WO2023083195A1 - 基于gas6构建的嵌合抗原受体免疫细胞制备及其应用 - Google Patents

基于gas6构建的嵌合抗原受体免疫细胞制备及其应用 Download PDF

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WO2023083195A1
WO2023083195A1 PCT/CN2022/130701 CN2022130701W WO2023083195A1 WO 2023083195 A1 WO2023083195 A1 WO 2023083195A1 CN 2022130701 W CN2022130701 W CN 2022130701W WO 2023083195 A1 WO2023083195 A1 WO 2023083195A1
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cells
car
gas6
chimeric antigen
axl
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French (fr)
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赵旭东
范加维
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四川大学华西医院
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Definitions

  • the invention belongs to the field of immune cell therapy, and in particular relates to the preparation and application of a chimeric antigen receptor immune cell constructed based on GAS6.
  • Cancer is the second largest disease threatening human health. In 2018, there were 18.1 million new cancer patients and 9.5 million cancer deaths worldwide. It is estimated that by 2040, there will be 29,500,000 new cancer cases and 16,400,000 deaths per year. Although traditional tumor treatment methods such as radiotherapy, chemotherapy, and surgical resection can delay the survival of tumor patients, the characteristics of patients' poor quality of life and easy recurrence still restrict traditional tumor treatment methods.
  • CAR-T Chimeric Antigen Receptor-T cell
  • AXL a member of the receptor tyrosine kinase (RTK) TAM family
  • RTK receptor tyrosine kinase
  • solid tumors such as lung cancer, pancreatic cancer, glioma, prostate cancer, breast cancer, liver cancer, colon cancer, ovarian cancer, gastric cancer, nasopharyngeal cancer, etc.
  • hematological tumors such as acute Myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), etc.
  • AXL is highly expressed and promotes tumor formation .
  • AXL is upregulated in macrophages in inflammatory diseases or in macrophages associated with the tumor microenvironment. Therefore, targeting AXL has anti-inflammatory or anti-tumor effects.
  • AXL is highly expressed in primary tumors and metastases compared with normal tissues, which is associated with poor prognosis of tumors and chemoresistance in many cancers; novel role of AXL in biological processes, becoming a tumor biomarker and marker molecules.
  • Pancreatic ductal adenocarcinoma is the most common type of pancreatic cancer and is projected to become one of the leading causes of cancer death in the United States by 2030. Despite advances in systemic chemotherapy for metastatic PDAC, overall survival for the disease remains poor. The main features of pancreatic cancer are metastasis, therapy resistance, and immunosuppression. AXL, a member of the TAM family of receptor tyrosine kinases, has been implicated in plasticity, chemoresistance, immunosuppression, and metastasis of pancreatic cancer cells.
  • AXL is an important target for tumor therapy; targeting AXL can effectively inhibit tumor growth, invasion, metastasis and drug resistance, thereby prolonging the survival rate of patients.
  • the object of the present invention is to provide a chimeric antigen receptor immune cell targeting GAS6 receptor and its preparation and application method.
  • a kind of chimeric antigen receptor is provided, it is characterized in that, described CAR contains an extracellular binding domain, and described extracellular binding domain has based on such as SEQ ID NO: The structure of the GAS6 protein or its fragments with the amino acid sequence shown in 1, and the extracellular binding domain can specifically bind the GAS6 receptor.
  • the extracellular binding domain has an amino acid sequence derived from GAS6.
  • the extracellular binding domain comprises GAS6 protein or a fragment thereof.
  • the extracellular binding domain comprises the extracellular region of GAS6 protein.
  • the extracellular binding domain contains a laminin G-like (LG) domain of GAS6 protein.
  • the LG domain includes LG1 and LG2 domains.
  • the LG domain includes wild-type and mutant LG domains.
  • the amino acid sequence of the LG domain is as shown in the sequence shown in SEQ ID NO: 1 to the 678th position, wherein, X is any positive integer of 250-285, preferably X is a positive integer of 260-279, most preferably 261.
  • amino acid sequence of the LG domain is shown in positions 279-678 of the sequence shown in SEQ ID NO:1.
  • the GAS6 protein or its fragments include the laminin G-like (LG) region of the GAS6 protein, and the amino acid sequence of the LG domain is 279 corresponding to the sequence shown in SEQ ID NO:1 -678 bits.
  • LG laminin G-like
  • the GAS6 protein or fragment thereof also includes a partial fragment of the epidermal growth factor (EGF)-like domain of the GAS6 protein, and the amino acid sequence of the EGF-like domain is shown in SEQ ID NO:1 118-278 bits of the sequence.
  • EGF epidermal growth factor
  • amino acid sequence of the partial fragment of the EGF-like domain is the n-278 position of the sequence shown in SEQ ID NO:1,
  • n is a positive integer ranging from 118 to 275, preferably a positive integer ranging from 250 to 270, most preferably 261.
  • the GAS6 protein or its fragments include the extracellular domain of the GAS6 protein, and the amino acid sequence of the extracellular domain of the GAS6 protein is 31- corresponding to the sequence shown in SEQ ID NO:1. 678 bits.
  • the extracellular binding domain of the CAR in addition to the first extracellular domain targeting GAS6 receptor, also includes a second extracellular domain targeting additional targets.
  • the additional target is a tumor-specific target.
  • the GAS6 protein or its fragments specifically bind to GAS6 receptors including AXL protein.
  • the GAS6 receptor is a receptor tyrosine kinase.
  • the receptor tyrosine kinase is selected from the group consisting of AXL, Tyro3, MERTK, or combinations thereof.
  • the GAS6 receptor is selected from the group consisting of AXL, Tyro3, and MERTK.
  • the receptor tyrosine kinase AXL is located on the cell membrane (or transmembrane, cytoplasm).
  • the receptor tyrosine kinase is AXL.
  • the receptor tyrosine kinase AXL is derived from human or non-human mammal.
  • the non-human mammals include: rodents (such as rats, mice), primates (such as monkeys); preferably primates.
  • the receptor tyrosine kinase AXL is of human or monkey origin.
  • said AXL is of human origin.
  • the GAS6 protein or its fragment has the amino acid sequence shown in SEQ ID NO: 1, or has the 1st to 678th (preferably the 1st to 678th) of the sequence shown in SEQ ID NO: 1 31 to 678, more preferably 118 to 678, more preferably 261-678) amino acid sequence.
  • amino acid sequence of the GAS6 protein or a fragment thereof is selected from the following group:
  • Each "-" is independently a connecting peptide or a peptide bond
  • L is nothing or a signal peptide sequence
  • EB is the extracellular binding domain
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is none or a co-stimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • RP is none or a reporter protein.
  • the reporter protein RP also includes a self-cleavage recognition site at its N-terminus, preferably a T2A sequence.
  • the reporter protein RP is a fluorescent protein.
  • the reporter protein RP is mKate2 red fluorescent protein.
  • amino acid sequence of the mKate2 red fluorescent protein is shown in SEQ ID NO:2.
  • said L is a signal peptide of a protein selected from the following group: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
  • said L is a signal peptide derived from CD8.
  • amino acid sequence of said L is shown in SEQ ID NO:3.
  • said H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.
  • said H is a hinge region derived from CD8.
  • amino acid sequence of the H is shown in SEQ ID NO:4.
  • the TM is a transmembrane region of a protein selected from the following group: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
  • the TM is a transmembrane region derived from CD8.
  • amino acid sequence of the TM is shown in SEQ ID NO:5.
  • said C is a co-stimulatory signal molecule selected from the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.
  • said C is a co-stimulatory signal molecule derived from 4-1BB.
  • amino acid sequence of C is shown in SEQ ID NO:6.
  • amino acid sequence of the cytoplasmic signaling sequence derived from CD3 ⁇ is shown in SEQ ID NO:7.
  • amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO:8.
  • nucleic acid molecule encoding the chimeric antigen receptor according to the first aspect of the present invention is provided.
  • a vector in the third aspect of the present invention, characterized in that the vector contains the nucleic acid molecule as described in the second aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
  • the vector is a lentiviral vector.
  • the vector is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV and the like.
  • the vector is pTomo lentiviral vector.
  • the vector further includes a promoter, a transcriptional enhancer element WPRE, a long terminal repeat sequence LTR, etc. selected from the group.
  • the vector comprises the nucleotide sequence shown in SEQ ID NO:9.
  • a host cell contains the vector as described in the third aspect of the present invention or the exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated in the chromosome Or express the chimeric antigen receptor as described in the first aspect of the present invention.
  • an engineered immune cell containing the vector as described in the third aspect of the present invention or the exogenous vector as described in the second aspect of the present invention integrated in the chromosome.
  • the engineered immune cells are selected from the group consisting of T cells, NK cells, NKT cells, or macrophages.
  • the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).
  • the engineered immune cells are CAR-T cells.
  • the sixth aspect of the present invention there is provided a method for preparing the engineered immune cell as described in the fifth aspect of the present invention, comprising the following steps: the nucleic acid molecule as described in the second aspect of the present invention or the nucleic acid molecule as described in the first aspect of the present invention
  • the vectors described in the three aspects are transduced into immune cells, so as to obtain the engineered immune cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • a pharmaceutical composition which contains the chimeric antigen receptor as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, such as The carrier according to the third aspect of the present invention, the host cell according to the fourth aspect of the present invention, and/or the engineered immune cell according to the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the dosage form of the preparation is injection.
  • the concentration of the engineered immune cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml ml.
  • a chimeric antigen receptor as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a carrier as described in the third aspect of the present invention, Or the host cell as described in the fourth aspect of the present invention, and/or the use of the engineered immune cell as described in the fifth aspect of the present invention, for the preparation of drugs for preventing and/or treating diseases related to abnormal expression of GAS6 receptors or preparations.
  • the GAS6 receptors include but not limited to Tyro3, AXL, MERTK and so on.
  • the abnormal expression of the GAS6 receptor refers to the overexpression of the GAS6 receptor.
  • the excessive GAS6 receptor means that the expression level of the GAS6 receptor is ⁇ 1.5 times, preferably ⁇ 2 times, more preferably ⁇ 2.5 times the expression level under normal physiological conditions.
