优选实施方式中，所述药物治疗包括化学药治疗或生物药治疗。
在一优选实施方式中，所述化学药物治疗包括安罗替尼，阿帕替尼，乐伐替尼，呋喹替尼，瑞格菲尼。
在一优选实施方式中，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。
在一优选实施方式中，所述的指示肿瘤负荷的指标为：所述受试者的肿瘤细胞总数、或者所述受试者的器官中肿瘤细胞总数、或者所述受试者的组织中的肿瘤细胞总数、或者肿瘤的质量或体积，或者肿瘤转移的程度、或者肿瘤数量。
在一优选实施方式中，基于所述评价的结果，确定细胞治疗产品的给药剂量。
在一优选实施方式中，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。
在一优选实施方式中，在给予细胞治疗产品后，细胞治疗产品在所述受试者体内增殖。
在本发明的第二方面，还提供了一种外源受体修饰的免疫效应细胞治疗肿瘤的方法，所述方法包括在给予肿瘤患者免疫效应细胞之前，先给予预处理药物，所述预处理包括环磷酰胺、氟达拉滨和微管蛋白抑制剂。
在一优选实施方式中，所述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。
在一优选实施方式中，氟达拉滨的给予量约为30-60mg/天、或约30-50mg/m 2/天。
在一优选实施方式中，所述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。
在一优选实施方式中，环磷酰胺的给予量约为300-700mg/天、或约300-550mg/天、或约300-500mg/天。
在一优选实施方式中，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90-120mg/天。
在一优选实施方式中，每种预处理药物连续使用不超过4天。
在一优选实施方式中，所述环磷酰胺给予2-3次；
在一优选实施方式中，所述氟达拉滨给予1-2次。
在一优选实施方式中，所述微管蛋白抑制剂给予一次。
在一优选实施方式中，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。
在一优选实施方式中，所述外源受体包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域，所述胞外抗原结合结构域包含特异性识别肿瘤抗原的抗体或其片段，其中，所述抗体或其片段具有：
SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3、SEQ ID NO：4所示的LCDR1、SEQ ID NO：5所示的LCDR2、SEQ ID NO：6所示的LCDR3；或者
SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；或者
SEQ ID NO：27所示的HCDR1、SEQ ID NO：28所示的HCDR2、SEQ ID NO：29所示的HCDR3、SEQ ID NO：30所示的LCDR1、SEQ ID NO：31所示的LCDR2、SEQ ID NO：32所示的LCDR3。
优选的，所述抗体或其片段具有：
SEQ ID NO：7所示的重链可变区和SEQ ID NO：9所示的轻链可变区；或者
SEQ ID NO：22所示的重链可变区和SEQ ID NO：23所示的轻链可变区；或者
SEQ ID NO：33所示的重链可变区和SEQ ID NO：34所示的轻链可变区。
在一优选实施方式中，所述外源受体的胞外抗原结合结构域具有SEQ ID NO：14、24、35、或37所示的序列。
在一优选实施方式中，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、或干细胞衍生的免疫效应细胞。
在一优选实施方式中，所述免疫效应细胞是选自T细胞，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)和T细胞抗原耦合器(TAC)。
在一优选实施方式中，所述外源受体选自嵌合抗原受体(CAR)；优选的，所述的嵌合抗原受体具有SEQ ID NO：11、12、13、25、26、36、38、52任一所示氨基酸序列。
在一优选实施方式中，所述免疫效应细胞是选自T细胞、NK细胞、NKT细胞。
在一优选实施方式中，所述的外源受体修饰的免疫效应细胞特异性识别肿瘤抗原，优选的，所述肿瘤抗原选自CD19、CD20、CD22、CD30、Mesothelin、BCMA、EGFR、EGFRvIII、PSMA、Muc1、claudin18.2、GPC3、IL13RA2、SLAMF7、GPRC5D，LILRB4；更优选的，所述肿瘤抗原选自CD19、BCMA、Claudin18.2。
在一优选实施方式中，所述肿瘤选自胃癌、胰腺癌、胆囊癌、黑色素瘤、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、头颈部鳞状细胞癌(HNSCC)、经典霍奇金淋巴瘤(cHL)、原发性纵膈大B细胞淋巴瘤(PMBCL)、尿路上皮癌(UC)、食管癌、宫颈癌、肝细胞癌、Merkel细胞癌、肾细胞癌(RCC)、结直肠癌(mCRC)、乳腺癌。
在一优选实施方式中，所述肿瘤选自胃癌、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、尿路上皮癌(UC)、肝癌、黑色素瘤。
在一优选实施方式中，给予所述免疫效应细胞之前，所述受试者没有接受过细胞治疗。
在一优选实施方式中，在给予所述免疫效应细胞之前，所述受试者还接受了药物治疗、手术治疗、放疗、或任意的组合。
在一优选实施方式中，所述药物治疗包括化学药治疗或生物药治疗；优选的，所述化学药物治疗包括安罗替尼，阿帕替尼，乐伐替尼，呋喹替尼，瑞格菲尼。
在一优选实施方式中，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，或细胞治疗产品中细胞剂量不超过约1ⅹ10 11个细胞/人。优选的，每个周期给予的免疫效应细胞的剂量不超过约2ⅹ10 8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 10个细胞/人，更优选地，每个周期给予的免疫效应细胞的剂量不超过约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 9个细胞/人。
在一优选实施方式中，每个周期给予的免疫效应细胞的剂量为约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免疫效应细胞剂量是1ⅹ10 7个细胞至1ⅹ10 9个细胞，优选的，为1ⅹ10 8个细胞至1ⅹ10 9个细胞，更优选的，为2.5ⅹ10 8个细胞至5ⅹ10 8个细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在 此不再一一累述。
附图说明
图1是肿瘤负荷相对基线最佳变化瀑布图
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现当癌症患者的在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体并且治疗失败时(依照RECIST 1.1的进展性疾病PD)，并不会对细胞治疗产品的疗效产生影响，仍有至少40％的患者对细胞治疗产品有响应。示例性，实体瘤中胃癌/食管胃结合部腺癌、krukenberg瘤、肝癌、胆囊癌患者在抗PD-1抗体和/或抗PD-L1抗体治疗失败后，对细胞治疗产品(例如，CAR-T细胞)有响应。其中对抗PD-1抗体和/或抗PD-L1抗体治疗失败后的胃癌/食管胃结合部腺癌患者，施用CAR-T细胞治疗后的疾病控制率达约79.2％。对抗PD-1抗体和/或抗PD-L1抗体治疗失败后的肝癌患者，施用CAR-T细胞治疗后的疾病控制率达约54.5％。
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发明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。
本发明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。
本申请中，有些请求保护的主题以范围形式呈现，应当理解，范围形式的描述仅仅是为了方便和简洁，并且不应被解释为对所要求保护的主题的范围的硬性限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，在提供有范围上限和范围下限的范围中，在该范围的上限和下限之间的每个中间值均被包括在要求保护的主题内，所述范围的上下限也属于请求保护的主题的范围。
本文中所述任何浓度范围、百分比范围、比例范围或整数范围应理解为包括在所述范围内的任何整数，以及在合适情况下，其分数(例如整数的十分之一与百分之一)的数值，除非另外指出。
本文使用的术语“约”是指本技术领域技术人员容易知晓的各值的通常误差范围。本文中述及“约”值或参数，包括(并描述)指向该值或参数本身的实施方式。例如，关于“约X”的描述包括“X”的描述。例如，“约”可意指按照在该领域中的实际的标准偏差在1以内 或多于1。或者“约”可意指至多20％、或至多10％，或至多5％、或至多1％的范围。例如，约5mg可包括在4.5mg与5.5mg之间的任何数目。当在申请案与申请专利范围中提供特定值或组成时，除非另外指出，否则“约”应假定为在该特定值或组成的可接受误差范围内。
在描述氨基酸或者核酸序列时，本专利中“具有”特定序列应当被理解为涵盖了特定序列的变体。本专利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。
本文所述的“在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败”是指在先治疗时，患者有接受PD-1和/或PD-L1的抗体治疗，或者有接受包含有PD-1和/或PD-L1的抗体的疗法的治疗，在先治疗不能控制疾病进展(如依照实体肿瘤的疗效评价标准RECIST1.1，疾病发展为疾病进展(PD)的情况)。
本文所述的“包含有PD-1和/或PD-L1的抗体的疗法”是指在治疗中根据治疗需要，除了使用PD-1和/或PD-L1的抗体，还使用有其他的药物或疗法。例如，在治疗中除了使用PD-1和/或PD-L1的抗体，还使用了放疗、干细胞移植等；或者在治疗中除了使用PD-1和/或PD-L1的抗体，还使用了其他化学药或生物药，如奥拉帕利、乐伐替尼、卡博替尼、阿西替尼、伊匹木单抗、铂类的化疗药物、培美曲塞、依托泊苷、紫杉烷类化合物、贝伐珠单抗等或其组合。
肿瘤患者的治疗会是一个漫长的过程。所谓的一线、二线、三线、后线(包括四线、五线等)治疗，其实指的是肿瘤治疗方案的选择和应用顺序。一线治疗，一般指的是肿瘤诊断以后，对于不能进行手术切除的肿瘤患者而言的第一轮系统性化学药和/或生物药治疗；对于能进行手术切除的肿瘤患者术后(加或不加辅助治疗)复发的第一轮系统性化学药和/或生物药治疗。二线治疗，一般指的是在一线治疗失败后(包括一线治疗有效后患者再次出现肿瘤进展)，需要更换不同的治疗方案。三线治疗，一般指的是二线治疗失败后，再次换用其它方案的治疗。大多数肿瘤一般到三线时，可选择的药物和有效的治疗方案就越来越少了。
PD1和/或PD-L1抗体治疗在不同肿瘤中已经被批准于不同线数治疗。现在临床上对于PD-L1阳性或阴性的肿瘤患者都有给予PD1和/或PD-L1抗体治疗的情况，在肿瘤治疗的一线、二线、三线、或后线(四线、五线等)都有单独给予PD1和/或PD-L1抗体治疗、或PD1和/或PD-L1抗体治疗联合其他药物的临床应用。
现有治疗胃癌的方案，如施用化疗药物(紫杉类、伊立替康等)、靶向药物(阿帕替尼)和/或免疫检查点抑制剂(纳武利尤单抗)的客观缓解率在约2％-11.2％，mPFS在约2-3个月左右，mOS在约6个月。本发明针对在PD-1和/或PD-L1抗体治疗失败的胃癌/食管胃结合部腺癌患者的二线、三线或后线治疗中，施用细胞治疗产品(示例性，CAR-T细胞)，显示了更高的缓解率约50％、疾病控制率约79.2％、及更长的无进展生存期和总生存期，使患者获得显著临床受益：CAR-T治疗后达PR的12例患者中，受试者mDOR为6.3月；其中，6例患者总生存期达约11个月以上，并且其中4例患者仍在生存随访期。本发明还观察到在PD-1和/或PD-L1抗体治疗失败的胃癌/食管胃结合部腺癌患者，施用CAR-T细胞治疗后疗效评估为完全缓解(CR)，并在持续的临床观察中。
Krukenberg瘤是一种主要来源于胃、肠道的转移性恶性卵巢癌，恶性度高，临床特征缺乏特异性，发现时常伴全身转移，且常合并腹水及弥漫型腹膜转移，对化疗不敏感，治疗效果不佳，预后差。本发明针对PD-1和/或PD-L1抗体治疗失败的Krukenberg瘤患者，施用细胞治疗产品(示例性，CAR-T细胞)，显示了更长的无进展生存期和总生存期，使患者获得显著临床受益：CAR-T细胞治疗后达到PR，无疾病进展生存期已超过4个月，并且仍在持续缓解期。
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囊癌的无进展生存期均低于约6个月(1.8-5.6个月)。本发明针对PD-1和/或PD-L1抗体治疗失败的胆囊癌患者，施用细胞治疗产品(示例性，CAR-T细胞)，显示了更长的无进展生存期和总生存期，使患者获得显著临床受益：CAR-T细胞治疗达到SD，无疾病进展生存期已达7个半月，并且仍在持续稳定期。
原发性肝癌是目前我国第4位常见恶性肿瘤及第2位肿瘤致死病因。由于大多数患者疾病处于晚期，肿瘤的恶化程度较高，治疗效果很不理想。本发明针对PD-1和/或PD-L1抗体治疗失败的肝癌患者，施用细胞治疗产品(示例性，CAR-T细胞)，显示了更长的无进展生存期和总生存期，使患者获得显著临床受益：CAR-T细胞治疗后疾病控制率约54.5％；达到疾病控制的6例患者的无疾病进展生存期均值约4.4月，其中4例患者总生存其超过1年，这4例中的3例仍在生存随访期。
本文所述的“细胞治疗产品”是指具有医学上治疗作用的细胞产品，如免疫细胞治疗产品，是采用人体自身或供体来源的免疫细胞，经过体外操作，包括但不限于分离、纯 化、培养、扩增、诱导分化、活化、遗传操作、细胞(系)的建立、冻存复苏等，再输入(或植入)到患者体内，通过诱导、增强或抑制机体的免疫功能而治疗疾病的产品。包括过继性细胞治疗(Adoptive cell therapy,ACT)产品、治疗性疫苗等。免疫细胞治疗产品类型主要包括但不限于细胞因子诱导的杀伤细胞(Cytokine-Induced Killer，CIK)、肿瘤浸润淋巴细胞(Tumor Infiltrating Lymphocyte，TIL)、嵌合抗原受体T细胞(Chimeric Antigen Recptor T-cell，CAR-T)、T细胞受体修饰的T细胞(T Cell Receptor-engineering T-cell，TCR-T)、自然杀伤细胞(Natural Killer，NK)细胞、树突状细胞(Dendritic cell，DC)、巨噬细胞等。
术语“抗PD-1的抗体”是指能够识别或结合PD-1的抗体，例如是能够识别或结合人PD-1(胞外段SEQ ID NO：39)的抗体。“识别或结合人PD-1的抗体”可以是以KD值1.0ⅹ10 -8mol/l或更低的结合亲和力特异性结合人PD1抗原。在一个实施方案中，抗PD-1的抗体可以是Nivolumab、Pembrolizumab、Cemiplimab、卡瑞利珠单抗、特瑞普利单抗、信迪利单抗、替雷利珠单抗或其组合。
术语“抗PD-L1的抗体”是指能够识别或结合PD-L1的抗体，例如是能够识别或结合人PD-L1(胞外段SEQ ID NO：40)的抗体。“识别或结合人PD-L1的抗体”可以是以KD值1.0ⅹ10 -8mol/l或更低的结合亲和力特异性结合人PD-L1抗原。在一个实施方案中，抗PD-L1的抗体可以是Atezolizumab、Durvalumab、Avelumab、舒格利单抗(CS1001)或其组合。
术语“结合”指选择性地结合靶标。表达与肿瘤抗原结合的受体的免疫效应细胞可以识别肿瘤抗原。
“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，例如表面等离振子共振技术(GE-Healthcare，Uppsala，Sweden)。
本文中所述的“剂量”，可以是以重量为基础计算的剂量，也可以是以体表面积(BSA)为基础计算的剂量，也可以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患者体重为基础所计算出的对于患者给予的剂量，例如mg/kg，细胞个数/kg等。以BSA为基础计算的剂量是以患者的表面积为基础所计算出的对患者给予的剂量，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患者每个周期或每次给予的剂量，例如mg/人，细胞个数/人。
本文中所述的“给药次数”是指每个周期内给予细胞治疗产品或每个预处理药物的频率。例如，可以按照下列来给予氟达拉滨：连续4日每日给予一次、连续3日每日给予一次、 连续2日每日给予一次、或1日给予一次。可以按照下列来给予环磷酰胺：连续4日每日给予一次、连续3日每日给予一次、连续2日每日给予一次、或1日给予一次。可以按照下列来给予白蛋白结合型紫杉醇：连续4日每日给予一次、连续3日每日给予一次、连续2日每日给予一次、或1日给予一次。示例性，每个周期内给予的细胞治疗产品可以一次性给予；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。
本文所述的“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(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)：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)。客观缓解率(ORR)的定义为最佳疗效达到确认的CR或PR的受试者占所在分析人群的百分比，疾病控制率(DCR)定义为最佳疗效获得确认的缓解(CR+PR)和疾病稳定(SD)的病例数占分析集的百分比。无进展生存期(PFS)被定义为从本专利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。在数据截止日期既没有进展也没有死亡的受试者将在他们最后一次充分的肿瘤评估之日被删除。将使用用于治疗人群的描述性统计(平均值、标准偏差、中值、最小值和最大值)来总结PFS。除了使用Kaplan-Meier方法基于观察值和删除值二者计算的中值，所有其他统计量(平均值、标准偏差、最小值和最大值)将仅基于观察值计算。总生存期(OS)被测量为从本专利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。
在一些实施例中，有应答包括本专利治疗方法处理后出现CR或PR。在一些实施例中，有应答包括本专利治疗方法处理后出现CR、PR或SD；在一些实施例中，有应答是指接受本专利治疗方法的受试者能获得明显的临床获益，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。在一些实施例中，有应答是指ORR达到10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或以上、或100％。在一些实施例中，有应答是指DCR达到10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或以上、或100％。
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。
本文中所述的细胞或细胞群针对某个标志物呈“阳性”，是指细胞的某个标志物(通常是表面标志物)的可通过检测证明其存在的，如该标志物通过流式细胞术检测到存在表面表达，例如，通过用特异性地结合至所述标志物的抗体染色，并检测所述抗体，其中，所述染色可被流式细胞术以一定水平检测到，所述水平显著高于采用同种型匹配的对照在其它条件相同的情况下进行相同步骤时检测到的染色的水平，和/或基本相似于已知对所述标志物呈阳性的细胞的水平，和/或显著高于已知对所述标志物呈阴性的细胞的水平。具体实施例中，所述标志物包括但不限于肿瘤抗原。示例性的，针对标志物(肿瘤抗原CLDN18.2或GPC3)为阳性的胃癌/食管胃结合部腺癌、krukenberg瘤、肝癌、胆囊癌患者在PD-1和/或PD-L1抗体治疗失败后，再接受CAR-T细胞治疗。
本文中所述的细胞或细胞群针对某个标志物呈“阴性”指，所述细胞不具有某个标志物或者尽管具有但检测不到，如该标志物通过流式细胞术检测不到表面表达，或者尽管检测到存在表面表达，例如，当通过用特异性地结合至所述标志物的抗体染色，并检测所述抗体来检测表面表达时，所述染色可被流式细胞术以一定水平检测到，所述水平显著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/或显著低于已知对所述标志物呈阳性的细胞的水平，和/或基本相似于已知对所述标志物呈阴性的细胞的水平。具体实施例中，肿瘤细胞中的标志物PD-L1呈阳性或阴性的患者，在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体失败后，都可以施用CAR-T细胞治疗。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。还可以包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。
术语“细胞”可以指人或非人的、来源于动物的细胞。
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，并且通常是人。在给予入组细胞治疗研究之前，受试者已经采用PD-1抗体和/或PD-L1抗体的治疗，对PD-1抗体或者PD-L1抗体是响应或非响应的。
术语“免疫效应细胞”是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、树突细胞、CIK细胞、巨噬细胞、肥大细胞、或干细胞衍生的免疫效应细胞等。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是天然的T细胞和/或经多能干细胞诱导产生的T细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是自体NK细胞或同种异体NK 细胞。
术语“经人工改造的具有免疫效应细胞功能的细胞”是指不具有免疫效应的细胞，或经人工改造或接受刺激物刺激后，该细胞获得了免疫效应细胞功能的细胞。如293T细胞，经人工改造，使其具有免疫效应细胞的功能；如干细胞，经体外诱导，使其分化成免疫效应细胞。
在一些情况下，“T细胞”可以是来自骨髓的多能干细胞，在胸腺内分化成熟成为具有免疫活性的成熟的T细胞。在一些情况下，“T细胞”可以是具有特定表型特征的细胞群，或不同表型特征的混合细胞群体，如“T细胞”可以是包含至少一种T细胞亚群的细胞：记忆性干细胞样T细胞(stem cell-like memory T cells，Tscm细胞)、中心记忆T细胞(Tcm)、效应性T细胞(Tef、Teff)、调节性T细胞(tregs)和/或效应记忆T细胞(Tem)。在一些情况下，“T细胞”可以是某种特定亚型的T细胞，如γδT或αβT细胞。
T细胞可以从许多来源获得，包括但不限于PBMC(外周血单个核细胞)、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下，可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从个体收集的血液获得T细胞。在一个实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆分子并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。或者，可以从健康供体或来自诊断患有癌症的患者衍生细胞。T细胞可以是任何类型的T细胞，可以是任何发育阶段，包括但不限于CD4+/CD8+双阳性T细胞、CD4+辅助T细胞，例如Th1和Th2细胞、CD8+T细胞(例如细胞毒T细胞)、肿瘤浸润细胞、记忆T细胞、天真T细胞等。
本文所用的术语“外源”指的是一个核酸分子或多肽、细胞、组织等没有在生物体自身内源性表达，或表达水平不足以实现过表达时具有的功能。
术语“内源”是指一个核酸分子或多肽等来自生物体自身。
术语“嵌合受体”，即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。嵌合受体包括但不限于：嵌合抗原受体(CAR)、嵌合T细胞受体(TCR)、T细胞抗原耦合器(TAC)。
术语“嵌合抗原受体”(CAR)包括胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域。在具体实施例中，CAR的胞外结合结构域包含scFv。胞内信号传导结构域包括刺激性分子和/或共刺激性分子的功能信号传导结构域；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结构域、或其功能片段或衍生物。在一个方面，刺激性分子包括与T细胞受体复合体结合的CD3ζ链；在一个方面，胞内信 号传导结构域进一步包括一种或多种共刺激性分子的功能性信号传导结构域，例如4-1BB(即CD137)、CD27和/或CD28。在某些实施例中，多肽组彼此连接。示例性，所述CAR的胞外抗原结合结构域包括具有SEQ ID NO：14、24、35、37、58、59、60、61、62、63、64、65、66、67、70、71、72、73、74、75、76、77、78或79所示的序列。
术语“T细胞受体(T cell receptor，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，V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3。具体实施例中，所述修饰的TCR胞外抗原结合结构域包括识别肿瘤抗原的抗体。示例性，所述TCR包括具有SEQ ID NO：14、24、35、37、58、59、60、61、62、63、64、65、66、67、70、71、72、73、74、75、76、77、78或79所示序列。
术语“TCR融合蛋白”或“TFP”包括来源于TCR的各种蛋白的重组蛋白，包含抗原结合结构域，一般能够：i)结合至靶细胞上的表面抗原；ii)当定位于T细胞时与完整TCR复合物的其它多肽组分相互作用。在一些实施方案中，该T细胞是CD4+T细胞、CD8+T细胞或CD4+/CD8+T细胞。在一些实施方案中，该TFP T细胞是NKT细胞。在一些实施方案中，该TFP T细胞是γδT或αβT细胞。具体实施例中，所述TFP的抗原结合结构域包括识别肿瘤抗原的抗体。示例性，所述TFP包括具有SEQ ID NO14、24、35、37、58、59、60、61、62、63、64、65、66、67、70、71、72、73、74、75、76、77、78或79所示序列。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、抗原结合结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。具体实施例中，所述TAC抗原结合结构域包括识别肿瘤抗原的抗体。示例性，所述TAC包括具有14、24、35、37、58、59、60、61、62、63、64、65、66、67、70、71、72、73、74、75、76、77、78或79所示序列。
术语“抗原结合结构域”是指免疫球蛋白分子(与“抗体”可互换使用)或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，可以含有抗体，如全人抗体，嵌合抗体，人源化抗体，鼠源抗体，也可以是识别抗原的配体。
术语“抗体”在本文中以最广义使用并且包括各种抗体结构，包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要其显示所需的抗原结合活性即可。本文中的术语“抗体”指免疫系统的抗原结合蛋白。“抗体”包括具有抗 原结合区域的完整的全长抗体及其中“抗原结合部分”或“抗原结合区域”保留的其任何片段、或其单链例如单链可变片段(scFv)。每条重链由重链可变区(本文缩写为VH)和重链恒定区组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区组成。术语“可变区或可变结构域”是指参与抗体抗原结合的抗体重链或轻链的结构域。重链和轻链的可变区含有与抗原相互作用的结合结构域。
抗体片段包括但不限于：(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段(Ward等，1989，Nature 341：544-546)；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点(Bird等，1988，Science 242：423-426；Huston等，1988，Proc.Natl.Acad.Sci.U.S.A 85:5879-5883)；(vii)双特异性单链Fv二聚体(PCT/US92/09965)；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段(Tomlinson等，2000，Methods Enzymol.326:461-479；WO94/13804；Holliger等，1993,Proc.Natl.Acad.Sci.U.S.A 90:6444-6448)；和(ix)与相同或不同抗体遗传融合的scFv(Coloma&Morrison,1997,Nature Biotechnology 15,159-163)。
在具体实施例中，抗原结合结构域可以含有scFv、Fv、Fab、Fab'、Fab'-SH、F(ab')2、单结构域片段、或结合抗原的天然配体，以及它们的任何衍生物。在一些方面，细胞外抗原结合结构域(例如scFv)，可以包含对抗原特异性的轻链CDR和/或重链CDR。。
在一优选实施方式中，抗原结合结构域包含的抗体或其片段可以具有以下CDR序列：SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3、SEQ ID NO：4所示的LCDR1、SEQ ID NO：5所示的LCDR2、SEQ ID NO：6所示的LCDR3；或者SEQ ID NO：16所示的HCDR1、SEQ ID NO：17所示的HCDR2、SEQ ID NO：18所示的HCDR3、SEQ ID NO：19所示的LCDR1、SEQ ID NO：20所示的LCDR2、SEQ ID NO：21所示的LCDR3；或者SEQ ID NO：27所示的HCDR1、SEQ ID NO：28所示的HCDR2、SEQ ID NO：29所示的HCDR3、SEQ ID NO：30所示的LCDR1、SEQ ID NO：31所示的LCDR2、SEQ ID NO：32所示的LCDR3。