  • the diseases related to the abnormal expression of GAS6 receptor include but not limited to tumor, venous thromboembolic disease, systemic lupus erythematosus, chronic renal failure and pre-eclampsia.
  • the diseases related to abnormal expression of GAS6 receptor include: drugs or preparations for diseases related to abnormal expression of receptor tyrosine kinase AXL.
  • the diseases related to the abnormal expression of AXL include: tumors, venous thromboembolic diseases, systemic lupus erythematosus, chronic renal failure and pre-eclampsia.
  • the disease is a malignant tumor with high expression of AXL.
  • the tumor includes blood tumors and solid tumors.
  • the blood tumor is selected from the group consisting of acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), diffuse massive B-cell lymphoma (DLBCL), or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • DLBCL diffuse massive B-cell lymphoma
  • the solid tumor is selected from the group consisting of pancreatic cancer, breast cancer, gastric cancer, liver and gallbladder cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer and esophageal cancer, glioma , lung cancer, prostate cancer, nasopharyngeal cancer, or a combination thereof.
  • the tumor is selected from the group consisting of pancreatic cancer, liver cancer, glioma, gastric cancer, and prostate cancer.
  • the ninth aspect of the present invention there is provided a use of the engineered immune cell according to the fifth aspect of the present invention, or the pharmaceutical composition according to the seventh aspect of the present invention, for preventing and/or treating cancer or tumors.
  • a method for treating diseases comprising administering an effective amount of the engineered immune cells as described in the fifth aspect of the present invention, or as described in the seventh aspect of the present invention, to a subject in need of treatment.
  • pharmaceutical composition comprising administering an effective amount of the engineered immune cells as described in the fifth aspect of the present invention, or as described in the seventh aspect of the present invention, to a subject in need of treatment.
  • the disease is a disease related to abnormal expression of GAS6 receptor.
  • the disease is cancer or tumor.
  • the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from the subject (autologous cells).
  • the engineered immune cells or the CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).
  • the above method can be used in combination with other treatment methods.
  • the other treatment methods include chemotherapy, radiotherapy, targeted therapy and other methods.
  • Figure 1 shows a schematic diagram of GAS6-CAR vector construction.
  • A is a schematic diagram of the GAS6 sequence, wherein 1-30AA is the signal peptide, 31-678AA is the extracellular domain, 49-90AA is the ⁇ -carboxyglutamic acid (Gla) domain, 91-117AA is the disulfide bridge, 118-278AA is an epidermal growth factor (EGF)-like domain, 279-678AA is two laminin G-like (LG) domains;
  • B is a schematic diagram of the structure of the control plasmid Mock-CAR and GAS6-CAR, in which the signal peptide, Both the hinge region and the transmembrane region are derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3 ⁇ is derived from human CD3, mKate2 is a fluorescent marker used to detect CAR expression;
  • C is pTomo-GAS6-CAR carrier HindIII enzyme digestion identification .
  • FIG. 1 shows the results of CAR transfection efficiency detection.
  • A is the result of cell fluorescence expression after Mock-CAR and GAS6-CAR infection of T cells for 72 hours, where BF is the bright field, and mKate2 is the fluorescence expression of CAR; B is the result of flow cytometry detection of fluorescence expression.
  • Figure 3 shows the killing effect of GAS6-CAR on different tumor cell lines.
  • A pancreatic cancer.
  • B Glioma.
  • C Non-small cell lung cancer.
  • D Prostate cancer.
  • E Liver cancer.
  • F breast tumor.
  • G gastric cancer.
  • Figure 4 shows the results of detection of AXL expression in different pancreatic cancer cell lines.
  • A is the expression of AXL protein detected by WB.
  • B is the detection of AXL mRNA level by qPCR.
  • C is flow cytometric detection of AXL expression.
  • D is the expression level of AXL on the cell membrane detected by immunofluorescence.
  • Figure 5 shows the gradient killing results of GAS6-CAR on different pancreatic cancer cell lines.
  • Figure 6 shows the results of IFN ⁇ release after killing different pancreatic cancer cell lines by GAS6-CAR.
  • FIG. 7 shows that overexpression of AXL in the pancreatic cancer cell line BXPC3 enhances the killing effect of GAS6-CAR.
  • A is the expression of AXL protein detected by WB.
  • B is the detection of AXL mRNA level by qPCR.
  • C is flow cytometric detection of AXL expression.
  • D is the expression level of AXL on the cell membrane detected by immunofluorescence.
  • E is the killing detection of BXPC3-AXL by GAS6-CAR.
  • F is the detection result of cytokine IFN ⁇ release.
  • FIG 8 shows that knockdown of AXL expression in the pancreatic cancer cell line PANC1 reduces the killing effect of GAS6-CAR.
  • A is the expression of AXL protein detected by WB.
  • B is the detection of AXL mRNA level by qPCR.
  • C is flow cytometric detection of AXL expression.
  • D is the expression level of AXL on the cell membrane detected by immunofluorescence.
  • E is the killing detection of GAS6-CAR to PANC1-shAXL.
  • F is the detection result of cytokine IFN ⁇ release.
  • Figure 9 shows the killing results of GAS6-CAR on the normal cell line HEK-293T.
  • A is the expression of AXL protein detected by WB.
  • B is the detection of AXL mRNA level by qPCR.
  • C is flow cytometric detection of AXL expression.
  • D is the expression level of AXL on the cell membrane detected by immunofluorescence.
  • E is the killing detection of GAS6-CAR on HEK-293T.
  • F is the detection result of cytokine IFN ⁇ release.
  • the inventor After extensive and in-depth research and a large number of screenings, the inventor first developed a GAS6-based chimeric antigen receptor immune cell preparation and application.
  • the experimental results show that the CAR-T targeting the GAS6 receptor of the present invention has a significant killing effect on target cells and a specific anti-tumor cell effect.
  • the present invention has been accomplished on this basis.
  • any positive integer of X1-X2 means any positive integer between X1 and X2 (inclusive), for example, a positive integer of 1-5 includes 1, 2, 3, 4 and 5, and so on.
  • the terms “comprising” or “comprising (comprising)” can be open, semi-closed and closed. In other words, the term also includes “consisting essentially of” or “consisting of”.
  • Transduction refers to the process of delivering exogenous polynucleotides into host cells, transcription and translation to produce polypeptide products, including the use of plasmid molecules to introduce exogenous The polynucleotide is introduced into a host cell (eg, E. coli).
  • a host cell eg, E. coli
  • Gene expression or “expression” refers to the process of gene transcription, translation and post-translational modification to produce the gene's RNA or protein product.
  • Polynucleotide refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and the like.
  • a polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs.
  • the term polynucleotide is used herein to refer to single- and double-stranded molecules interchangeably. Unless otherwise stated, polynucleotides in any of the embodiments described herein include a double-stranded form and two complementary single strands known or predicted to constitute the double-stranded form.
  • amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine, and proline; aspartic acid, glutamic acid amino acids; asparagine, glutamine; serine, threonine, lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine .
  • the present invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.
  • administration refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or Other parenteral routes of administration, for example by injection or infusion.
  • TAM family is receptor tyrosine kinase (Receptor tyrosine kinase, RTK), the family contains members AXL, Tyro3, MERTK, which play an important role in tumorigenesis and development.
  • the AXL gene is located on the long arm of chromosome 19 and consists of 20 exons. It was originally discovered in patients with chronic myelogenous leukemia.
  • This protein is encoded by the AXL gene (or UFO or ARK or Tyro7 or JTK11), the full-length AXL protein contains 894 amino acids and has a molecular weight of 98 kDa; however, the actual AXL protein is 120 kDa due to post-translational glycosylation (partially glycosylated ) or 140kDa (fully glycosylated).
  • AXL is composed of extracellular, transmembrane and intracellular domains, and the extracellular structure is composed of two immunoglobulin (IgL)-like repeats and two fibronectin type III (Fro III)-like repeats;
  • the Fro III domain is responsible for the binding of AXL to its ligand GAS6.
  • the intracellular domain of AXL plays an important role in autophosphorylation and downstream kinase activity.
  • AXL is highly expressed in a variety of clinical tumors (such as lung cancer, pancreatic cancer, glioma, prostate cancer, breast cancer, liver cancer, colon cancer, ovarian cancer, gastric cancer, etc.), including protein levels and RNA levels; and in It is highly expressed in the corresponding tumor cell lines; at the same time, studies have shown that AXL is highly expressed in macrophages associated with inflammatory diseases and tumor microenvironment.
  • AXL The high expression of AXL is closely related to the survival, proliferation, metastasis, invasion, epithelial-mesenchymal transformation of tumor cells and the stemness of cancer cells, and is also related to the prognosis and survival rate of patients; the expression of AXL even affects tumor microbiology environment to promote tumor immunosuppression. Reducing the expression of AXL by gene knockdown can significantly inhibit the invasion and metastasis of tumor cells and improve the prognosis.
  • T cells can produce anti-tumor effects through AXL (such as triple-negative breast cancer). Compared with AXL-negative tumor cells, AXL-expressing tumor cells are more likely to be killed by AXL-CAR T cells. The above results indicate that AXL is an important target molecule for tumor therapy.
  • Growth arrest-specific protein 6 (Growth arrest-specific protein 6, GAS6 or AXSF, AXLLG)
  • GAS6 is a vitamin K-dependent multi-domain protein with a molecular weight of 75 kDa; GAS6 consists of 678 amino acids, including: N-terminal ⁇ -carboxyglutamic acid (Gla) domain (amino acids 49-90) which endows GAS6 Ability to bind to anionic phospholipids on the cell surface, associated with stimulation of cell proliferation; four epidermal growth factor (EGF)-like domains (amino acids 118-278) and two laminin G-like (LG) domains (amino acids 279–678) .
  • GEF epidermal growth factor
  • LG laminin G-like domains
  • the GAS6 gene is highly expressed in a variety of cancers and has been recognized as a poor prognostic marker, and elevated protein levels are also associated with a variety of disease states, including venous thromboembolic disease, systemic lupus erythematosus, chronic renal failure and preeclampsia etc.
  • GAS6 is the ligand of the TAM family (including Tyro3, AXL, MERTK). Besides AXL, it can also bind other members of the TAM family. Among them, GAS6 has the highest binding affinity to AXL, and GAS6 is the only activating ligand of AXL found so far.