在一优选实施方式中，抗原结合结构域包含的抗体或其片段可以具有以下重链可变区和轻链可变区序列：SEQ ID NO：7所示的重链可变区和SEQ ID NO：9所示的轻链可变区；或者SEQ ID NO：22所示的重链可变区和SEQ ID NO：23所示的轻链可变区；或者SEQ ID NO：33所示的重链可变区和SEQ ID NO：34所示的轻链可变区。
在具体实施例中，抗原结合结构域包含的抗体或其片段可以具有SEQ ID NO：58、59、60、61、62、63、64、65、66、67、70、71、72、73、74、75、76、77、78或79所示 的scFv序列。
在一些情况下，CAR的胞外区包括识别肿瘤抗原的抗原结合结构域和连接片段(也称为铰链、间隔区)。连接片段可被认为是用于向细胞外抗原结合结构域提供柔性的CAR的一部分。连接片段可以是任何长度的。示例性，在一个实施例中，CAR包括一个铰链域，示例性，CD8α铰链，具有SEQ ID NO：41所示。
术语“跨膜域”可以将外源受体锚定在细胞的质膜上。例如，可以采用CD28、CD8的跨膜域，具有SEQ ID NO：42或43所示序列。术语“信号传导结构域”是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用，经由确定的信号传导途径调节细胞的活性。胞内信号传导结构域可以包括分子的全部细胞内部分、或全部天然胞内信号传导结构域、或其功能片段或衍生物。示例性，胞内信号传导结构域具有SEQ ID NO：44、45、或46所示的序列。
刺激性分子的功能信号传导结构域，也称为“初级信号域”，以刺激性方式调节CAR或TCR复合物的初始活化。一方面，初级信号域由例如TCR/CD3复合物与加载了肽的MHC分子的结合而引发，由此介导T细胞反应(包括但不限于，增殖、活化、分化等)。以刺激性方式起作用的初级信号域可以包含免疫受体酪氨酸激活基序或ITAM的信号传导基序。在本申请中尤其有用的包含ITAM的初级信号域的例子包括但不限于，源自CD3ζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε，CD5，CD22，CD79a，CD79b，CD278(也称作“ICOS”)和CD66d的序列。
术语“共刺激分子”指与细胞刺激信号分子，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号，具有如SEQ ID NO：44或45所示的序列。为T细胞上的关联结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，包括但不限于细胞增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激分子包括但不限于，MHC I类分子、BTLA和Toll配体受体、以及OX40、CD2、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)和4-1BB(CD137)。
在某些情况下，胞内信号传导结构域可被设计成包含若干可能的刺激性分子和/或共刺激信号结构域：示例性，第一代CAR包含单个刺激结构域，例如CD3ζ链；第二代CAR包含一个共刺激结构域，例如包含CD3ζ链与CD28、或包含CD3ζ链与4-1BB；第三代CAR包含两个共刺激结构域(第三代CAR)，例如包含CD3ζ链与CD28和OX40、或包含CD3ζ链与CD28和4-1BB。
具体实施例中，CAR包含可选的前导序列。一方面，CAR在胞外抗原识别结构域的N端还包含前导序列，其中前导序列任选地在CAR的细胞加工和定位至细胞膜的过程中从抗原识别结构域(例如scFv)切下。在一实施例中，前导序列包含具有SEQ ID NO:53所示 序列。
具体实施例中，本发明靶向GPC3-CAR的抗原结合结构域包括具有SEQ ID NO：70、71、72、73、74、75、76、77、78或79所示的序列，跨膜域包括具有SEQ ID NO：42或43所示的序列，胞内信号传导结构域包括具有SEQ ID NO：44和46、或SEQ ID NO：45和46、或SEQ ID NO：45、44和46所示的序列。示例性的，针对PD1和/或PD-L1抗体治疗失败的肝癌患者，施用所述GPC3-CAR-T细胞，显示了更长的无进展生存期和总生存期，使患者获得显著临床受益。本发明所述实施例中，针对PD1和/或PD-L1抗体治疗失败的肝癌患者，施用所述GPC3-CAR-T细胞和转录因子RUX3多肽，部分患者治疗后评估为PR，显示显著临床受益。具体实施例中，所述GPC3-CAR-T表达RUNX3多肽。
术语“RUNX3(Runt-related transcription factor 3)”，也称为成骨相关转录因子3，是转录因子runt结构域家族的成员，GenBank Accession No.NP_004341.1的氨基酸序列。在细胞生长、发育、凋亡和信号转导及其生物学效应有重要作用。
术语“激活”和“活化”可互换使用，可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指T细胞逐步活化的过程。该活化过程受第一刺激信号和共刺激信号的共同调控。“T细胞活化”或“T细胞激活”指被刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。使用CD3/CD28磁珠，体外抗原刺激或者体内抗原刺激都会对T细胞的活化程度和持续时间造成影响。在一个实施例中，所述工程化T细胞与含特定靶抗原肿瘤细胞共孵育或病毒感染后活化。
术语“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，预防疾病的发生或复发、减轻症状、减少疾病的任何直接或间接病理学结果、防止转移、减缓疾病进展速率、改善或减轻疾病状态，和减轻或改善预后。本文中所用的“延迟肿瘤的发展"表示推迟、阻碍、减缓、减慢、稳定、遏制和/或拖延所述肿瘤的发展。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(对于未发展所述肿瘤的个体而言)。例如，晚期癌症，例如转移的发展，可被延迟。
在一些实施方式中，所提供的细胞治疗产品用于延迟肿瘤发展或减缓肿瘤进展。
本文中所用的“抑制”指在与其它情况下的相同病症相比时或者相较于另一病症，功能或活性的减少。
“有效量”或“治疗有效量”是指足以预防或治疗个体疾病(癌症)的剂量。治疗性或预防性使用的有效剂量取决于所治疗疾病的阶段和严重程度、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轮或多轮、或多次给予本申请所述细胞治疗产品。
术语“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本发明的过度增生性病症是指癌症(包括肿瘤)。本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
术语“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。
术语“预处理”是指在给予产品(如细胞治疗产品)之前，先给予患者其他的药物或治疗，使得患者的身体状态更适合产品治疗。如在给予细胞治疗产品(如CAR-T细胞)之前，先给予化学药、生物药、放疗或其组合进行预处理。优选的，先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。
在一些实施方式中，进行预处理之后，患者的淋巴细胞相比预处理之前减少约50％、55％、60％、65％或者70％。
术语“化学药”或“化学药物”是指通过化学合成的方法制备的抗癌药物，可以是传统的化疗药，例如烷化剂、抗代谢药、抗癌抗生素等，也可以是靶向药，如阿法替尼、爱乐替尼等。
术语“生物药”或“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。
术语“化疗药”是指一种治疗肿瘤的药物。化疗药物可杀灭肿瘤细胞、或抑制肿瘤细胞生长、迁移或侵袭等。这些药物能作用在肿瘤细胞生长繁殖的不同环节上，抑制或杀死肿瘤细胞。化疗药物治疗是目前治疗肿瘤的主要手段之一。
在一些实施方式中，本文所述的化疗药，是指化学治疗使用的药物，是指对恶性肿瘤有防治作用的化学药物。化疗药包括但不限于烷化剂、抗代谢物、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。在一些实施方式中，所述抗代谢物例如包括但不限于抗代谢物如卡莫氟、替加氟、喷司他丁、去氧氟尿苷、曲美沙特、氟达拉滨、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。在一些实施方式中，所述烷化剂例如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。
细胞治疗产品的用途或方法
本发明提供了细胞治疗产品用于在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败的癌症(与“肿瘤”互换使用)治疗的用途或方法。。
在一些实施方式中，至少40％的在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败的患者对细胞治疗产品有应答。
在一些实施方式中，至少50％的在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败的患者对细胞治疗产品有应答。
在一些实施方式中，给予细胞治疗产品后，患者的ORR大于40％；优选的，大于50％。
就本文而言，“剂量”是指给予的药品的量，如，在一个周期内给予的细胞治疗产品的量，或在一个周期内每次给予的细胞治疗产品的量，或在一个周期内，每次给予的预处理药物的量。
在一些实施方式中，给予患者至少一个周期的细胞治疗产品进行治疗。在具体实施方式中，每个周期的剂量可以是相同的，也可以是不同的。
在一些实施方式中，在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，在一个周期内的细胞治疗产品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。
在一些实施方案中，当一个周期内的剂量分多次给予受试者时，每次给予的细胞治疗产品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、以及医师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。在一些实施方式中，每次给予的剂量呈递增趋势。在一些实施方式中，每次给予的剂量呈递减趋势。在 一些实施方式中，每次给予的剂量呈先递增后递减的趋势。在一些实施方式中，每次给予的剂量呈先递减后递增的趋势。
给予多周期的细胞治疗产品是指具有多个时间段，在每个时间段内给予一定总量的细胞治疗产品。在一些实施方式中，每个时间段的间隔时间是一致的。在一些实施方式中，每个时间段的间隔时间是不一致的。在一些实施方式中，给予至少2个周期的细胞治疗产品，如给予2、3、4、5、6、7、8、9或10个周期的细胞治疗产品。
在一些实施方式中，每个周期给予的细胞剂量及给予次数，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，也可以是改善治疗效果。
在一些实施方案中，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，或细胞治疗产品中细胞剂量不超过约1ⅹ10 11个细胞/人。优选的，每个周期给予的细胞治疗产品中细胞的剂量不超过约2ⅹ10 8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 10个细胞/人。更优选地，每个周期给予的细胞治疗产品中细胞的剂量不超过约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。
在一些实施方案中，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。
在一些实施方案中，每个周期给予的细胞治疗产品中细胞剂量是约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。
在一些实施方案中，每个周期给予的细胞治疗产品之前，先对患者进行预处理，所述预处理包括给予所述受试者化疗剂和/或放疗。在一些实施方案中，所述放疗可以时全身辐射治疗，也可以是局部辐射治疗。
在一些实施方案中，预处理的化疗剂可以是环磷酰胺、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。
本文所述的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。
术语“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。在一些实施 方式中，所述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。在一些实施方式中，所述紫杉烷类化合物的所述桥亚甲基苯并环癸烯母核结构中不含不饱和键。在一些实施方式中，所述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。在一些实施方式中，所述紫杉烷类化合物的给药方式为注射给药。
在一些实施方案中，预处理的化疗剂可以是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。
在一些实施方案中，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是白蛋白结合型紫杉醇。
在一些实施方案中，，所述嘧啶类抗肿瘤药物选自5-氟尿嘧啶、吉美嘧啶、奥替拉西钾、双呋氟尿嘧啶、卡莫氟、去氧氟尿苷、卡培他滨。
在一些实施方案中，氟达拉滨的给予量约为20-60mg/天、或约30-55mg/天、或约30-50mg/天。
在一些实施方案中，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。
在一些实施方案中，环磷酰胺的给予量约为200-700mg/天、或约300-600mg/天、或约300-560mg/天、或约300-550mg/天、或约300-500mg/天。。
在一些实施方案中，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。
在一些实施方案中，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。
在一些实施方案中，每种化疗剂连续使用不超过4天。
在一些实施方案中，环磷酰胺给予2-3次。在一些实施方案中，氟达拉滨给予1-2次。在一些实施方案中，紫杉烷类化合物给予一次。
在一些实施方式中，本文所提供的方法或用途是基于申请人观察到的受试者对所给予的细胞治疗产品的响应和治疗结果得出。如对在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败的CLDN18A2阳性癌症的受试者给予不同的剂量、或不同周期数的靶向CLDN18A2的CAR-T细胞，CAR-T细胞依然能够呈现较好的治疗结果。如对在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败的GPC3阳性癌症的受试者给予不同的剂量、或不同周期数的靶向GPC3的CAR-T细胞，CAR-T细胞依然能够呈现较好的治疗结果。如 对在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败的GPC3阳性癌症的受试者给予不同的剂量、或不同周期数的表达RUNX3的GPC3-CAR-T细胞，依然能够呈现较好的治疗结果。例如，这些治疗结果包括患者的生存、缓解或病情的稳定。