  • AXL binds to its ligand GAS6 with high affinity
  • the intracellular kinase domain of the AXL receptor undergoes homodimerization and trans-autophosphorylation, thereby recruiting adapter molecules and Src homology 2 (Src homology 2, SH2 ) or other phosphotyrosine binding domains (Phosphotyrosine binding domains, PTB), thereby activating downstream signaling pathways RAS, RAF, MAPK, ERK1/2, PI3K, TGF- ⁇ 1/2, etc., and then play a role.
  • the GAS6/AXL signaling pathway is closely related to tumor cell growth, metastasis, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, drug resistance, immune regulation and stem cell maintenance.
  • EMT epithelial-mesenchymal transition
  • the present invention integrates a specific fragment of GAS6 into a CAR vector through genetic engineering, and modifies related immune cells, so as to achieve specific killing of GAS6-binding protein-positive cells, which can be used for the treatment of related diseases.
  • Chimeric antigen receptor is composed of extracellular antigen recognition region, transmembrane region and intracellular co-stimulatory signal region.
  • the design of CAR has gone through the following process: the first-generation CAR has only one intracellular signaling component CD3 ⁇ or Fc ⁇ RI molecule, and because there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion , but can not provide long-term T cell proliferation signal and sustained anti-tumor effect in vivo, so it has not achieved good clinical efficacy.
  • the second-generation CAR introduces a co-stimulatory molecule based on the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CAR, the function is greatly improved, and the persistence of CAR-T cells and the protection against tumor cells are further enhanced. lethality.
  • some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series to develop into the third-generation and fourth-generation CAR.
  • the extracellular segment of CAR can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity, and secretion of cytokines, thereby eliminating target cells.
  • isolate the patient's own cells (or a heterologous donor) activate and genetically modify immune cells that produce CAR, and then inject them into the same patient. In this way, the probability of suffering from graft-versus-host disease is extremely low, and the antigen is recognized by immune cells in a non-MHC-restricted manner.
  • CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. Such a high response rate was unattainable by any previous treatment method, and has triggered an upsurge of clinical research all over the world.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements.
  • the extracellular domain can be the ScFv of an antibody based on the specific binding of antigen-antibody, or it can be a natural sequence or a derivative thereof based on the specific binding of ligand-receptor.
  • the extracellular domain of the chimeric antigen receptor is a GAS6 protein or a fragment thereof that can specifically bind to the AXL target of the CAR of the present invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence at positions 261 to 678 of the sequence shown in SEQ ID NO:1.
  • the intracellular domain includes the co-stimulatory signaling region and the zeta chain portion.
  • a co-stimulatory signaling region refers to a portion of an intracellular domain that includes co-stimulatory molecules.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigens.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the CAR of the present invention when expressed in T cells, is capable of antigen recognition based on antigen binding specificity. When it binds its cognate antigen, it affects tumor cells, causing them not to grow, being induced to die, or otherwise affected, and resulting in a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the co-stimulatory molecule and the zeta chain.
  • the antigen binding domain is fused to the CD8 signaling domain, and the intracellular domain in combination with the CD3 ⁇ signaling domain.
  • the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which refer to the amino acid sequence at positions 261 to 678 (or positions 279-678) of SEQ ID NO: 1, At most 10, preferably at most 8, more preferably at most 5 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • the number of amino acids added, deleted, modified and/or substituted is preferably no more than 40% of the total amino acid number of the original amino acid sequence, more preferably no more than 35%, more preferably 1-33%, More preferably 5-30%, more preferably 10-25%, more preferably 15-20%.
  • the number of added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, Optimally 1.
  • the CAR can be designed to include the transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with the receptor complex. interactions with other members.
  • the intracellular domain in the CAR of the present invention includes a 4-1BB co-stimulatory domain and a CD3 ⁇ signaling domain.
  • the CAR is a CAR that can specifically target AXL.
  • a chimeric antigen receptor immune cell which comprises the chimeric antigen receptor specifically targeting GAS6 receptor (preferably AXL) of the present invention.
  • the chimeric antigen receptor immune cells of the present invention may be CAR-T cells, or CAR-NK cells, or CAR-macrophages.
  • the chimeric antigen receptor immune cells of the present invention are CAR-T cells.
  • CAR-T cell As used herein, the terms "CAR-T cell”, “CAR-T” and “CAR-T cell of the present invention” all refer to the CAR-T cell described in the fifth aspect of the present invention.
  • CAR-T cells Compared with other T-cell-based therapies, CAR-T cells have the following advantages: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, targeting a certain Once the CAR gene construction of tumor markers is completed, it can be widely used; (3) CAR can use both tumor protein markers and glycolipid non-protein markers, expanding the target range of tumor markers; ( 4) Using the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • CAR-NK cell As used herein, the terms “CAR-NK cell”, “CAR-NK” and “CAR-NK cell of the present invention” all refer to the CAR-NK cell of the fifth aspect of the present invention.
  • the CAR-NK cells of the present invention can be used for tumors with high expression of AXL receptors (preferably AXL).
  • NK cells are a major type of immune effector cells, which protect the body from virus infection and tumor cell invasion through non-antigen-specific pathways. NK cells through engineering (gene modification) may obtain new functions, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with CAR-T cells, CAR-NK cells also have the following advantages, for example: (1) directly kill tumor cells by releasing perforin and granzymes, but have no killing effect on normal cells of the body; (2) they release very A small amount of cytokines reduces the risk of cytokine storm; (3) It is very easy to expand in vitro and develop into "off-the-shelf" products. Other than that, it is similar to CAR-T cell therapy.
  • CAR-macrophage (M) cell As used herein, the terms "CAR-macrophage (M) cell", “CAR-macrophage (M)” and “CAR-macrophage (M) cell of the present invention” all refer to the CAR-macrophage described in the fifth aspect of the present invention.
  • Phage cells The CAR-macrophages of the present invention can be used for inflammatory diseases and tumors with high expression of AXL receptors (preferably AXL).
  • Macrophages are phagocytic cells derived from monocytes and play an important role in the body's innate immunity and cellular immunity. Macrophages through engineering (gene modification) can enhance the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxicity.
  • a nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology, directly isolated from cells and tissues containing the gene.
  • the gene of interest can be produced synthetically.
  • the invention also provides a vector comprising a nucleic acid molecule of the invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have an advantage over vectors derived from oncogenic retroviruses, such as murine leukemia virus, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector.
  • This vector is suitable for replication and integration in eukaryotic cells.
  • a typical cloning vector contains transcriptional and translational terminators, an initial sequence and a promoter useful for regulating the expression of the desired nucleic acid sequence.
  • the expression constructs of the invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the present invention provides gene therapy vectors.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • vectors of interest include expression vectors, replication vectors, probe production vectors and sequencing vectors.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and US Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • promoter elements can regulate the frequency of transcription initiation. Typically these are located in the 30-110 bp region upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible in order to preserve promoter function when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can act cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
  • an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired.
  • inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
  • the expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene, so as to seek transfected or infected cell populations from viral vectors. Identification and selection of expressing cells.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences.
  • a reporter gene is a gene that is absent from or expressed by a recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82).
  • the reporter gene is the gene encoding mKate2 red fluorescent protein.
  • Suitable expression systems are known and can be prepared using known techniques or obtained commercially.
  • the construct with the minimum of 5 flanking regions showing the highest level of reporter gene expression was identified as a promoter.
  • Such a promoter region can be linked to a reporter gene and used to assess the ability of the agent to regulate promoter-driven transcription.
  • the vector can be easily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method in the art.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg human, cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).
  • an exemplary delivery vehicle is liposomes.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be associated with a lipid.
  • Lipid-associated nucleic acids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides
  • linker molecules associated with both liposomes and oligonucleotides
  • entrapped in liposomes complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complexes with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are fatty substances, which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets, which occur naturally in the cytoplasm as well as compounds comprising long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides a chimeric antigen receptor CAR according to the first aspect of the present invention, the nucleic acid molecule according to the second aspect of the present invention, the vector according to the third aspect of the present invention, or the CAR according to the fourth aspect of the present invention.
  • the host cell or the engineered immune cell according to the fifth aspect of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives e.g, aluminum hydroxide
  • the invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding an expression cassette of the invention.
  • the transduced T cells can target the marker receptor tyrosine kinase of tumor cells (preferably AXL), activate T cells synergistically, and cause an immune response of immune cells, thereby significantly improving the killing efficiency of tumor cells.
  • the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cells of the present invention to the mammal.
  • the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • a patient's own T cells or a heterologous donor
  • CAR-T can treat all cancers that express that antigen.
  • CAR-cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific for the antigen-binding domain in the CAR.
  • receptor tyrosine kinase (preferably AXL) CAR-T cells elicit a specific immune response of receptor tyrosine kinase expressing (preferably AXL) cells.
  • the data disclosed herein specifically discloses lentiviral vectors comprising the GAS6 protein or fragments thereof, the hinge and transmembrane regions, and the 4-1BB and CD3 ⁇ signaling domains
  • the invention should be construed as including the inclusion of the GAS6 protein as part of the construct. Any number of variations of each.
  • Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors.
  • Cancer may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors.
  • Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignancies, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such as
  • Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas).
  • solid tumors such as sarcomas and carcinomas
  • solid tumors such as sarcomas and carcinomas
  • fibrosarcoma myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, liver and gallbladder cancer, colorectal cancer, bladder cancer, non-small cell lung cancer , ovarian cancer and esophageal cancer, glioblastoma, lung cancer, prostate cancer, nasopharyngeal cancer, etc.
  • the CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is a human.
  • cells are isolated from a mammal (preferably a human) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing a CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be a human, and the CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • the invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.
  • the CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; Agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives eg, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's disease - although appropriate dosages may be determined by clinical trials.
  • compositions of the invention to be administered can be determined by a physician, It takes into account individual differences in age, weight, tumor size, degree of infection or metastasis, and condition of patients (subjects). It may generally be stated that pharmaceutical compositions comprising T cells as described herein may be dosed at a dose of 104 to 109 cells/kg body weight, preferably at a dose of 105 to 106 cells/kg body weight (including all integers within those ranges value) applied. T cell compositions can also be administered multiple times at these doses.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.