在一些实施方式中，本文所述的用途或方法还包括监控受试者对细胞治疗产品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。在一些实施方式中，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。
在一些实施方式中，通常在给予细胞治疗产品之后，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。在一些实施方案中，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。在一些实施方案中，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％。
在一些实施方式中，在基于在先周期的细胞治疗产品之后，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。
在一些实施方式中，选择给予在后周期的时机，以避免已经给予的在先周期所产生的宿主免疫应答可能诱导使得在后周期的功效降低。在一些方面，在出现宿主免疫应答，例如针对给予的细胞和/或其表达的嵌合抗原受体的适应性或特异性，例如体液或细胞介导的免疫应答之前给予在后周期。在一些实施方式中，在例如通过一种或多种专门的检测方法可以检测到这种响应之前给予在后周期。通常在针对细胞的宿主适应性免疫应答未被检测到、尚未建立和/或尚未达到一定水平或程度或阶段时给予一个或多个在后周期。
在一些实施方式中，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，在一些实施方案中，在后周期是疾病巩固剂量。
在一些实施方式中，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。
术语“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。在一些实施方案中，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。
所述肿瘤标志物又称肿瘤标记物，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。
本发明提供的用途或方法包括向在先治疗中使用抗PD-1抗体和/或抗PD-L1抗体治疗失败的癌症患者，给予至少一个周期的细胞治疗产品，如表达CAR、TCR、TFP、或TAC的免疫效应细胞或其组合。
本文中所述的“肿瘤抗原”包括但不限于促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2，Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏 死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P4501B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。在一些实施方案中，所述肿瘤抗原为EGFR、EGFRvIII，磷脂酰肌醇蛋白聚糖3、claudin 18.2或BCMA。
在一些实施方式中，肿瘤抗原可以是CD19、CD20、CD22、CD30、Mesothelin、BCMA、EGFR、EGFRvIII、PSMA、Muc1、claudin18.2、GPC3、IL13RA2中的至少一种。
在一些实施方式中，肿瘤抗原可以是CD19、BCMA、Claudin18.2、GPC3中的至少一种。
在一些实施方式中，肿瘤或癌症可以是结肠癌，胆囊癌，krukenberg瘤，直肠癌，肾细胞癌，肝癌，肺癌，小细胞性肺癌，小肠癌，食道癌，黑色素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，霍奇金氏病，非霍奇金淋巴瘤，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤，肿瘤 血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症的转移性病灶。在一些实施例中，胃癌包括食管胃结合部腺癌。在一些实施例中，肝癌包括肝细胞癌。
CLD18和CLD18阳性肿瘤
密蛋白18(Claudin 18，CLD18)分子(Genbank登记号：剪接变体1(Claudin 18.1、CLD18A1)：NP_057453、NM016369，以及剪接变体2(Claudin 18.2、CLD18A2)：NM_001002026、NP_001002026)是分子量约为27,9/27,72kD的内在跨膜蛋白。密蛋白是位于上皮和内皮的紧密连接中的内在膜蛋白。具体实施例中，是CLD18A2包括具有SEQ ID NO:56所示序列。CLD18A2在若干癌症类型中强烈表达，包括胃癌/食管胃结合部腺癌、食管、胆囊癌、胰腺癌、肺癌、krukenberg瘤。表达主要是在这些适应症的腺癌亚型中。
GPC3和GPC3阳性肿瘤
本文所用的“GPC3”或“磷脂酰肌醇蛋白聚糖3”是Glypican家族的一员，其在调控细胞生长和分化方面起重要作用。具体实施例中，是GPC3包括具有SEQ ID NO:68所示序列。GPC3异常表达与多种肿瘤的发生、发展关系密切，如在肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌等中呈现异常表达。
给予细胞治疗产品的方法是已知的。在一些实施方式中，细胞治疗产品(例如CAR-T细胞)可以通过自体回输的方式给予患者。因此，在一些方面中，所述细胞治疗产品源自需要所述细胞治疗产品的受试者，并在体外进行基因修饰后给予同一受试者。在一些实施方式中，细胞治疗产品(例如CAR-T细胞)可以通过同种异体回输的方式给予患者。因此，在另一些方面中，所述细胞治疗产品源自健康供者，供者和回输细胞的受试者不同。在这些方面中，将细胞治疗产品给予遗传学组织相容性高的受试者，如供者和受试者是遗传学相同或相似的。在一些实施方式中，所述供者和受试者具有相同的HLA类别或超类型。
所述细胞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(subconjectval)注射、结膜下(subconjuntival)注射、巩膜上腔注射、球后注射、眼周注射或球周递送。在一些实施方式中，它们通过胃肠外、肺内的和鼻内，以及如果希望局部治疗，也可以病灶内给予。腹膜外输注包括肌内、静脉内、动脉内、腹膜内或皮下给予。
对于预防或治疗疾病，合适剂量可取决于治疗的疾病类型，嵌合抗原受体或细胞类型，疾病的严重程度和疾病病程、该细胞是否给予用于预防性或治疗性目的、先前治疗、受试者的临床史和对所述细胞的响应，以及主治医师的判断。在一些实施方式中，所述组 合物和细胞适合在一个时间点或以一系列治疗来给予受试者。
在给予细胞治疗产品之前给予预处理，可改善免疫效应细胞治疗的效果。将每个周期中给予免疫效应细胞(例如CAR-T细胞)第一次输注之日定为第0日，在给予免疫效应细胞输注前给予预处理。在一些实施方式中，在过继细胞或者免疫效应细胞给予前至少2、3、4、5、6、7、8、9、或10天进行预处理。在一些实施方式中，在给予细胞治疗产品前2-8天实施预处理；优选的，在给予细胞治疗产品前3-6天实施预处理。如上所述，将对患者每个周期给予CAR-T细胞疗法之日指定为第0日。在一些实施方式中，在给予CAR-T细胞疗法之前的任何时候可给予预处理。
在一些实施方式中，可以通过调整预处理中给予的化疗药或治疗的给予时间或次数，使细胞治疗产品的疗效达到最大。
在一些实施方式中，所述环磷酰胺给予2-3次；或所述氟达拉滨给予1-2次；或所述白蛋白结合型紫杉醇给予一次。
在一些实施方式中，每种化疗剂连续使用不超过4天。
在一些实施方式中，氟达拉滨的给予量约为20-60mg/天、或约30-60mg/天、或约30-55mg/天、或约30-50mg/天。
在一些实施方式中，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。
在一些实施方式中，环磷酰胺的给予量约为200-700mg/天、或约300-700mg/天、或约300-600mg/天、或约300-560mg/天、或约300-550mg/天、或约300-500mg/天。
在一些实施方式中，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。
在一些实施方式中，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。
将对患者每轮给予CAR-T细胞疗法之日指定为第0日。在一些实施方式中，在第-6和-5日对患者给予氟达拉滨。在一些实施方式中，在第-5和-4日对患者给予氟达拉滨。在一些实施方式中，在第-4和-3日对患者给予氟达拉滨。在一些实施方式中，在第-6、-5、-4和-3日对患者给予氟达拉滨。在一些实施方式中，在第-7、-6、-5和-4日对患者给予氟达拉滨。在一些实施方式中，在第-6、-5、-4和-3日对患者给予环磷酰胺。在一些实施方式中，在第-6、-5和-4日对患者给予环磷酰胺。在一些实施方式中，在第-5、-4和-2日对患者给予环磷酰胺。在一些实施方式中，在第-5、-4和-3日对患者给予环磷酰胺。在一些实施方式中，在第-4、-3和-2日对患者给予环磷酰胺。在一些实施方式中，在第-4和-3日对患 者给予环磷酰胺。在一些实施方式中，在第-6和-5日对患者给予环磷酰胺。在一些实施方式中，在第-5和-4日对患者给予环磷酰胺。在一些实施方式中，在第-2日、第-3日、第-4日、第-5日、或第-6日，对患者给予紫杉烷类化合物(如白蛋白结合型紫杉醇)。
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。
在某些实施方式中，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。在一些实施方式中，在给予氟达拉滨前对患者给予环磷酰胺。在一些实施方式中，在给予氟达拉滨后对患者给予环磷酰胺。在一些实施方式中，在给予氟达拉滨前对患者给予白蛋白结合型紫杉醇。在一些实施方式中，在给予氟达拉滨后前对患者给予白蛋白结合型紫杉醇。在一些实施方式中，在给予环磷酰胺前对患者给予白蛋白结合型紫杉醇。在一些实施方式中，在给予环磷酰胺后对患者给予白蛋白结合型紫杉醇。
可利用任何途径(包括静脉注射(IV))给予氟达拉滨、环磷酰胺和白蛋白结合型紫杉醇。
在此处所述的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患者的不良时间。本发明的范围包括对患者给予组成物以使这些不良事件中的某些事件减少。在某些实施方式中，该方法包含对患者给予生理盐水。可在给予环磷酰胺和/或氟达拉滨前或后，或在给予环磷酰胺和/或氟达拉滨前与后对患者给予生理盐水。在一些实施方式中，在每个输注日给予环磷酰胺和/或氟达拉滨前，及在给予环磷酰胺和/或氟达拉滨后对患者给予生理盐水。另外，还可对患者给予佐剂与赋形剂。例如，美司钠(2-巯基乙烷磺酸钠)。此外，还可对患者给予外源性细胞激素。
给予受试者细胞治疗产品后可以多种已知方法中的任一种来测量工程改造的免疫效应细胞群的生物活性。用以评估的参数包括：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)。在某些实施方式中，工程改造的免疫效应细胞破坏靶细胞的能力可采用本领域已知的任何合适的方法来检测，例如描述于如下文献的细胞毒性试验，例如，Kochenderfer等，J.Immunotherapy,32(7):689-702(2009)和Herman等J.Immunological Methods,285(1):25-40(2004)。在某些实施方式中，也可通过测定某些细胞因子，例如CD107a，IFNγ，IL-2和TNF的表达和/或分泌，来测量免疫效应细胞的生物活性。在一些方面中，生物活性通过评估临床结果，例如肿瘤负荷或负载的减少。在一些方面中，评估肿瘤标志物的降低。在一些方面，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。
如本文所用，“在先周期”是相对“在后周期”而言，在在后周期给药前的给药周期，如在完成第一周期给药后，又给予了第二周期的给药和第三周期的给药，第二周期相对于第一周期来说，第一周期是在先周期，第二周期是在后周期，第三周期相对于第二周期来说，第二周期是在先周期，第三周期是在后周期。
在具体实施方式中，细胞治疗产品的剂量是指细胞治疗产品中含有的细胞数量。
在一些实施方式中，在后周期的量或大小足以减少肿瘤负荷或其指标，和/或疾病或病症的一种或多种症状。在一些实施方式中，剂量具有有效改善受试者生存的大小，例如，改善受试者存活，例如，诱导受试者存活、无复发存活或无事件存活持续至少1个月或至少1、2、3、4或5年。
在一些实施方式中，与给予在先周期或在后周期之前时刻相比，在在后周期后，肿瘤负荷，包括肿瘤大小、肿瘤体积和/或肿瘤质量减少至少等于或约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％或更多。
在其他实施方式中，以在后周期给予的免疫效应细胞的数量低于在在先周期中给予的免疫效应细胞的数量。
在一些方面，基于一个或多个标准来确定是否给予在后周期的治疗，例如根据受试者对在先周期治疗的反应，例如，化疗，受试者的肿瘤负荷，例如肿瘤体积，大小或度，程度，或转移类型，阶段和/或中晚期癌症常见的并发症，例如癌性胸腹水和/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。
基于本发明的教导，本领域技术人员应该理解，本发明具体公开的剂量是本发明人经过研究得到是安全有效的剂量，本领域技术人员，例如临床医师可以根据各种实际情况，例如患者的肿瘤负荷、患者本身的身体状况等因素决定每个周期的给药剂量；如果需要进一步给予在后周期，本领域技术人员可以根据，例如给予免疫效应细胞之后的肿瘤负荷变化来决定在后周期的剂量。
在一些方面，基于在给予在先周期的治疗之前的肿瘤负荷来确定在后周期给予的细胞治疗产品的剂量。在一些实施方式中，例如，其中在先周期导致肿瘤负荷减少或降低，或已经低于特定阈值量或水平，例如高于其表示毒性结果的风险增加，在后周期的剂量增加。
在一些情况下，即使在接受在先周期治疗后，受试者的肿瘤负荷没有降低，在后周期的细胞治疗产品的剂量也可以增加。
可利用任何途径(包括静脉注射(IV))给予T细胞(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。
在一些实施方式中，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。
在一些实施方式中，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。
在一些实施方式中，在后周期给药的时间是从在先周期的细胞治疗产品完成(在先周期的总剂量输注完成日定为0日)开始起算。
在一些实施方式中，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。