  • a T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., previously , simultaneously or subsequently) to the patient in a form of treatment including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for specific tumor patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for specific tumor patients.
  • T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cell composition of the invention is administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1 ⁇ 10 6 to 1 ⁇ 10 10 CAR-T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion.
  • AXL is basically not expressed on the cell membrane of normal cells, but is highly expressed on the cell membrane of tumor tissue and macrophages, so that this CAR specifically kills tumor cells and macrophages that highly express AXL on the membrane, It has no obvious killing effect on normal cells.
  • the present invention utilizes the ligand-receptor binding mode instead of scfv in the traditional sense.
  • the conservation of receptor-ligand interaction determines that safety tests in animals, especially primates, can better reflect its safety in humans.
  • GAS6 can also bind to other members of the TAM family, and its multi-target effect can prevent immune escape of tumor cells.
  • Reagents, plasmids, and cells in the examples of the present application are commercially available unless otherwise stated.
  • MDA-MB231 breast cancer cells NUGC4 gastric cancer cells
  • MKN28 gastric cancer cells AGS gastric cancer cells
  • Sequence the recombinant plasmid compare the sequencing results to confirm whether the plasmid is correct, and the sequencing primers are universal sequencing primers. Both the sequencing and enzyme digestion results showed that the coding sequence of CAR was correctly inserted into the predetermined position of the plasmid (Fig. 1C).
  • All plasmids were extracted with QIAGEN’s endotoxin-free large extraction kit, and the purified plasmids were transfected into HEK-293T cells with Biyuntian lipo6000 for lentiviral packaging.
  • HEK-293T cells were cultured in 15 cm dishes for virus packaging. Transfect HEK-293T cells when the confluence is around 80%-90%, prepare 2ml OPTIMEM dissolved plasmid mixture (core plasmid 20ug, pCMV ⁇ R8.9 10ug, PMD2.G 4ug); in another centrifuge tube 2ml OPTIMEM and 68ul of lipo6000. After standing still at room temperature for 5 minutes, the plasmid complex was added to the liposome complex, and left standing at room temperature for 20 minutes. The above mixture was added dropwise to HEK-293T cells, incubated at 37°C for 6 hours, and then the medium was removed. Refill with pre-warmed complete medium.
  • the virus supernatants After collecting the virus supernatants for 48 hours and 72 hours, they were centrifuged at 3000 rpm for 20 minutes at 4°C. After filtering with a 0.45um filter membrane, centrifuge at 25,000rpm at 4°C for 2.5 hours to concentrate the virus. After the concentrated virus was dissolved overnight with 30ul virus lysate, the virus titer was detected by QPCR. The results showed that the virus titer reached the requirement.
  • Example 4 Positive rate of infected CAR-T cells detected by flow cytometry
  • CAR-T cells and NTD cells 72 hours after virus infection were collected by centrifugation, washed once with PBS, supernatant was discarded, cells were resuspended in PBS containing 2% FBS, and the positive rate was detected by flow cytometry.
  • Figure 2B shows that the detection by flow cytometry shows that the positive expression rate of CAR or mKate2CAR-T is about 30%.
  • (1) Cellular immunofluorescence Spread the target cells on a disc of a 24-well plate, fix the cells with 4% paraformaldehyde (PFA) for 20 minutes after 24 hours, wash with PBST three times for 5 minutes each time; use 10% goat serum Block at room temperature for 1 hour, and incubate overnight at four degrees with an antibody that specifically recognizes AXL. The next day, wash three times with PBST, five minutes each time. Incubate with CY3-labeled secondary antibody that specifically recognizes the primary antibody for 1 hour at room temperature. After washing three times with PBS, nuclei were stained with DAPI. Confocal microscopy imaging.
  • PFA paraformaldehyde
  • FIG. 4 The results of AXL expression detection in each cell line are shown in FIG. 4 .
  • the expression of AXL in target cells was detected by WB ( Figure 4A), qPCR ( Figure 4B), and flow cytometry (Figure 4C), and the results consistently showed that PANC1 and ASPC1 highly expressed AXL, while BXPC3 expressed low AXL. Further immunofluorescence localization verified the high expression of AXL on the cell membranes of PANC1 and ASPC1, and the high expression of AXL on the cell membranes of PANC1 and BXPC3 (Fig. 4D).
  • Embodiment 6 Target cell construction carrying luciferase
  • the target cells used include: target cells with high expression of AXL: PANC1, ASPC1, U251, U87, H1299, PC3, HepG2, MDA-MB231, AGS; target cells with no or low expression of AXL: BXPC3, A549, MHCC-97H , SMMC-7721, MDA-MB468, NUGC4, MKN28.
  • the cell supernatant was collected and frozen at -80°C to detect the release of IFN ⁇ (see Example 8).
  • Cell killing was detected with a promega fluorescence detection kit.
  • the cells were treated with 20ul 1*PLB lysate for 20 minutes, and 100ul substrate was added to each well for immediate detection with a BioTek microplate reader.
  • Cytotoxic killer cells% (1-fluorescence value of target cells with effector cells/fluorescence value of target cells without effector cells) ⁇ 100%
  • GAS6-CAR The killing effect of GAS6-CAR on different tumor cell lines is shown in Figure 3. Pancreatic cancer cells (A); Glioma cells (B); Non-small cell lung cancer cells (C); Prostate cancer cells (D); Liver cancer cells (E); Breast tumor cells (F); Gastric cancer cells (G) .
  • the results showed that GAS6-CAR had good killing effect on multiple tumor cell lines.
  • the gradient killing results of GAS6-CAR on different pancreatic cancer cell lines are shown in Figure 5.
  • the results showed that the killing effect of GAS6CAR-T cells on tumor cells with high expression of AXL was gradually enhanced as the effector-to-target ratio (E:T) increased.
  • the method is as follows: take the cell supernatant of the CAR-T cells of the present invention co-incubated with PANC1, BXPC3, ASPC1, HEK-293T target cells (E:T ratio is 4:1) in Example 7 according to the IFN gamma Human ELISA Kit (life technology) to detect IFN ⁇ .
  • Example 9 The effect of overexpression of AXL on the killing effect of GAS6-CAR-T
  • the AXL overexpression plasmid (EX-Z7835-Lv105-B, ORF lentiviral expression clone) was purchased from Yijin Biotechnology, and the AXL overexpression plasmid was transiently transfected.
  • BXPC3-Vector and BXPC3-AXL-luciferase were digested and counted, and the cell density was adjusted to 2 ⁇ 10 4 /ml.
  • 100ul luciferase cells were inoculated in a 96-well plate, the CAR-T/NT cells were adjusted to a cell density of 1 ⁇ 10 5 , and inoculated into a black 96-well plate according to the E:T ratio of 4:1, and 100ul was inoculated in each well.
  • FIG. 7-A is the detection of AXL protein expression by WB.
  • Figure 7-B is the detection of AXL mRNA level by qPCR.
  • Figure 7-C is flow cytometric detection of AXL expression.
  • Figure 7-D shows the expression level of AXL on the cell membrane detected by immunofluorescence.
  • Figure 7-E shows the killing effect of GAS6-CAR on BXPC3 after overexpressing AXL.
  • Figure 7-F shows the release of IFN ⁇ in the killing effect of GAS6-CAR-T on BXPC3 overexpressing AXL.
  • Example 10 Effect of specific knockdown of AXL on the killing effect of GAS6-CAR-T
  • shRNA sequence library provided by Sigma, select MISSION shRNA Lentiviral Transduction Particles, select the shRNA that has been validated in the CDS region, and BLAST each shRNA selected on NCBI to ensure the specificity of the target.
  • HEK-293T cells per dish were inoculated into 6cm dish for culture. Before transfection, replace the 6cm dish with 5ml fresh culture medium (containing serum, no antibiotics); take two clean and sterile centrifuge tubes, add 250 ⁇ l Medium, then add 5 ⁇ g of plasmid DNA to one of the tubes, and gently blow and mix with a gun; add 10 ⁇ l Lipo6000 TM transfection reagent to the other tube, and gently blow and mix with a gun.
  • fresh culture medium containing serum, no antibiotics
  • PANC1-shCOO2 PANC1-shAXL#1, PANC1-shAXL#2-luciferase cells were digested and counted, and the cell density was adjusted to 2 ⁇ 10 4 /ml. 100ul luciferase cells were inoculated in a 96-well plate, the CAR-T/NT cells were adjusted to a cell density of 1 ⁇ 10 5 , and inoculated into a black 96-well plate according to the E:T ratio of 4:1, and 100ul was inoculated in each well. After mixing the above target cells and T cells, they were incubated in an incubator for 24 hours, and then the killing effect was detected.
  • Figure 8-A is the detection of AXL protein expression by WB.
  • Figure 8-B is the detection of AXL mRNA level by qPCR.
  • Figure 8-C is flow cytometric detection of AXL expression.
  • Figure 8-D shows the expression level of AXL on the cell membrane detected by immunofluorescence.
  • Figure 8-E shows the killing effect of GAS6-CAR on PANC1 knockdown AXL.
  • Figure 8-F shows the release of IFN ⁇ after the killing effect of GAS6-CAR on PANC1 silencing AXL.
  • Example 11 The killing effect of GAS6-CAR-T on non-tumor cells
  • HEK-293T cells are human embryonic kidney cell lines. They were inoculated into black 96-well plates according to the effect-to-target ratio of 0.5:1, 1:1, 2:1, and 4:1. GAS6-CAR-T cells and HEK-293T- Luciferase cells were co-incubated, and the killing effect of GAS6-CAR-T on HEK-293T cells was detected by the change of fluorescence value.
  • Figure 9-A is the detection of AXL protein expression by WB.
  • Figure 9-B is the detection of AXL mRNA level by qPCR.
  • Figure 9-C is flow cytometric detection of AXL expression.
  • Figure 9-D shows the expression level of AXL on the cell membrane detected by immunofluorescence.
  • Figure 9-E shows the killing effect of GAS6-CAR on HEK-293T.
  • Figure 9-F shows the release of IFN ⁇ from the killing effect of GAS6-CAR on HEK-293T.