在一些实施方式中，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。
在一些实施方式中，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。
在一些实施方式中，在在先周期和在后周期之间监测肿瘤负荷。
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。例如，在一些实施方式中，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。在一些这样的测定中，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。
在一些方面，例如，在给予在先周期或在后周期的治疗后，检测或测量免疫应答的数量和程度。
在一些方面，在给予在先周期的治疗之前，或给予在先周期和在后周期的治疗之间，或在给予在后周期的治疗之后，测量或检测疾病负荷。
在一些实施方案中，在给予在先周期的治疗后，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％。在一些方面，给予在后周期的治疗后会进一步降低疾病负荷，例如在后周期治疗前后的疾病负荷相比，治疗之后的降低约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％。
在一些实施方式中，与其他方法相比，通过该方法改善受试者的无进展存活率或总体存活率。例如，在一些实施方式中，在先周期治疗后1个月，受试者的无进展存活率大于约10％、大于约20％、大于约30％、大于约40％、大于约50％、大于约60％、大于约70％、大于约80％、大于约90％、或大于约95％。在一些方面，总存活率大于约10％、大于约20％、大于约30％、大于约40％，大于约50％，大于约60％，大于约70％，大于约80％，大于约90％或大于约95％。在一些实施方式中，受试者表现出无进展生存，无复发生存或总生存期至少约1个月，或至少约2、3、4、5、6、7、8、9、10、11个月、或1、2、3、4、5、6、7、8、9或10年。在一些实施方式中，无进展生存期的时间，例如大于或约1个月，或至少约2、3、4、5、6、7、8、9、10、11个月、或1、2、3、4、5、6、7、8、9或10年。
在一些实施方式中，通过该方法进行的治疗，与其它方法相比，复发的概率降低。例如，在一些实施方式中，在先周期后1个月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％。
由细胞表达的外源受体
细胞可以通过已知的生物学技术进行外源修饰，表达外源性的受体，例如外源表达TCR受体，嵌合抗原受体(CAR)和T细胞融合蛋白(T cell fusionprotein，TFP)和T细胞抗原耦合器(T cell antigen coupler，TAC)。
示例性的外源受体，如包括CAR的序列可以具有如SEQ ID NO：11、12、13、25、26、36、38、52任一所示氨基酸序列。示例性，本申请的CAR包括具有SEQ ID NO：58、59、60、61、62、63、64、65、66或67所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合。示例性，如SEQ ID NO：58所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：58所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：58所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：59所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：59所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：59所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：60所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：60所示序列与SEQ ID NO： 81所示序列顺序连接，SEQ ID NO：60所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：61所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：61所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：61所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：62所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：62所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：62所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：63所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：63所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：63所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：64所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：64所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：64所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：65所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：65所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：65所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：66所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：66所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：66所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：67所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：67所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：67所示序列与SEQ ID NO：82所示序列顺序连接。
示例性，本申请的CAR包括具有如SEQ ID NO：70所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：70所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：70所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：71所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：71所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：71所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：72所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：72所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：72所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：73所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：73所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：73所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：74所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：74所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：74所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：75所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：75所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：75所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：76所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：76所示序列与SEQ ID NO：81所示序 列顺序连接，SEQ ID NO：76所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：77所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：77所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：77所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：78所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：78所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：78所示序列与SEQ ID NO：82所示序列顺序连接；如SEQ ID NO：79所示序列与SEQ ID NO：80所示序列顺序连接，或SEQ ID NO：79所示序列与SEQ ID NO：81所示序列顺序连接，SEQ ID NO：79所示序列与SEQ ID NO：82所示序列顺序连接。示例性，如SEQ ID NO：83、84或85所示的序列。
术语“多肽”、“肽”、“蛋白”和“蛋白质”可互换使用，表示氨基酸残基的聚合物，并且不限于最小长度。包括提供的受体和其他多肽(例如接头或肽)的多肽可以包括氨基酸残基，包括天然和/或非天然氨基酸残基。该术语还包括多肽的表达后修饰，例如糖基化、唾液酸化、乙酰化和磷酸化。在一些方面中，所述多肽可包含对原始或天然序列进行的修饰，只要该蛋白质保留所需活性即可。这些修饰可以是有意的，例如通过定点诱变或者可以是意外的，例如通过产生蛋白质的宿主突变或PCR扩增所致的误差。
在本发明中，表达外源受体的细胞一般是真核细胞，例如哺乳动物细胞，并且通常是人细胞。在一些实施方式中，细胞衍生自血液、骨髓、淋巴或淋巴器官，是免疫系统的细胞，如先天或适应性免疫的细胞，例如，骨髓或淋巴样细胞，包括淋巴细胞，一般是T细胞、NKT细胞和/或NK细胞。其他示例性的细胞包括干细胞，如专能(multipotent)和多能(pluripotent)干细胞，包括诱导性多能干细胞(iPSC)。细胞一般是原代细胞，如直接从受试者分离和/或从受试者分离并冷冻的细胞。在一些实施方式中，细胞包括T细胞或其他细胞类型的一个或多个亚组，如全T细胞群、CD4+细胞T细胞、CD8+细胞T细胞及其亚群，如由功能、活化状态、成熟、分化潜力、扩增、再循环、定位，和/或持久能力、抗原特异性、抗原受体类型、特定器官或隔室中的存在、标志物或细胞因子分泌概况，和/或分化程度限定的T细胞。关于待治疗的受试者，细胞可以是同种异体和/或自体的。制备所述表达外源受体的细胞的方法包括现成的方法。在一些实施方式中，该方法包括从受试者分离细胞，对其进行制备、处理、培养和/或工程改造，并且在冷冻保存前或后将其再导入同一受试者。
T细胞和/或CD4+T细胞和/或CD8+T细胞的亚型和亚群包括：原初T(TN)细胞、效应T细胞(TEFF)、记忆T细胞及其亚型，例如干细胞记忆T(TSCM)、中心记忆T(TCM)、效应记忆T(TEM)、或最终分化的效应记忆T细胞、肿瘤-浸润性淋巴细胞(TIL)、不成熟T细胞、成熟T细胞、辅助性T细胞、细胞毒性T细胞、粘膜相关的非变体T(MAIT)细胞、天然产生的和过继性调节T(Treg)细胞、辅助性T细胞，例如TH1细胞、TH2细胞、TH3细胞、TH17 细胞、TH9细胞、TH22细胞、滤泡辅助性T细胞、α/βT细胞，和δ/γT细胞或其组合。
在一些实施方式中，所述细胞是自然杀伤(NK)细胞。在一些实施方式中，所述细胞是单核细胞或粒细胞，例如，骨髓细胞、巨噬细胞、嗜中性粒细胞、树突细胞、肥大细胞、嗜酸性粒细胞，嗜碱性粒细胞或其组合。
在一些实施方式中，所述细胞包括通过遗传工程改造导入的一种或多种核酸，并由此表达所述核酸的重组或经遗传工程改造的产物。在一些实施方式中，核酸是异源性的，通常不存在于细胞或从该细胞获得的样品中，例如从另一个生物体或细胞获得的样品，或例如通常不在工程改造中的细胞和/或这类细胞来源的生物体中发现。在一些实施方式中，核酸不是天然存在的，如核酸不是自然中发现的，包括包含编码来自多种不同细胞类型的各种结构域的核酸的嵌合组合，如CAR，嵌合细胞因子受体。
用于遗传工程改造的方法和载体
本发明还提供了用于产生表达嵌合抗原受体的遗传工程改造的细胞的方法、组合物和试剂盒。遗传工程改造一般涉及将编码所述重组或经工程改造的部分的核酸导入细胞，例如，通过病毒(例如慢病毒、逆转录病毒、AAV)转导、转染或转化进行。
术语“工程化”是指应用细胞生物学和分子生物学的原理和方法，通过某种工程学手段，在细胞整体水平或细胞器水平上，按照人们的意愿来改变细胞内的遗传物质或获得细胞产品的一门综合科学技术。在一个实施例中，所述工程化是指核酸的一个或多个改变，例如生物体基因组内的核酸。在一个实施例中，所述工程化是指基因的改变、添加和/或缺失。在一个实施例中，所述工程细胞或所述工程化细胞还可以指具有加入、缺失和/或改变的基因的细胞。
在一些实施方式中，基因转移通过如下方式进行：首先，刺激细胞，例如，通过将其与刺激物合并，所述刺激物诱导响应，例如增殖、存活和/或活化，例如，通过细胞因子或活化标志物的表达来检测，然后转导该活化的细胞，并且在培养物中扩增至足以供于临床应用的数量。
在一些方面中，细胞还经工程改造以促进细胞因子或其他因子的表达。
在一些实施方式中，采用重组感染性病毒颗粒将重组核酸转移进入细胞，例如，源自猿病毒40(SV40)、腺病毒、腺相关病毒(AAV)的载体。在一些实施方式中，采用重组慢病毒载体或逆转录病毒载体，例如γ-逆转录病毒载体，将重组核酸转移进入T细胞。