Abstract

本发明提供了一种基于生长停滞特异蛋白6构建的嵌合抗原受体免疫细胞制备及其应用。具体地，本发明提供了一种基于GAS6改造的嵌合抗原受体(CAR)，所述的CAR含有一胞外结合域，所述的胞外结合域能够特异性地靶向GAS6受体。本发明的CAR免疫细胞具有较强的特异性和靶点亲和力，因此对靶细胞的杀伤能力较强且安全性高。

Description

基于GAS6构建的嵌合抗原受体免疫细胞制备及其应用 技术领域
本发明属于免疫细胞治疗领域，具体涉及一种基于GAS6构建的嵌合抗原受体免疫细胞制备及其应用。
背景技术
肿瘤是威胁人类健康的第二大疾病，2018年全球有18,100,000新增肿瘤患者，肿瘤死亡病例9,500,000。据估计，到2040年肿瘤每年新增29,500,000病例，死亡病例16,400,000。传统的肿瘤治疗手段比如放疗、化疗、手术切除等虽能延缓肿瘤患者的生存期，但患者的生存质量下降、易复发等特点仍制约着传统肿瘤治疗手段。
生物免疫疗法已成为继手术、放疗、化疗后的第四种肿瘤治疗手段，并将成为未来肿瘤治疗必选手段。嵌合抗原抗体受体T细胞(Chimeric Antigen Receptor-T cell,CAR-T)是指经基因修饰后，能以MHC非限制性方式识别特定目的抗原，并且持续活化扩增的T细胞。CAR的结构包括肿瘤相关抗原结合区、胞外铰链区、跨膜区和胞内信号区。目前CAR-T疗法在血液恶性肿瘤中显示出良好治疗效果，但实体瘤由于存在肿瘤异质性、缺少肿瘤特异性抗原、肿瘤免疫抑制微环境等限制了CAR-T疗法在实体瘤中的应用。
大量研究表明，受体酪氨酸激酶(Receptor tyrosine kinase，RTK)TAM家族成员AXL涉及多种细胞过程，包括细胞生长、增殖、存活、凋亡和粘附。从实体瘤到血液瘤的各种恶性肿瘤中，实体瘤诸如肺癌、胰腺癌、胶质瘤、前列腺癌、乳腺癌、肝癌、结肠癌、卵巢癌、胃癌、鼻咽癌等；血液瘤诸如急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)等，AXL均高表达，促进肿瘤形成。同时，研究发现，AXL在炎症性疾病中的巨噬细胞或肿瘤微环境相关的巨噬细胞中表达上调。因此，靶向AXL具有抑制炎症或抗肿瘤的作用。与正常组织相比，AXL在原发性肿瘤和转移灶中高表达，这与肿瘤的不良预后和多种癌症的化学耐药性有关；AXL在生物过程中的新颖作用，成为了肿瘤生物标志物和发生标记分子。
胰腺导管腺癌(Pancreatic ductal adenocarcinoma，PDAC)是最常见的胰腺癌类型，预计到2030年将成为美国癌症死亡的主要原因之一。虽然在转移性PDAC的系统化学疗法方面取得了进展，但该病的总体生存率仍然很低。胰腺癌的主要特征是转移、治疗耐药性和免疫抑制。受体酪氨酸激酶TAM家族成员AXL与胰腺癌细胞的可塑性、化学抗性、免疫抑制和转移有关。研究表明，70％左右的胰腺癌患者肿瘤组织中受体酪氨酸激酶AXL的表达明显增高，在正常的胰导管上皮中几 乎没有表达。在肿瘤患者中，AXL蛋白过表达与淋巴结转移、肿瘤耐药、远处转移、病情恶化、较差的总生存率和复发率等成正相关。研究表明，敲降AXL的表达增加了胰腺癌细胞对吉西他滨的敏感性。
以上研究表明AXL是肿瘤治疗的一个重要靶点；靶向AXL可以有效抑制肿瘤的生长、侵袭、转移和耐药，从而延长患者的生存率。
因此，本领域迫切需要开发一种以AXL为靶点的嵌合抗原受体免疫细胞及其治疗方法。
发明内容
本发明的目的就是提供一种以GAS6受体为靶点的嵌合抗原受体免疫细胞及其制备和应用方法。
在本发明的第一方面，提供了一种嵌合抗原受体(CAR)，其特征在于，所述的CAR含有一胞外结合域，所述的胞外结合域具有基于如SEQ ID NO:1所示氨基酸序列的GAS6蛋白或其片段的结构，并且所述的胞外结合域能够特异性地结合GAS6受体。
在另一优选例中，所述的胞外结合域具有来源于GAS6的氨基酸序列。
在另一优选例中，所述的胞外结合域包含GAS6蛋白或其片段。
在另一优选例中，所述的胞外结合域包含GAS6蛋白的胞外区。
在另一优选例中，所述的胞外结合域含有GAS6蛋白的层粘连蛋白G样(LG)结构域。
在另一优选例中，所述的LG结构域包括LG1和LG2结构域。
在另一优选例中，所述LG结构域包括野生型和突变型的LG结构域。
在另一优选例中，所述LG结构域的氨基酸序列如SEQ ID NO:1所示序列的第X位至第678位所示，其中，X为250-285的任一正整数，较佳地X为260-279的正整数，最优选为261。
在另一优选例中，所述LG结构域的氨基酸序列如SEQ ID NO:1所示序列的279-678位所示。
在另一优选例中，所述的GAS6蛋白或其片段包括GAS6蛋白的层粘连蛋白G样(LG)区域，所述LG结构域的氨基酸序列为对应于SEQ ID NO:1所示序列的279-678位。
在另一优选例中，所述的GAS6蛋白或其片段还包括GAS6蛋白的表皮生长因子(EGF)样结构域的部分片段，所述EGF样结构域的氨基酸序列为SEQ ID NO:1所示序列的118-278位。
在另一优选例中，所述EGF样结构域的部分片段的氨基酸序列为SEQ ID NO:1所示序列的n-278位，
其中n为118至275的正整数，优选为250至270的正整数，最优选为261。
在另一优选例中，所述的GAS6蛋白或其片段包括GAS6蛋白的胞外结构域，所述GAS6蛋白的胞外结构域的氨基酸序列为对应于SEQ ID NO:1所示序列的31-678位。
在另一优选例中，所述的CAR的胞外结合域除了含有针对GAS6受体的第一胞外结构域之外，还包括针对额外靶点的第二胞外结构域。
在另一优选例中，所述的额外靶点为肿瘤特异性靶点。
在另一优选例中，所述的GAS6蛋白或其片段与AXL蛋白在内的GAS6受体具有特异性结合。
在另一优选例中，所述的GAS6受体为受体酪氨酸激酶。
在另一优选例中，所述的受体酪氨酸激酶选自下组：AXL，Tyro3,MERTK，或其组合。
在另一优选例中，所述的GAS6受体选自下组：AXL，Tyro3,MERTK。
在另一优选例中，所述的受体酪氨酸激酶AXL是位于细胞膜上(或跨膜、胞质)。
在另一优选例中，所述的受体酪氨酸激酶是AXL。
在另一优选例中，所述的受体酪氨酸激酶AXL来源于人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括：啮齿动物(如大鼠、小鼠)、灵长动物(如猴)；优选为灵长动物。
在另一优选例中，所述的受体酪氨酸激酶AXL是人源的或猴源的。
在另一优选例中，所述的AXL是人源的。
在另一优选例中，所述GAS6蛋白或其片段具有如SEQ ID NO:1所示的氨基酸序列，或具有如SEQ ID NO:1所示序列的第1至678位(较佳地为第31至678位，更佳地第118至678位,更佳地为第261-678位)的氨基酸序列。
在另一优选例中，所述的GAS6蛋白或其片段的氨基酸序列选自下组：
(i)如SEQ ID NO:1所示序列的第31至678位所示的序列；和
(ii)在如SEQ ID NO:1所示序列的第261至678位所示序列的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至30个氨基酸残基，较佳地1至10个氨基酸残基，更佳地1至5个氨基酸残基，从而获得的氨基酸序列；并且所述获得的氨基酸序列与如SEQ ID NO:1所示序列的第261至678位所示序列具有≥85％(优选地≥90％，更优选地≥95％，例如≥96％、≥97％、≥98％或≥99％)的序列同一性；并且所获得的氨基酸序列与(i)所示的序列具有相同或相似的功能。
在另一优选例中，所述嵌合抗原受体的结构如下式I所示：
L-EB-H-TM-C-CD3ζ-RP    (I)
式中，
各“-”独立地为连接肽或肽键；
L是无或信号肽序列；
EB是胞外结合域；
H是无或铰链区；
TM是跨膜结构域；
C是无或共刺激信号分子；
CD3ζ是源于CD3ζ的胞浆信号传导序列；
RP是无或报告蛋白。
在另一优选例中，所述的报告蛋白RP中还包括位于其N端的自剪切识别位点，优选地为T2A序列。
在另一优选例中，所述的报告蛋白RP为荧光蛋白。
在另一优选例中，所述的报告蛋白RP为mKate2红色荧光蛋白。
在另一优选例中，所述的mKate2红色荧光蛋白的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，所述的L是选自下组的蛋白的信号肽：CD8、CD28、GM-CSF、CD4、CD137、或其组合。
在另一优选例中，所述的L是CD8来源的信号肽。
在另一优选例中，所述L的氨基酸序列如SEQ ID NO:3所示。
在另一优选例中，所述的H是选自下组的蛋白的铰链区：CD8、CD28、CD137、或其组合。
在另一优选例中，所述的H是CD8来源的铰链区。
在另一优选例中，所述H的氨基酸序列如SEQ ID NO:4所示。
在另一优选例中，所述的TM是为选自下组的蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述的TM是CD8来源的跨膜区。
在另一优选例中，所述TM的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述的C是选自下组的蛋白的共刺激信号分子：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2，或其组合。
在另一优选例中，所述的C是4-1BB来源的共刺激信号分子。
在另一优选例中，所述C的氨基酸序列如SEQ ID NO:6所示。
在另一优选例中，所述的源于CD3ζ的胞浆信号传导序列的氨基酸序列如SEQ ID NO:7所示。
在另一优选例中，所述的嵌合抗原受体CAR的氨基酸序列如SEQ ID NO:8所示。
在本发明的第二方面，提供了一种核酸分子，所述核酸分子编码如本发明第一方面所述的嵌合抗原受体。
在本发明的第三方面，提供了一种载体，其特征在于，所述的载体含有如本发明第二方面所述的核酸分子。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、或其组合。
在另一优选例中，所述载体为慢病毒载体。
在另一优选例中，所述载体选自下组：pTomo慢病毒载体、plenti、pLVTH、pLJM1、pHCMV、pLBS.CAG、pHR、pLV等。
在另一优选例中，所述的载体是pTomo慢病毒载体。