在一些实施方式中，通过电穿孔将重组核酸转移进入T细胞。在一些实施方式中，通过转位将重组核酸转移进入T细胞(参见例如，Manuri等，(2010)Hum Gene Ther 21(4):427-437；Sharma等，(2013)Molec Ther Nucl Acids 2,e74；和Huang等，(2009)Methods Mol Biol 506:115-126)。在免疫细胞中引入和表达遗传物质的其它方法包括磷酸钙转染(例如， 描述于《分子生物学方案新编》(Current Protocols inMolecular Biology)，约翰威利父子公司(John Wiley&Sons)，纽约州纽约)、原生质体融合、阳离子脂质体介导的转染；钨颗粒促进的微粒轰击(Johnston，Nature,346:776-777(1990))；和磷酸锶DNA共沉淀(Brash等，Mol.Cell Biol.,7:2031-2034(1987))。
免疫效应细胞的制备
在一些实施方式中，经工程化改造的细胞的制备包括一种或多种培养和/或一个或多个制备步骤。用于导入编码转基因受体(例如，CAR)的核酸的细胞可从样品(例如生物样品，例如获自或源自受试者的样品)分离。在一些实施方式中，从中分离细胞的受试者是患有一定疾病或病症或需要细胞治疗或将给予细胞治疗的受试者。在一些实施方式中，受试者是需要特定治疗性介入的人，例如，需要过继细胞疗法或免疫效应细胞疗法的人，用于该疗法的细胞是经分离、加工和/或经工程改造的。在一些实施方式中，所述细胞是原代细胞，例如，原代人细胞。所述样品包括直接取自受试者的组织、体液和其它样品，以及获自一个或多个处理步骤，例如分离、离心、遗传工程改造(例如采用病毒载体的转导)、清洗和/或孵育的样品。所述生物样品可以是直接获自生物来源的样品或经处理的样品。生物样品包括但不限于，体液，例如血液、血浆、血清、脑脊髓液、滑膜液、尿液和汗液，组织和器官样品，包括源自它们的经处理的样品。
在一些方面中，从中衍生或分离细胞的样品是血液或血液源性的样品或者，是或源自单采或白细胞去除术产物。示例性样品包括全血、PBMC、白细胞、骨髓、胸腺、组织活检物、肿瘤、白血病、淋巴瘤、淋巴结、肠相关的淋巴样组织、粘膜相关的淋巴样组织、脾、其它淋巴样组织、肝、肺、胃、肠、结肠、肾、胰腺、乳腺、骨、前列腺、子宫颈、睾丸、卵巢、扁桃体或其它器官，和/或源自其中的细胞。在细胞治疗(例如，过继细胞治疗或免疫效应细胞治疗)的情况中，样品包括来自自体和同种异体来源的样品。
在一些实施方式中，所述细胞源自细胞系，例如，T细胞系。在一些实施方式中，细胞获自异种异体来源，例如，获自小鼠、大鼠、非人灵长类，或猪。
在一些实施方式中，细胞的分离包括一个或多个制备和/或不基于亲和性的细胞分离步骤。在一些实例中，细胞在一种或多种物质的存在下经清洗、离心和/或孵育，例如，以移除不需要的组分，富集所需组分，裂解或移除对具体物质敏感的细胞。在一些实例中，细胞基于一种或多种性质，例如密度、粘附性质、尺寸、对具体组分的敏感性和/或抗性被分离。在一些实例中，例如，通过单采或白细胞去除术，获得来自受试者循环血液的细胞。在一些方面中，所述样品包括淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其它有核血液白细胞、血红细胞，和/或血小板和在一些方面中包括与血红细胞和血小板不同的细胞。
在一些实施方式中，所述方法包括基于密度的细胞分离方法，例如，通过裂解血红细胞或者不裂解红细胞并通过Percoll或Ficoll梯度离心外周血或者单采样品或白细胞去除术样品制备获得外周血单个核细胞(PBMC)。
在一些实施方式中，所述分离方法包括，基于细胞中一种或多种特定分子，例如表面标志物，例如，表面蛋白质、胞内标志物或核酸的表达或存在来分离不同的细胞类型。在一些实施方式中，可采用基于此类标志物进行分离的任何已知方法。在一些实施方式中，所述分离是基于亲和性或基于免疫亲和性的分离。例如，在一些方面中，所述分离包括基于细胞的一种或多种标志物(通常是细胞表面标志物)的表达或表达水平来分离细胞和细胞群，例如，通过与特异性地结合至此类标志物的抗体或结合伴侣孵育，随后通常是清洗步骤，和从尚未结合至所述抗体或结合伴侣的那些细胞分离已结合所述抗体或结合伴侣的细胞。
所述分离不需导致具体细胞群或表达具体标志物的细胞的100％的富集或移除。
例如，在一些方面中，T细胞的特定亚群，例如一种或多种表面标志物阳性细胞或表达高水平的一种或多种表面标志物的细胞，例如，CD3+、CD28+、CD62L+、CCR7+、CD27+、CD127+、CD4+、CD8+、CD45RA+和/或CD45RO+T细胞，通过正或负选择技术分离。
例如，CD3+、CD28+T细胞可以采用CD3/CD28连接的磁珠(例如，DYNA珠M-450CD3/CD28T细胞扩增器)来正选择。
在一些实施方式中，T细胞通过对在非T细胞(例如B细胞、单核细胞或其它血液白细胞)上表达的标志物(例如CD14)进行负选择来从PBMC样品分离。在一些方面中，使用CD4+或CD8+选择步骤来分离辅助性CD4+和CD8+细胞毒性T细胞。通过针对一种或多种原初、记忆和/或效应T细胞亚群上表达或以相对较高的程度表达的标志物进行正或负选择，可将此类CD4+和CD8+群进一步分选成亚群。
在一些实施方式中，CD8+细胞针对原初、中心记忆、效应记忆和/或中心记忆干细胞进行进一步富集或消耗，例如通过基于与对应亚群相关联的表面抗原进行正或负选择。在一些实施方式中，进行针对中心记忆T(TCM)细胞的富集，以增加功效，例如以改善长期存活、扩增和/或给予后的植入，在一些方面中，其在此类亚群中特别强健。
在一些实施方式中，制备方法包括：冷冻步骤，例如，在分离、孵育和/或工程改造之前或之后，冻存所述细胞。在一些实施方式中，所述冷冻和后续融化步骤移除粒细胞，并且，在某种程度上，移除细胞群中的单核细胞。在一些实施方式中，例如，在清洗步骤以移除血浆和血小板之后，将所述细胞悬浮于冷冻溶液中。在一些方面中，可采用任何各种已知冷冻溶液和参数。一个实例涉及采用包含20％DMSO和8％人血清白蛋白(HAS)的PBS或其它合适的细胞冷冻培养基。然后，其用培养基1:1稀释，从而DMSO和HSA的终浓度分别是10％和4％。然后，一般以程控降温装置按照既定的程序或者原理如1°/分钟的速 率将细胞冷冻至-80℃或者-90℃，并贮存在液氮储罐的汽相中。
在一些实施方式中，在遗传工程改造之前或与遗传工程改造一起孵育和/或培养细胞。孵育步骤可包括培养、培育、刺激、活化，和/或增殖。在一些实施方式中，在刺激条件或刺激性试剂存在下孵育细胞或组合物。这类条件包括设计成在群中诱导细胞增殖、繁殖、活化，和/或存活以模拟抗原接触，和/或引发细胞用于遗传工程改造，如用于导入重组抗原受体的那些。
在一些实施方式中，刺激条件或试剂包括一种或多种物质，例如，配体，其能够活化TCR复合物的胞内信号转导结构域。在一些方面中，所述物质开启或启动T细胞中的TCR/CD3胞内信号转导级联反应。此类物质可包括抗体，例如对TCR组分和/或共刺激受体具有特异性的那些，例如，抗CD3、抗CD28，例如，其结合至固体支持物，例如珠，和/或一种或多种细胞因子。任选地，扩增方法还可包括如下步骤：向培养基(例如，以至少约0.5ng/ml的浓度)添加抗CD3和/或抗CD28抗体。在一些实施方式中，刺激剂包括1L-2和/或IL-15和/或IL-7和/或IL-21，例如，至少约10单位/mL浓度的IL-2。
在某实施方式中，通过用抗原刺激天然或抗原特异性T淋巴细胞来获得抗原特异性T细胞，如抗原特异性CD4+和/或CD8+T细胞。例如，抗原特异性T细胞系或克隆可针对巨细胞病毒抗原产生，通过从感染的受试者分离T细胞并采用相同抗原体外刺激细胞来进行。
用表达CAR的自体同源T细胞治疗癌症患者
本发明的方法可以总结为：
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，从患者获得血液，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患者特异性的，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。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明 而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如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：
本发明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，参考例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN108884459A、CN108610420A、CN108341872A、CN108456250A、CN109796532A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN109880803A、CN110055275 A、CN110123837A、CN 110438082 A、CN 110468105 A，国际专利申请公开号WO2018006882A1、WO2015172339A8、WO2016086813A1、WO2017032293A1、WO2017020812A1、WO2017080377A1、WO2018108106A1、WO2018/133877 A1、WO2018/149358 A1、WO 2018/219299 A1、WO2019/096261A1、WO2018/210279 A1、WO2019/024933 A1、WO2020118634A1、WO2019052562A1、WO2019047932A1、WO2019109980A1、WO2019/141270 A1、WO2019/149279 A1、WO2019/170147A1、WO 2019/210863 A1、WO2019/219029A1、WO2020020210A1、WO2020057666A1、WO2020057641A1、WO 2020/083406A1、WO2020114518A1、WO2020143631A1、WO2020156554A1、WO2021/027785 A1、WO2021/052496 A1、WO2021/057906 A1、WO2021/136550 A1、WO2021148019A1、WO2021232864A1、WO2016036973A1、WO2016049459A1中公开的全文内容。
示例性的，本发明下述实施例中，嵌合抗原受体的scFv能够识别肿瘤抗原claudin18.2，具有SEQ ID NO：14所示的序列，嵌合抗原受体具有SEQ ID NO：11所示的序列，具有的CDR区为SEQ ID NO：1所示的HCDR1，SEQ ID NO：2所示的HCDR2，SEQ ID NO：3所示的HCDR3，SEQ ID NO：4所示的LCDR1，SEQ ID NO：5所示的LCDR2，SEQ ID NO：6所示的LCDR3。
CAR-T细胞的构建方法：首先构建CAR基因，从5’端到3’端依次包括：CD8α信号肽(核苷酸序列如SEQ ID NO:47所示)、scFv(核苷酸序列如SEQ ID NO:15所示)、CD8铰链区(核苷酸序列如SEQ ID NO:48所示)、CD28跨膜区(核苷酸序列如SEQ ID NO:49所示)和CD28的胞内信号传导结构域(核苷酸序列如SEQ ID NO:50所示)以及CD3ζ的胞内信号传导结构域CD3ζ(核苷酸序列如SEQ ID NO:51所示)。然后将CAR基因克隆至质粒PRRLSIN-cPPT.EF-1α，获得含有CAR基因的目的质粒。将构建好的目的质 粒和包装质粒共转染293T细胞，制备得到慢病毒载体，将慢病毒载体转导从患者外周血获得的T细胞，得到CAR-T细胞。
抗原(如claudin18.2)阳性的强度通过免疫组化的染色强度免来定义，为部分或全部的细胞膜或细胞浆染色，染色强度分为：0(阴性，无染色)，+(弱阳性染色)，++(阳性染色，)及+++(强阳性染色)。染色的肿瘤细胞百分比指各染色强度的肿瘤细胞分别占全部肿瘤细胞的百分比。
实施例1：CAR-T细胞的治疗
对12例CLDN18.2阳性的采用PD-1和/或PD-L1抗体治疗失败的胃癌患者进行CAR-T细胞治疗，采用既往手术或活检的肿瘤组织切片进行CLDN 18.2表达检测，均为CLDN18.2表达阳性(≥+，10％)，所有受试者均有远处转移，12例患者的情况如表1所示，其中，患者之前使用的PD-1和/或PD-L1的抗体是：卡瑞利珠单抗、帕博利珠单抗、信迪利单抗、特瑞普利单抗、及一些正在临床试验中的单抗。
在给予CAR-T细胞之前，患者接受“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CLDN18A2CAR的病毒载体转导和扩增，得到CAR-T细胞。所得的CAR-T细胞用冷冻袋在冷冻液中冷冻保存。
在给予CAR-T细胞之前，给予患者化疗药或者放疗进行淋巴细胞清除。
在给予CAR-T细胞治疗之前，可以通过例如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细胞治疗时，在后周期的CAR-T细胞给药，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细胞给药，可以一次给予，也可以分两次或两次以上静脉(IV)滴注给予，每次给予的细胞产品在约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细胞给药完毕后，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。检查可以是从患者获得血液，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IL6治疗，或给予其他CRS治疗，以减少CRS的症状。
每个周期在CAR-T细胞给药完毕后，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细胞数量。
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。
将氟达拉滨、环磷酰胺、白蛋白结合型紫杉醇三者联合作为CAR-T细胞治疗的预处理，在CAR-T细胞输注前2-10天给予受试者。12例患者均接受了氟达拉滨、环磷酰胺和白蛋白结合型紫杉醇的预处理。其中，氟达拉滨给予两次，分别在CAR-T细胞输注前第6，5天(D-6,-5)、或第5，4天(D-5,-4)、或第4，3天(D-4,-3)静脉滴注氟达拉滨30-50mg/天，其中7例在30-40mg/天，5例在41-50mg/天，日平均剂量为39.8(±4.08)mg，按体表面积的日平均剂量为25.0(±0.30)mg/m 2。环磷酰胺给予2次或3次，其中，当给予两次环磷酰胺时，分别在CAR-T细胞给药前第6，5天(D-6,-5)、或第5，4天(D-5,-4)、或第4，3天(D-4,-3)给予，当给予三次环磷酰胺时，分别在CAR-T细胞给药前第6，5天(D-6,-5)、或第5，4天(D-5,-4)、或第4，3天(D-4,-3)给予，在CAR-T细胞给药前第6，5，4天(D-6,-5，-4)、或第5，4，3天(D-5,-4，-3)、或第4，3，2天(D-4,-3，-2)给予，给予的环磷酰胺的剂量为300-600mg/天，其中，患者给予的环磷酰胺量是300-400mg/天或401-500mg/天，日平均剂量为395.8(±47.81)mg，按体表面积为249.6(±16.38)mg/m 2。白蛋白紫杉醇给予1次，在给予CAR-T细胞前第3-5天给予，如在D-4给予白蛋白结合型紫杉醇90-200mg/天。白蛋白紫杉醇的日平均剂量为102.2(±8.60)mg。