在另一优选例中，所述载体中还包括选自下组的：启动子、转录增强元件WPRE、长末端重复序列LTR等。
在另一优选例中，所述载体包含如SEQ ID NO:9所示的核苷酸序列。
在本发明的第四方面，提供了一种宿主细胞，所述的宿主细胞含有如本发明第三方面所述的载体或染色体中整合有外源的如本发明第二方面所述的核酸分子或表达如本发明第一方面所述的嵌合抗原受体。
在本发明的第五方面，提供了一种工程化免疫细胞，所述的免疫细胞含有如本发明第三方面所述的载体或染色体中整合有外源的如本发明第二方面所述的核酸分子或表达如本发明第一方面所述的嵌合抗原受体。
在另一优选例中，所述的工程化免疫细胞选自下组：T细胞、NK细胞、NKT细胞，或巨噬细胞。
在另一优选例中，所述的工程化的免疫细胞是嵌合抗原受体T细胞(CAR-T细胞)或嵌合抗原受体NK细胞(CAR-NK细胞)。
在另一优选例中，所述的工程化免疫细胞是CAR-T细胞。
在本发明的第六方面，提供了一种制备如本发明第五方面所述的工程化免疫细胞的方法，包括以下步骤：将如本发明第二方面所述的核酸分子或如本发明第三方面所述的载体转导入免疫细胞内，从而获得所述工程化免疫细胞。
在另一优选例中，所述的方法还包括对获得的工程化免疫细胞进行功能和有效性检测的步骤。
在本发明的第七方面，提供了一种药物组合物，所述药物组合物含有如本发明第一方面所述的嵌合抗原受体、如本发明第二方面所述的核酸分子、如本发明第三方面所述的载体、如本发明第四方面所述的宿主细胞，和/或如本发明第五方面所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂的剂型为注射剂。
在另一优选例中，所述制剂中所述工程化的免疫细胞的浓度为1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
在本发明的第八方面，提供了一种如本发明第一方面所述的嵌合抗原受体、如本发明第二方面所述的核酸分子、如本发明第三方面所述的载体、或如本发明第四方面所述的宿主细胞，和/或如本发明第五方面所述的工程化免疫细胞的用途，用于制备预防和/或治疗GAS6受体异常表达相关的疾病的药物或制剂。
在另一优选例中，所述的GAS6受体包括但不限于Tyro3,AXL，MERTK等。
在另一优选例中，所述的GAS6受体异常表达是指所述GAS6受体过度表达。
在另一优选例中，所述GAS6受体过度是指所述GAS6受体表达量为正常生理状况下表达量的≥1.5倍，较佳地≥2倍，更佳地≥2.5倍。
在另一优选例中，所述的GAS6受体异常表达相关的疾病包括但不限于肿瘤、静脉血栓栓塞性疾病，系统性红斑狼疮，慢性肾功能衰竭和先兆子痫等。
在另一优选例中，所述的GAS6受体异常表达相关的疾病包括：受体酪氨酸激酶AXL异常表达相关的疾病的药物或制剂。
在另一优选例中，所述的AXL异常表达相关的疾病包括：肿瘤、静脉血栓栓塞性疾病，系统性红斑狼疮，慢性肾功能衰竭和先兆子痫等。
在另一优选例中，所述的疾病是AXL高表达的恶性肿瘤。
在另一优选例中，所述肿瘤包括血液肿瘤和实体瘤。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)，或其组合。
在另一优选例中，所述的实体瘤选自下组：胰腺癌、乳腺癌、胃癌、肝胆癌、结直肠癌、膀胱癌、非小细胞肺癌、卵巢癌和食道癌、胶质细胞瘤、肺癌、前列腺癌、鼻咽癌或其组合。
在另一优选例中，所述的肿瘤选自下组：胰腺癌、肝癌、胶质细胞瘤、胃癌、前列腺癌。
在本发明的第九方面，提供了一种如本发明第五方面所述的工程化免疫细胞、或 如本发明第七方面所述的药物组合物的用途，用于预防和/或治疗癌症或肿瘤。
在本发明的第十方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用有效量的如本发明第五方面所述的工程化免疫细胞、或如本发明第七方面所述的药物组合物。
在另一优选例中，所述疾病为GAS6受体异常表达相关的疾病。
在另一优选例中，所述疾病为癌症或肿瘤。
在另一优选例中，所述的工程化免疫细胞或药物组合物中所包含的CAR免疫细胞是来源于所述对象的细胞(自体细胞)。
在另一优选例中，所述的工程化免疫细胞或药物组合物中所包含的CAR免疫细胞是来源于健康个体的细胞(异体细胞)。
在另一优选例中，所述的方法可与其他治疗方法联合使用。
在另一优选例中，所述的其他治疗方法包括化疗、放疗、靶向治疗等方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了GAS6-CAR载体构建示意图。
其中，A为GAS6序列示意图，其中1-30AA为信号肽、31-678AA为胞外结构域、49-90AA为γ-羧基谷氨酸(Gla)结构域、91-117AA为二硫化合物桥、118-278AA为表皮生长因子(EGF)样结构域、279-678AA为两个层粘连蛋白G样(LG)结构域；B为对照组质粒Mock-CAR及GAS6-CAR结构示意图，其中信号肽、铰链区、跨膜区均来源于人CD8分子，4-1BB来自于人CD137,CD3ζ来源于人CD3,mKate2为荧光标记，用于检测CAR表达；C为pTomo-GAS6-CAR载体HindIII酶切鉴定。
图2显示了CAR转染效率检测结果。
其中，A为Mock-CAR及GAS6-CAR感染T细胞72小时后细胞荧光表达结果，其中BF为明场，mKate2为CAR荧光表达；B为流式检测荧光表达结果。
图3显示了GAS6-CAR对不同肿瘤细胞系的杀伤作用。
其中，A,胰腺癌。B,胶质细胞瘤。C,非小细胞肺癌。D,前列腺癌。E,肝癌。F,乳腺瘤。G,胃癌。
图4显示了不同胰腺癌细胞系AXL表达检测的结果。
其中，A为WB检测AXL蛋白表达。B为qPCR检测AXL mRNA水平。C为流式检测AXL表达。D为免疫荧光检测AXL在细胞膜上的表达水平。
图5显示了GAS6-CAR对不同胰腺癌细胞系的梯度杀伤结果。
图6显示了GAS6-CAR对不同胰腺癌细胞系杀伤后的IFNγ释放结果。
图7显示了在胰腺癌细胞系BXPC3中过表达AXL增强GAS6-CAR杀伤作用。
其中，A为WB检测AXL蛋白表达。B为qPCR检测AXL mRNA水平。C为流式检测AXL表达。D为免疫荧光检测AXL在细胞膜上的表达水平。E为GAS6-CAR对BXPC3-AXL的杀伤检测。F为细胞因子IFNγ释放检测结果。
图8显示了在胰腺癌细胞系PANC1中敲降AXL表达降低GAS6-CAR杀伤作用。
其中，A为WB检测AXL蛋白表达。B为qPCR检测AXL mRNA水平。C为流式检测AXL表达。D为免疫荧光检测AXL在细胞膜上的表达水平。E为GAS6-CAR对PANC1-shAXL的杀伤检测。F为细胞因子IFNγ释放检测结果。
图9显示了GAS6-CAR对正常细胞系HEK-293T的杀伤结果。
其中，A为WB检测AXL蛋白表达。B为qPCR检测AXL mRNA水平。C为流式检测AXL表达。D为免疫荧光检测AXL在细胞膜上的表达水平。E为GAS6-CAR对HEK-293T的杀伤检测。F为细胞因子IFNγ释放检测结果。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种基于GAS6构建的嵌合抗原受体免疫细胞制备及其应用。实验结果表明，本发明的靶向GAS6受体的CAR-T具有对靶细胞杀伤效果显著、特异性抗肿瘤细胞的效果。在此基础上完成了本发明。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
此外，X1-X2的任一正整数表示介于X1和X2之间(包括端点)的任何正整数，例如1-5的正整数包括1、2、3、4和5，依此类推。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。
如本文所用，术语“含有”或“包括(包含)”可以使开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”或“由…构成”。
“转导”、“转染”、“转化”或本文用到的术语指的是将外源多核苷酸传递导至宿主细胞，转录和翻译产生多肽产物的过程，包括利用质粒分子将外源多核苷酸引入宿主细胞(例如大肠杆菌)。
“基因表达”或“表达”指的是基因转录，翻译和翻译后修饰产生基因的RNA或蛋白产物的过程。
“多核苷酸”指的是任意长度的核苷酸的聚合形式，包括脱氧核苷酸(DNA)，核糖核苷酸(RNA)，其杂合序列和类似物。多核苷酸可包括修饰的核苷酸，比如甲基化或加帽的核苷酸或核苷酸类似物。本文使用的术语多核苷酸指可互换的单链和双链分子。除非另有说明，本文描述的任意实施例里的多核苷酸包括双链的形式和已知的或可预测的构成双链形式的两条互补的单链。
保守氨基酸的取代是本领域已知的。在一些实施例中，潜在的取代氨基酸在以下组的一个或多个内：甘氨酸，丙氨酸；和缬氨酸，异亮氨酸，亮氨酸和脯氨酸；天冬氨酸，谷氨酸；天冬酰胺，谷氨酰胺；丝氨酸，苏氨酸赖氨酸，精氨酸和组氨酸；和/或苯丙氨酸，色氨酸和酪氨酸；蛋氨酸和半胱氨酸。此外，本发明还提供了允许来自不同基团的氨基酸取代的非保守的氨基酸取代。
本领域技术人员将容易理解本文所述的所有参数，尺寸，材料和构造的含义。实际参数，尺寸，材料和/或配置取决于使用本发明说明的特定应用。本领域技术人员能够理解，实施例或权利要求仅是通过示例的方式给出的，并且在等效物或权利要求的范围内，本发明的实施例可涵盖的范围不限于具体描述和要求的范围。
本文的定义和使用的所有定义应被理解为超过词典定义或通过引用并入的文档中的定义。
本文所发明的所有参考文献，专利和专利申请都相对于其所引用的主题通过引用并入，在某些情况下可能包含整个文档。
应当理解，对于本文所述的包括一个以上步骤的任何方法，步骤的顺序不一定限于这些实施例中描述的顺序。
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一 种将本发明的产品物理引入受试者，包括静脉内、肌内、皮下、腹膜内、脊髓或其它肠胃外给药途径，例如通过注射或输注。
TAM家族
TAM家族是受体酪氨酸激酶(Receptor tyrosine kinase，RTK)，家族含有成员AXL、Tyro3、MERTK，在肿瘤发生发展中起着重要作用。AXL基因位于第19号染色体长臂，由20个外显子组成，最初在慢性粒细胞白血病患者中发现。该蛋白质由AXL基因(或UFO或ARK或Tyro7或JTK11)编码，全长AXL蛋白包含894个氨基酸，分子量为98kDa；但是，由于翻译后糖基化，实际的AXL蛋白为120kDa(部分糖基化)或140kDa(完全糖基化)。
AXL由细胞外、跨膜和细胞内结构域组成，细胞外结构由两个免疫球蛋白(IgL)样重复序列和两个纤连蛋白III型(Fibronectin type III，Fro III)样重复序列组成；Fro III结构域负责AXL与其配体GAS6的结合。AXL细胞内结构域在自身磷酸化和下游的激酶活性中具有重要作用。
研究表明，AXL在多种临床肿瘤(如肺癌、胰腺癌、胶质瘤、前列腺癌、乳腺癌、肝癌、结肠癌、卵巢癌、胃癌等)组织中高表达，包括蛋白水平和RNA水平；且在对应的肿瘤细胞系中高表达；同时有研究表明，AXL在炎症性疾病和肿瘤微环境相关的巨噬细胞中高表达。AXL的高表达与肿瘤细胞的存活、增殖、转移、侵袭、上皮-间质化和癌细胞的干性有着密切的关系，还与患者的愈后、存活率有关；AXL的表达甚至影响肿瘤微环境以促进肿瘤免疫抑制。利用基因敲降手段降低AXL的表达，可显著抑制肿瘤细胞的侵袭、转移，并改善预后。
在肿瘤免疫治疗领域，T细胞可以通过AXL产生抗肿瘤作用(诸如三阴性乳腺癌)，与AXL阴性肿瘤细胞相比，AXL高表达肿瘤细胞更容易被AXL-CAR T细胞杀死。上述结果表明AXL是肿瘤治疗的重要靶标分子。
生长停滞特异蛋白6(Growth arrest-specific protein 6,GAS6或AXSF、AXLLG)
GAS6是一种维生素K依赖的多域蛋白，分子量为75kDa；GAS6包含678个氨基酸，包括：N末端的γ-羧基谷氨酸(Gla)结构域(氨基酸49-90)该结构域赋予了GAS6与细胞表面阴离子磷脂结合的能力，与细胞增殖的刺激有关；四个表皮生长因子(EGF)样域(氨基酸118-278)和两个层粘连蛋白G样(LG)域(氨基酸279–678)。AXL和GAS6晶体复合物显示，GAS6的C端LG结构域与AXL的胞外结构域IgL-1和IgL-2结合。
GAS6基因在多种癌症中高表达，并已被认为是不良的预后标志物，其蛋白质水平升高还与多种疾病状态相关，包括静脉血栓栓塞性疾病，系统性红斑狼疮， 慢性肾功能衰竭和先兆子痫等。
GAS6是TAM家族(包括Tyro3,AXL，MERTK)的配体，除AXL外还可结合TAM家族其他成员，其中GAS6与AXL的结合亲和力最高，而且GAS6是目前发现的AXL的唯一激活配体。AXL在与其配体GAS6高亲和力结合时，AXL受体的细胞内激酶结构域发生同源二聚化以及反式自磷酸化，进而募集衔接分子和含有Src同源性2(Src homology 2，SH2)的效应蛋白或其他磷酸酪氨酸结合域(Phosphotyrosine binding domains，PTB)，从而激活下游信号通路RAS、RAF、MAPK、ERK1/2、PI3K、TGF-β1/2等，进而发挥作用。GAS6/AXL信号通路与肿瘤细胞的生长、转移、侵袭、上皮-间质转化(Epithelial-mesenchymal transition，EMT)、血管生成、耐药、免疫调节和干细胞维持密切相关。
基于此，本发明首次将GAS6的特定片段通过基因工程方式整合入CAR载体中，并修饰了相关免疫细胞，从而实现对GAS6结合蛋白阳性的细胞特异杀伤，可用于相关疾病的治疗。
本发明嵌合抗原受体(CAR)
嵌合免疫抗原受体(Chimeric antigen receptor，CAR)由胞外抗原识别区域、跨膜区以及胞内共刺激信号区域组成。
CAR的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效。第二代CAR在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CAR相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CAR基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CAR。
CAR的胞外段可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子，进而清除靶细胞。首先分离病人自体细胞(或者异源供体)，激活并进行基因改造产生CAR的免疫细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被免疫细胞以非MHC限制方式识别。
CAR-免疫细胞治疗在血液恶性肿瘤治疗中取得了非常高的临床反应率，这样的高反应率是以往任何一种治疗手段都无法达到的，在世界各引发了临床研究的热潮。
具体地，本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。
胞外结构域包括靶-特异性结合元件。所述的胞外结构域可以是基于抗原-抗 体的特异性结合的抗体的ScFv，也可以是基于配体-受体的特异性结合的天然序列或其衍生物。
在本发明中，所述嵌合抗原受体的胞外结构域是一种可特异性结合本发明CAR的AXL靶点的GAS6蛋白或其片段。更加优选地，本发明嵌合抗原受体的胞外结合域具有如SEQ ID NO:1所示序列的第261至678位的氨基酸序列。
细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与CD8信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
在本发明中，本发明CAR的胞外结合域还包括基于序列的保守性变异体，指与SEQ ID NO:1的第261至678位(或第279-678位)的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、3、4或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
本发明的CAR中的胞内结构域包括4-1BB共刺激结构域和CD3ζ的信号传导结构域。
在本发明的一个实施方式中，所述的CAR是可以特异性靶向AXL的CAR。
嵌合抗原受体免疫细胞(CAR-免疫细胞)
在本发明中，提供了一种嵌合抗原受体免疫细胞，其包含本发明的具有特异性靶向GAS6受体(优选地为AXL)的嵌合抗原受体。
本发明的嵌合抗原受体免疫细胞可以是CAR-T细胞，也可以是CAR-NK细胞，CAR-巨噬细胞。优选地，本发明的嵌合抗原受体免疫细胞是CAR-T细胞。
如本文所用，术语“CAR-T细胞”、“CAR-T”、“本发明CAR-T细胞”均指本发明第五方面所述的CAR-T细胞。
CAR-T细胞较其它基于T细胞的治疗方式存在以下优势：(1)CAR-T细胞的作用过程不受MHC的限制；(2)鉴于很多肿瘤细胞表达相同的肿瘤标志物，针对某一种肿瘤标志物的CAR基因构建一旦完成，便可以被广泛利用；(3)CAR既可以利用肿瘤蛋白质标志物，又可利用糖脂类非蛋白质标志物，扩大了肿瘤标志物的靶点范围；(4)使用患者自体细胞降低了排异反应的风险；(5)CAR-T细胞具有免疫记忆功能，可以长期在体内存活。
如本文所用，术语“CAR-NK细胞”、“CAR-NK”、“本发明CAR-NK细胞”均指本发明第五方面所述的CAR-NK细胞。本发明CAR-NK细胞可用于AXL受体(优选地为AXL)高表达的肿瘤。
自然杀伤(NK)细胞是一类主要的免疫效应细胞，通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能，包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与CAR-T细胞相比，CAR-NK细胞还具有一下优点，例如：(1)通过释放穿孔素和颗粒酶直接杀伤肿瘤细胞，而对机体正常的细胞没有杀伤作用；(2)它们释放很少量的细胞因子从而降低了细胞因子风暴的危险；(3)体外极易扩增及发展为“现成的”产品。除此之外，与CAR-T细胞治疗类似。
如本文所用，术语“CAR-巨噬(M)细胞”“CAR-巨噬(M)”、“本发明CAR-巨噬(M)细胞”均指本发明第五方面所述的CAR-巨噬细胞。本发明CAR-巨噬细胞可用于AXL受体(优选地为AXL)高表达的炎性疾病和肿瘤。
巨噬细胞为吞噬细胞，来自单核细胞，在机体的先天性免疫和细胞免疫中伴有重要作用。通过工程化(基因修饰)的巨噬细胞可增强特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了包含本发明的核酸分子的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动 子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。在本发明的一个实施方式中，报告基因是编码mKate2红色荧光蛋白的基因。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒 载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选的实施方式中，所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明第一方面所述的嵌合抗原受体CAR、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面的宿主细胞或本发明第五方面所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用编码本发明表达盒的慢病毒载体(LV)转导的细胞(例如，T细胞)进 行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物受体酪氨酸激酶(优选地为AXL)，协同激活T细胞，引起免疫细胞免疫应答，从而显著提高其对肿瘤细胞的杀伤效率。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，受体酪氨酸激酶(优选地为AXL)的CAR-T细胞引起受体酪氨酸激酶表达(优选地为AXL)细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括GAS6蛋白或其片段、铰链和跨膜区、和4-1BB和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌、卵巢癌、乳腺癌、胃癌、肝胆癌、结直肠癌、膀胱癌、非小细胞肺癌、卵巢癌和食道癌、成胶质细胞瘤、肺癌、前列腺癌、鼻咽癌等。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“有效量”、“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细 胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对具体肿瘤患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明的CAR-T细胞，通过例如静脉回输的方式，施用于患者。