12例患者中，6例患者接受了两个周期的细胞输注，第一周期CAR-T细胞给药结束到开始给予第二周期的CAR-T细胞的间隔中位时间为64天。
表1 12例患者的情况
Figure PCTCN2022085921-appb-000001
Figure PCTCN2022085921-appb-000002
实施例2：治疗的评估
实施例1所涉及的12例受试者的治疗评估采用Kaplan Meier方法对PFS、DDC、DOR和OS进行统计分析，对TTR做描述性分析。
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。
在给予CAR-T细胞后，对受试者进行评估并监测神经毒性(神经系统并发症，包括混乱症状，失语症，癫痫发作，抽搐，嗜睡和/或改变的精神状态)，根据严重程度分级(使用1-5级量表例如，Guido Cavaletti和Paola Marmiroli Nature Reviews Neurology 6，657-666(2010年12月)，其中3级(严重症状)，4(危及生命的症状)或5(死亡)被认为是严重的神经毒性。
(一)确定并监测细胞因子释放综合征(CRS)
1级(轻微)-不威胁生命：仅需要全身治疗如退热剂和止吐剂(例如，发热、恶心、疲劳、头痛、肌痛、不适)。
2级(中等)-需要并响应中等介入：氧气需求<40％，或相应体液或低剂量单一血管升压类药物的低血压，或2级器官毒性(通过CTCAE v5.0)。
3级(严重)-需要并响应积极介入：氧气需求≥40％，或需要高剂量单一血管升压类药物(例如，去甲肾上腺素≥20ug/kg/分钟，多巴胺≥10ug/kg/分钟，苯肾上腺素≥200ug/kg/分钟，或肾上腺素≥10ug/kg/分钟)的低血压，或需要多种血管升压类药物(例如，抗利尿素+上述试剂之一，或等于≥20ug/kg/分钟去甲肾上腺素的血管升压类药物的组合)的低血压，或3级器官毒性或4级转氨酶炎(通过CTCAE v5.0)。
4级(威胁生命)-需要通气机支持，或4级器官毒性(排除转氨酶炎)。
5级(致命)-死亡。
(二)神经毒性的示例性分级标准
1级(无临床症状或轻微)-轻微或无临床症状。
2级(中等)-存在限制日常积极活动(ADL)的症状，如做饭、买菜或买衣服、使用电话、管理钱。
3级(严重)-存在限制性自我管理ADL,如洗澡、穿衣或脱衣、进食、使用厕所、服用药物的症状。
4级(威胁生命)-威胁生命，需要紧急介入的症状；
5级(致命)-死亡。
(三)治疗有效性
在给予第一周期的CAR-T细胞治疗后，对CAR-T治疗的安全性和疗效进行了评估，结果如表2所示。如表2所示，无论治疗前的疾病负荷如何，第一周期治疗中没有发生严重CRS、严重神经毒性，因此第一周期的治疗是安全的。另外，还观察到经过第一周期的治疗，5个患者处于疾病稳定(SD)，5个患者达到PR，2个患者疾病进展(PD)，说明CAR-T细胞对使用过PD-1或者PD-L1抗体的患者仍然可以产生明显的疗效，ORR达到41.6％(5/12)，疾病控制率达到83.3％(10/12)。
表2 CAR-T治疗的安全性和疗效
Figure PCTCN2022085921-appb-000003
Figure PCTCN2022085921-appb-000004
对部分患者进行了第二周期的治疗，第二周期开始治疗的时间及治疗后的疗效如表3所示。
表3 CAR-T治疗的安全性和疗效
Figure PCTCN2022085921-appb-000005
如图1所示，按照RECIST 1.1标准评估，12例既往经过PD-L1或PD-1抗体治疗失败的患者在接受2.5×10 8-5.0×10 8个CAR-T细胞治疗后，11例患者的靶病灶都出现了不同程度的缩小，6例达到部分缓解，客观缓解率(ORR)(95％CI)达到50％(21.09％,78.91％)，疾病控制率(DCR)(95％CI)为75％(42.81％,94.51％)。
达到客观缓解的6例受试者mDOR(中位缓解持续时间)为5.4月(1.9，NE)。其中，1例仅接受过1次治疗的受试者持续缓解亦达6.4个月；1例患者接受1次治疗后疗效评估时未检测到靶病灶，持续缓解时间已超过4个月。
无疾病进展生存期(PFS)与总生存期(OS)：采用反向K-M曲线方法计算12例受试者自单采和首次CAR-T细胞输注后的中位随访时间分别约6.4个月和5.6个月；采用K-M曲线法计算mPFS(95％CI)，所有受试者接受CAR-T细胞治疗的mPFS为4.2个月(2.2，NE)；采用K-M曲线法计算mOS(95％CI)为7.4个月(2.6，NE)。结果提示，受试者可以从CAR-T治疗中获得更为明显的临床获益，PFS与OS明显延长。
为了进一步评估在在先周期和在后周期中给予的细胞的功效，通过检测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(所用探针为： FAM-5’-CTGAGCAGCGTGACCGCCGC-3’TAMRA(SEQ ID NO:49)；上游引物序列为：5’-TGGAGTGGATCGGCTACATC-3’(SEQ ID NO:50)；下游引物序列为：5’-AGTAGTAGATGGCGGTGTCG-3’(SEQ ID NO:51))检测外周血中含有CAR-CLDN18DNA的拷贝数。
12例受试者细胞代谢结果显示，自第1次CAR-T细胞输注后，在体内扩增拷贝数中位达峰时间在7天左右，CAR拷贝峰值中位数(最小值，最大值)分别为7196(3422，39315)[拷贝数/μg gDNA]。首次输注后CAR-T细胞在体内扩增持续存在，最长可达203天。未见明显的剂量依赖关系。进行第二周期的细胞输注后，细胞扩增达峰中位时间提前至5天以内，幅值明显低于第一周期，中位Cmax为1074拷贝数/μg gDNA。
(四)安全性
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。
本试验94.6％受试者发生CRS，均为1-2级。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，对于进行第2周期输注的患者，血液学检测指标异常的AE发生率与首次治疗后大致接近，提示再输注并未出现蓄积毒性，未增加受试者的风险。
实施例3.在胃癌中在抗PD-1/PD-L1抗体治疗失败后CAR-T治疗
与上述实施例所述类似，新增12例CLDN18.2阳性胃癌患者在PD-1和/或PD-L1抗体治疗下显示进展(依照RECIST 1.1的进展性疾病PD)后接受CLDN18A2-CAR-T治疗。患者之前使用的PD-1和/或PD-L1的抗体是：卡瑞利珠单抗、帕博利珠单抗、信迪利单抗、特瑞普利单抗、及一些正在临床试验中的单抗。
按照RECIST 1.1标准评估，新增12例既往经过PD-L1或PD-1抗体治疗失败的患者在接受1个周期或2个周期的约2.5×10 8个-5.0×10 8个CAR-T细胞/周期治疗后，4例患者处于SD，6例达到PR，仅有2例为PD，说明CAR-T细胞对使用过PD-1或者PD-L1抗体的胃癌患者仍然可以产生明显的疗效。本试验受试者发生CRS均为1-2级，无神经毒性发生。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。
综合统计实施例1所述的12例胃癌患者和本实施例所述的新增12例胃癌患者在PD-1和/或PD-L1抗体治疗下显示进展(依照RECIST 1.1的进展性疾病PD)后接受 CLDN18A2-CAR-T治疗效果显示，12例达到部分缓解，ORR(95％CI)达到约50％(29.1％,70.9％),DCR(95％CI)达到约79.2％(57.8％，92.9％)。
CAR-T治疗后达PR的12例受试者mDOR为6.3月(1.9，9.5)。其中，6例患者总生存期达约11个月以上，且仍在生存随访期。
采用K-M曲线法计算mPFS(95％CI)，所有受试者接受CAR-T细胞治疗的mPFS为4.9个月(2.6，7.4)；采用K-M曲线法计算mOS(95％CI)为6.7个月(3.7，9.0)。结果提示，受试者可以从CAR-T治疗中获得更为明显的临床获益，PFS与OS明显延长。
24例受试者细胞代谢结果显示，自第1次CAR-T细胞输注后，在体内扩增拷贝数中位达峰时间在7天左右，CAR拷贝峰值中位数(最小值，最大值)分别为4536(433,39315)[拷贝数/μg gDNA]。
实施例4.在krukenberg瘤中在抗PD-1/PD-L1抗体治疗失败后CAR-T治疗
与上述实施例所述类似，CLDN18.2阳性krukenberg瘤患者在PD-1抗体(帕博利珠单抗)治疗下显示进展(依照RECIST 1.1的进展性疾病PD)后接受CLDN18A2-CAR-T治疗，1个周期，约2.5×10 8个CAR-T细胞治疗，达到PR，无疾病进展生存期约4个月，并且仍在持续稳定期。本受试者发生1级CRS，无神经毒性发生。
实施例5.在胆囊癌中在抗PD-1/PD-L1抗体治疗失败后CAR-T治疗
与上述实施例所述类似，CLDN18.2阳性胆囊癌患者在PD-1抗体(Pembrolizumab)治疗下显示进展(依照RECIST 1.1的进展性疾病PD)后接受CLDN18A2-CAR-T治疗，1个周期，约2.5×10 8个CAR-T细胞治疗，达到SD，无疾病进展生存期约7个半月，并且仍在持续稳定期。本受试者发生2级CRS，无神经毒性发生。
实施例6.在肝癌中抗PD-1/PD-L1抗体治疗失败后CAR-T治疗
与上述实施例所述类似，本实施例示例性的，对11例肿瘤抗原GPC3阳性肝癌患者在PD-1和/或PD-L1抗体治疗下显示进展(依照RECIST 1.1的进展性疾病PD)后接受GPC3-CAR-T细胞治疗。患者之前使用的PD-1和/或PD-L1的抗体是：卡瑞利珠单抗、帕博利珠单抗、信迪利单抗、特瑞普利单抗、及一些正在临床实验中的单抗。这11例患者既往均接受过至少1线标准治疗，其中1例接受了1线治疗，8例接受了2线治疗，1例接受了4线治疗，1例接受了6线治疗。
按照RECIST 1.1标准评估，本实施例11肝癌患者在接受1个周期或2个周期的约2.5×10 8-5.0×10 8个CAR-T细胞/周期治疗后，5例患者处于SD，1例达到PR，5例为PD，说 明CAR-T细胞对使用过PD-1或者PD-L1抗体的肝癌患者仍然可以产生明显的疗效，疾病控制率达到约54.5％(6/11)。达到疾病控制的6例患者的无疾病进展生存期均值约4.4月，其中4例患者总生存其超过1年，这4例中的3例仍在生存随访期。
本试验受试者均未发生严重不可控CRS，无神经毒性发生。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。
如实施例2所述方法采用Q-PCR的方法检测患者外周血中含有CAR-GPC3DNA的拷贝数。结果显示，自第1次CAR-T细胞输注后，在体内扩增拷贝数中位达峰值时间在7天左右。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
以下表格中列出了本申请中使用的序列：
Figure PCTCN2022085921-appb-000006
Figure PCTCN2022085921-appb-000007
Figure PCTCN2022085921-appb-000008
Figure PCTCN2022085921-appb-000009
Figure PCTCN2022085921-appb-000010
Figure PCTCN2022085921-appb-000011
Figure PCTCN2022085921-appb-000012
Figure PCTCN2022085921-appb-000013

Claims (58)

  1. 细胞治疗产品在制备用于治疗在先治疗癌症失败的患者中的癌症的药物中的用途，所述在先治疗包括使用抗PD-1抗体和/或抗PD-L1抗体治疗。
  2. 如权利要求1所述的用途，其中所述细胞治疗产品含有表达有外源受体的免疫效应细胞。
  3. 如权利要求2所述的用途，其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。
  4. 如权利要求2所述的用途，其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。
  5. 如权利要求2-4中任一项所述的用途，其中所述免疫效应细胞为自体/同种异体T细胞、或原代T细胞。
  6. 如权利要求3-5中任一项所述的用途，其中所述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。
  7. 如权利要求2-6中任一项所述的用途，其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)或其组合。
  8. 如权利要求2-7中任一项所述的用途，其中所述外源受体的抗原结合结构域特异性识别肿瘤抗原。
  9. 如权利要求8所述的用途，其中所述肿瘤抗原选自CD19、CD20、CD22、CD30、Mesothelin、BCMA、EGFR、EGFRvIII、PSMA、Muc1、claudin18.2、GPC3、IL13RA2、SLAMF7、GPRC5D、LILRB4或其组合；优选地，所述肿瘤抗原选自GPC3、CD19、BCMA、Claudin18.2或其组合。
  10. 如权利要求1-9中任一项所述的用途，其中所述癌症包括实体瘤和/或血液肿瘤；优选地所述实体瘤包括消化道肿瘤；更优选地所述消化道肿瘤包括胃癌/食管胃结合部腺癌、胆囊癌、肝癌、krukenberg瘤或其组合。
  11. 如权利要求1-10中任一项所述的用途，其中所述癌症选自krukenberg瘤、胃癌、胰腺癌、胆囊癌、黑色素瘤、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、头颈部鳞状细胞癌(HNSCC)、经典霍奇金淋巴瘤(cHL)、原发性 纵膈大B细胞淋巴瘤(PMBCL)、尿路上皮癌(UC)、食管癌、宫颈癌、肝癌、Merkel细胞癌、肾细胞癌(RCC)、结直肠癌(mCRC)、乳腺癌。
  12. 如权利要求1-11中任一项所述的用途，其中所述抗PD-1抗体/PD-L1抗体选自Nivolumab、Pembrolizumab、Cemiplimab、卡瑞利珠单抗、特瑞普利单抗、信迪利单抗、替雷利珠单抗、赛帕利单抗(GLS-010)、替雷利珠单抗或其组合。
  13. 如权利要求1-12中任一项所述的用途，其中所述在先治疗包括药物治疗、手术治疗、放疗或其组合；所述药物治疗包括给予化学药和/或生物药；优选的，所述在先治疗包括给予奥拉帕利、乐伐替尼、卡博替尼、阿西替尼、伊匹木、铂类的化疗药物、培美曲塞、依托泊苷、紫杉烷类化合物、贝伐珠单抗、瑞戈非尼、罗替尼、阿帕替尼、乐伐替尼、呋喹替尼、瑞格菲尼或其组合。
  14. 如权利要求1所述的用途，其中给予患有所述癌症的患者至少一个周期的细胞治疗产品进行治疗；优选的，给予患有所述癌症的患者1-3个周期的细胞治疗产品进行治疗。
  15. 如权利要求7-14中任一项所述的用途，其中所述嵌合抗原受体(CAR)包括：
    (i)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD3ζ；
    (ii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；
    (iii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
    (iv)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
  16. 如权利要求14或15所述的用途，其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、2ⅹ10 8个细胞/kg或2ⅹ10 7个细胞/kg患者体重；或细胞治疗产品中细胞的剂量不超过约1ⅹ10 11个细胞/患者、1ⅹ10 10个细胞/患者、5ⅹ10 9个细胞/患者、2ⅹ10 9个细胞/患者或1ⅹ10 9个细胞/患者。
  17. 如权利要求16所述的用途，其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患者体重至2ⅹ10 7个细胞/kg患者体重，或约1ⅹ10 6细胞/kg患者体重至2ⅹ10 7细胞/kg患者体重；或者
    每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 7个细胞至5ⅹ10 9个细胞/患者、约1ⅹ10 7个细胞至2ⅹ10 9个细胞/患者、或约1ⅹ10 7个细胞至1ⅹ10 9个细胞/患者；或者每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 8个细胞至5ⅹ10 9个细胞/患者、约1ⅹ10 8个细胞至2ⅹ10 9个细胞/患者、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的剂量为约2.5ⅹ10 8个细胞至5ⅹ10 8个细胞/患者。
  18. 如权利要求14-17中任一项所述的用途，其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患者化学药物、生物药物、放疗或其组合。
  19. 如权利要求18所述的用途，其中所述预处理在给予所述细胞治疗产品前1-8天实施；优选地，在给予所述细胞治疗产品前2-6天实施；优选地，化学药物、生物药物、放疗或其组合连续使用不超过4天。
  20. 如权利要求18或19所述的用途，其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。
  21. 如权利要求20所述的用途，其中所述微管蛋白抑制剂是紫杉烷类化合物；优选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；更优选所述紫杉烷类化合物为白蛋白结合型紫杉醇。
  22. 如权利要求20或21所述的用途，其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300-700mg/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。
  23. 如权利要求20-22中任一项所述的用途，其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。
  24. 如权利要求8-15中任一项所述的用途，其中所述抗原结合结构域具有：
    SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3、SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2、 SEQ ID NO:6所示的LCDR3；或者
    SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2、SEQ ID NO:18所示的HCDR3、SEQ ID NO:19所示的LCDR1、SEQ ID NO:20所示的LCDR2、SEQ ID NO:21所示的LCDR3；或者
    SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2、SEQ ID NO:29所示的HCDR3、SEQ ID NO:30所示的LCDR1、SEQ ID NO:31所示的LCDR2、SEQ ID NO:32所示的LCDR3；
    或所述抗原结合结构域具有：
    SEQ ID NO:7所示的重链可变区和SEQ ID NO:9所示的轻链可变区；或者
    SEQ ID NO:22所示的重链可变区和SEQ ID NO:23所示的轻链可变区；或者
    SEQ ID NO:33所示的重链可变区和SEQ ID NO:34所示的轻链可变区；
    或所述抗原结合结构域具有：SEQ ID NO:14、24、35、或37所示的序列。
  25. 如权利要求7-24中任一项所述的用途，其中所述的嵌合抗原受体具有SEQ ID NO:11、12、13、25、26、36、38、52任一所示氨基酸序列、或具有SEQ ID NO：58、59、60、61、62、63、64、65、66或67任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽、或具有SEQ ID NO：70、71、72、73、74、75、76、77、78或79任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽。
  26. 如权利要求14-25中任一项所述的用途，其中在每个周期给予所述细胞治疗产品之前，对所述患者的指示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。
  27. 如权利要求1-26中任一项所述的用途，其中在给予所述细胞治疗产品治疗后，所述患者没有显示严重CRS，或没有显示超过3级的神经毒性。
  28. 如权利要求1-27中任一项所述的用途，其中所述使用抗PD-1抗体或抗PD-L1抗体治疗癌症失败的患者中的至少一部分，优选至少40％，更优选至少50％对所述细胞治疗产品有应答。
  29. 如权利要求28所述的用途，其中所述应答是指ORR大于40％，优选大于50％。
  30. 一种使用细胞治疗产品治疗在先治疗癌症失败的患者中的癌症的方 法，其中所述在先治疗包括使用抗PD-1抗体和/或抗PD-L1抗体治疗。
  31. 如权利要求30所述的方法，其中所述细胞治疗产品含有表达有外源受体的免疫效应细胞。
  32. 如权利要求31所述的方法，其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。
  33. 如权利要求31所述的方法，其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。
  34. 如权利要求31-33中任一项所述的方法，其中所述免疫效应细胞为自体/同种异体T细胞、或原代T细胞。
  35. 如权利要求32-34中任一项所述的方法，其中所述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。
  36. 如权利要求31-35中任一项所述的方法，其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)或其组合。
  37. 如权利要求31-36中任一项所述的方法，其中所述外源受体的抗原结合结构域特异性识别肿瘤抗原。
  38. 如权利要求37所述的方法，其中所述肿瘤抗原选自CD19、CD20、CD22、CD30、Mesothelin、BCMA、EGFR、EGFRvIII、PSMA、Muc1、claudin18.2、GPC3、IL13RA2、SLAMF7、GPRC5D、LILRB4或其组合；优选地，所述肿瘤抗原选自GPC3、CD19、BCMA、Claudin18.2或其组合。
  39. 如权利要求30-38中任一项所述的方法，其中所述癌症包括实体瘤和/或血液肿瘤；优选地所述实体瘤包括消化道肿瘤；更优选地所述消化道肿瘤包括胃癌/食管胃结合部腺癌、胆囊癌、肝癌、krukenberg瘤或其组合。
  40. 如权利要求30-39中任一项所述的方法，其中所述癌症选自krukenberg瘤、胃癌、胰腺癌、胆囊癌、黑色素瘤、非小细胞肺癌(NSCLC)、小细胞肺癌(SCLC)、头颈部鳞状细胞癌(HNSCC)、经典霍奇金淋巴瘤(cHL)、原发性纵膈大B细胞淋巴瘤(PMBCL)、尿路上皮癌(UC)、食管癌、宫颈癌、肝癌、Merkel细胞癌、肾细胞癌(RCC)、结直肠癌(mCRC)、乳腺癌。
  41. 如权利要求30-40中任一项所述的方法，其中所述抗PD-1抗体/PD-L1 抗体选自Nivolumab、Pembrolizumab、Cemiplimab、卡瑞利珠单抗、特瑞普利单抗、信迪利单抗、替雷利珠单抗、赛帕利单抗(GLS-010)、替雷利珠单抗或其组合。
  42. 如权利要求30-41中任一项所述的方法，其中所述在先治疗包括药物治疗、手术治疗、放疗或其组合；所述药物治疗包括给予化学药和/或生物药；优选的，所述在先治疗包括给予奥拉帕利、乐伐替尼、卡博替尼、阿西替尼、伊匹木、铂类的化疗药物、培美曲塞、依托泊苷、紫杉烷类化合物、贝伐珠单抗、瑞戈非尼、罗替尼、阿帕替尼、乐伐替尼、呋喹替尼、瑞格菲尼或其组合。
  43. 如权利要求30所述的方法，其中给予患有所述癌症的患者至少一个周期的细胞治疗产品进行治疗；优选的，给予患有所述癌症的患者1-3个周期的细胞治疗产品进行治疗。
  44. 如权利要求36-43中任一项所述的方法，其中所述嵌合抗原受体(CAR)包括：
    (i)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD3ζ；
    (ii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域和CD3ζ；
    (iii)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD137的共刺激信号结构域和CD3ζ；和/或
    (iv)特异性结合肿瘤抗原的抗原结合结构域、CD28或CD8的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
  45. 如权利要求43或44所述的方法，其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、2ⅹ10 8个细胞/kg或2ⅹ10 7个细胞/kg患者体重；或细胞治疗产品中细胞的剂量不超过约1ⅹ10 11个细胞/患者、1ⅹ10 10个细胞/患者、5ⅹ10 9个细胞/患者、2ⅹ10 9个细胞/患者或1ⅹ10 9个细胞/患者。
  46. 如权利要求45所述的方法，其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患者体重至2ⅹ10 7个细胞/kg患者体重，或约1ⅹ10 6个细胞/kg患者体重至2ⅹ10 7个细胞/kg患者体重；或者
    每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 7个细胞至5ⅹ10 9个细胞/患者、约1ⅹ10 7个细胞至2ⅹ10 9个细胞/患者、或约1ⅹ10 7个细胞至1ⅹ10 9个细胞/患者；或者每个周期给予的细胞治疗产品中细胞的剂量为约1ⅹ10 8个细 胞至5ⅹ10 9个细胞/患者、约1ⅹ10 8个细胞至2ⅹ10 9个细胞/患者、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的剂量为约2.5ⅹ10 8个细胞至5ⅹ10 8个细胞/患者。
  47. 如权利要求43-46中任一项所述的方法，其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患者化学药物、生物药物、放疗或其组合。
  48. 如权利要求47所述的方法，其中所述预处理在给予所述细胞治疗产品前1-8天实施；优选地，在给予所述细胞治疗产品前2-6天实施；优选地，化学药物、生物药物、放疗或其组合连续使用不超过4天。
  49. 如权利要求47或48所述的方法，其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。
  50. 如权利要求49所述的方法，其中所述微管蛋白抑制剂是紫杉烷类化合物；优选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；更优选所述紫杉烷类化合物为白蛋白结合型紫杉醇。
  51. 如权利要求49或50所述的方法，其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300-700mg/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。
  52. 如权利要求49-51中任一项所述的方法，其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。
  53. 如权利要求37-44中任一项所述的方法，其中所述抗原结合结构域具有：
    SEQ ID NO:1所示的HCDR1、SEQ ID NO:2所示的HCDR2、SEQ ID NO:3所示的HCDR3、SEQ ID NO:4所示的LCDR1、SEQ ID NO:5所示的LCDR2、SEQ ID NO:6所示的LCDR3；或者
    SEQ ID NO:16所示的HCDR1、SEQ ID NO:17所示的HCDR2、SEQ ID  NO:18所示的HCDR3、SEQ ID NO:19所示的LCDR1、SEQ ID NO:20所示的LCDR2、SEQ ID NO:21所示的LCDR3；或者
    SEQ ID NO:27所示的HCDR1、SEQ ID NO:28所示的HCDR2、SEQ ID NO:29所示的HCDR3、SEQ ID NO:30所示的LCDR1、SEQ ID NO:31所示的LCDR2、SEQ ID NO:32所示的LCDR3；
    或所述抗原结合结构域具有：
    SEQ ID NO:7所示的重链可变区和SEQ ID NO:9所示的轻链可变区；或者
    SEQ ID NO:22所示的重链可变区和SEQ ID NO:23所示的轻链可变区；或者
    SEQ ID NO:33所示的重链可变区和SEQ ID NO:34所示的轻链可变区；
    或所述抗原结合结构域具有：SEQ ID NO:14、24、35、或37所示的序列。
  54. 如权利要求36-53中任一项所述的方法，其中所述的嵌合抗原受体具有SEQ ID NO:11、12、13、25、26、36、38、52任一所示氨基酸序列、或具有SEQ ID NO：58、59、60、61、62、63、64、65、66或67任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽、或具有SEQ ID NO：70、71、72、73、74、75、76、77、78或79任一所示的序列分别与SEQ ID NO：80、81或82所示的序列顺序连接组合而成的多肽。
  55. 如权利要求43-54中任一项所述的方法，其中在每个周期给予所述细胞治疗产品之前，对所述患者的指示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。
  56. 如权利要求30-55中任一项所述的方法，其中在给予所述细胞治疗产品治疗后，所述患者没有显示严重CRS，或没有显示超过3级的神经毒性。
  57. 如权利要求30-56中任一项所述的方法，其中所述使用抗PD-1抗体或抗PD-L1抗体治疗癌症失败的患者中的至少一部分，优选至少40％，更优选至少50％对所述细胞治疗产品有应答。
  58. 如权利要求57所述的方法，其中所述应答是指ORR大于40％，优选大于50％。
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