本发明的主要优点包括：
1)靶点特异：AXL在正常细胞的细胞膜上基本不表达，但在肿瘤组织细胞膜及巨噬细胞上高表达，从而本CAR特异性杀死膜上高表达AXL的肿瘤细胞和巨噬细胞，而对正常细胞无明显杀伤作用。
2)本发明利用配体与受体相结合作用方式，而非传统意义上的scfv。受体-配体相互作用的保守性决定了在动物特别是灵长类动物中的安全性试验更能反应其在人体的安全性。
3)GAS6也可结合TAM家族其他成员，多靶点作用可避免肿瘤细胞免疫逃逸。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本申请实施例中的试剂、质粒、和细胞，除非另外说明，均为可市售获得的。
表1序列
Figure PCTCN2022130701-appb-000001
Figure PCTCN2022130701-appb-000002
Figure PCTCN2022130701-appb-000003
表2细胞系
细胞系 类型
PANC1 胰腺癌细胞
BXPC3 胰腺癌细胞
ASPC1 胰腺癌细胞
U251 胶质瘤细胞
U87 胶质瘤细胞
H1299 非小细胞肺癌细胞
A549 非小细胞肺癌细胞
PC3 前列腺癌细胞
MHCC-97H 肝癌细胞
HepG2 肝癌细胞
SMMC-7721 肝癌细胞
MDA-MB468 乳腺癌细胞
MDA-MB231 乳腺癌细胞
NUGC4 胃癌细胞
MKN28 胃癌细胞
AGS 胃癌细胞
实施例1：制备GAS6-CAR载体
基于GAS6的核苷酸序列(NM_000820.4)、人CD8信号肽、人CD8α铰链区、人CD8跨膜区、人4-1BB胞内区以及人CD3ζ胞内区基因序列信息，通过人工合成方法或PCR法获得相应的核苷酸序列。合成CD8信号肽及GAS6胞外区域(为保证GAS6胞外域粘连蛋白G样结构的完整性，在其N端保留了表皮生长因子样域的18个氨基酸)，并通过AgeI(Thermo)和NheI(Thermo)双酶切该CAR分子的核苷酸序列，经T4DNA连接酶(NEB)连接插入已将CD8跨膜区、4-1BB共刺激结构域、CD3ζ信号传导区插入的慢病毒载体pTomo中。转化感受态大肠杆菌(Stbl3)。
将重组质粒进行测序，比对测序结果以确认质粒是否正确，测序引物为通用测序引物。测序和酶切鉴定结果均表明，CAR的编码序列正确地插入了质粒的预定位置(图1C)。
所有质粒均用QIAGEN公司的无内毒素大抽试剂盒抽提，纯化质粒用碧云天lipo6000转染HEK-293T细胞进行慢病毒包装。
实施例2：病毒包装
在15cm培养皿中培养HEK-293T细胞用于病毒包装。待HEK-293T细胞汇合度在80％-90％左右进行转染，准备2ml OPTIMEM溶解的质粒混合物(核心质粒20ug、pCMVΔR8.9 10ug、PMD2.G 4ug)；在另一离心管中2ml OPTIMEM以及68ul的lipo6000。室温静置5min后，将质粒复合物加入脂质体复合物中，室温静置20min。将上述混合物滴加入HEK-293T细胞中，37℃孵育6小时后去除培养基。重新加入预热的完全培养基。收集48小时和72小时病毒上清后，于4℃3000rpm离心20分钟。用0.45um滤膜过滤后，于25000rpm 4℃离心2.5小时进行病毒浓缩。浓缩的病毒用30ul病毒溶解液过夜溶解后，病毒滴度用QPCR检测。结果显示，病毒滴度达到要求。
实施例3：CAR-T细胞制备
用Ficool分离液从人外周血中分离单核细胞，由RosetteSep Human T Cell Enrichment Cocktail(Stemcell technologies)获得纯化的CD3+T细胞。T细胞用CD3/CD28磁珠进行活化(Life technology)，再加入200U/ml的IL2(PeproTech)，刺激培养48小时后进行病毒感染。慢病毒在lentiboost存在时按照MOI＝100感染T细 胞制备CAR-T细胞。感染一天后更换培养基。
实施例4：流式细胞仪检测感染CAR-T细胞的阳性率
分别离心收集病毒感染72小时后的CAR-T细胞和NTD细胞(对照组)，PBS洗涤一次后弃上清，用含有2％FBS的PBS重悬细胞，流式检测阳性率。
转染效率的结果如图2所示。
如图2A所示，CAR-T细胞表达的CAR-T2A-mKate2融合蛋白经切割后，形成的mKate2蛋白在胞内表现出红色荧光。
图2B显示，采用流式细胞术进行检测，表明CAR或mKate2CAR-T的阳性表达率为约30％。
实施例5：检测各靶细胞AXL的表达
(1)细胞免疫荧光：将靶细胞铺于24孔板的圆片上，24小时后用4％多聚甲醛(PFA)固定细胞20分钟，PBST洗三次，每次5分钟；用10％山羊血清室温封闭1小时，用特异性识别AXL的抗体四度孵育过夜。第二天用PBST洗三次，每次五分钟。用CY3标记的特异性识别一抗的二抗室温孵育1小时。PBS洗三次后，DAPI染核。共聚焦显微镜成像。
(2)流式细胞术：收集100万个细胞，用4％PFA室温固定细胞15min，1XPBS离心洗涤；用100％甲醇冰上通透处理细胞15min，1XPBS离心洗涤；100μl稀释的一抗(1：300)重悬细胞，室温孵育1小时；1XPBS离心洗涤。丢弃上清液。重复操作。用100μl稀释的荧光物质偶联的二抗(FITC-488anti-rabbit)重悬细胞，室温避光孵育30分钟，1X PBS进行离心洗涤。丢弃上清液。重复操作。用300μl 1X PBS重悬细胞，流式细胞分析仪。
(3)免疫印迹：收集6cm培养皿细胞，5500r/min，4℃离心5min；去上清后，根据细胞数目加入含蛋白酶抑制剂PMSF的RIPA细胞裂解液，冰上裂解20min，14,000r/min，4℃离心30min，收集上清溶液测浓度；上样蛋白质样品量50μg，进行蛋白电泳，检测靶细胞AXL的表达。
(4)qPCR：收集6孔板细胞，去除培养基，加入1ml Trizol裂解细胞，室温静置5min，加入200μl/1ml Trizol的氯仿，颠倒混匀6-8次，室温静置5min；12000g，4℃离心15分钟，吸取上层清液到另一离心管中；加入等体积的异丙醇，颠倒混合，室温放置10min；12000g，4℃离心10分钟，弃上清；加入1ml 70％乙醇(用RNase free H2O配制)洗涤，7500g，室温离心5min；弃上清，室温放置10min干燥RNA，加30ul RNase free水溶解RNA；Nandrop 2000测定RNA的浓度，并用1％琼脂糖凝胶电泳检测RNA的完整性及定量的准确性。按RevertAidTM First Strand cDNA Synthesis Kit(Thermo Scientific)的说明书合成CDNA，并进行mRNA 水平的检测。
各细胞系AXL表达检测的结果见图4。通过WB(图4A)、qPCR(图4B)、流式细胞术(图4C)检测靶细胞AXL的表达，结果一致表明PANC1、ASPC1高表达AXL,BXPC3低表达AXL。进一步通过免疫荧光定位验证了PANC1、ASPC1细胞膜上AXL高表达，PANC1、BXPC3细胞膜上AXL高表达(图4D)。
实施例6：携带luciferase的靶细胞构建
pTomo-CMV-Luciferase-IRES-Puro慢病毒包装步骤与实施例2中相同。
病毒感染PANC1、BXPC3、ASPC1、U251、U87、H1299、A549、PC3、MHCC-97H、HepG2、SMMC-7721、MDA-MB468、MDA-MB231、NUGC4AGS、MKN28、HEK-293T细胞后用Puromycin(1ug/ml)筛选2周，成功获得PANC1、BXPC3、ASPC1、U251、U87、H1299、A549、PC3、MHCC-97H、HepG2、SMMC-7721、MDA-MB468、MDA-MB231、NUGC4AGS、MKN28-luciferase、HEK-293T-luciferase细胞。
实施例7：CAR-T细胞杀伤
在本实施例中，检测本发明CAR-T细胞对不同靶细胞的杀伤能力。采用的靶细胞包括：高表达AXL的靶细胞：PANC1、ASPC1、U251、U87、H1299、PC3、HepG2、MDA-MB231、AGS；不表达或低表达AXL的靶细胞：BXPC3、A549、MHCC-97H、SMMC-7721、MDA-MB468、NUGC4、MKN28。
将PANC1、BXPC3、ASPC1、U251、U87、H1299、A549、PC3、MHCC-97H、HepG2、SMMC-7721、MDA-MB468、MDA-MB231、NUGC4AGS、MKN28-luciferase细胞消化计数后调整细胞密度为2×10 4/ml。将100ul luciferase细胞接种于96孔板中，将CAR-T和NT细胞调整细胞密度为1×10 5/ml,按照E:T为5:1接种至黑色96孔板中，每孔接种100ul。将上述靶细胞和T细胞混匀后至于培养箱孵育24小时。
将PANC1、BXPC3、ASPC1-luciferase细胞消化计数后调整细胞密度为2×10 4/ml。将100ul PANC1、BXPC3、ASPC1-luciferase细胞接种于96孔板中，将CAR-T和NT细胞调整细胞密度为1×10 5/ml,按照E:T为0.5:1、1:1、2:1、4:1接种至黑色96孔板中，每孔接种100ul。将上述靶细胞和T细胞混匀后至于培养箱孵育24小时。
收集细胞上清冻存于-80℃检测IFNγ释放量(见实施例8)。细胞杀伤用promega荧光检测试剂盒检测，首先细胞用20ul 1*PLB裂解液处理细胞20分钟，每孔加入100ul底物后立即用BioTek酶标仪检测。
细胞毒性杀伤细胞％＝(1-含效应细胞时靶细胞荧光值/无效应细胞时靶细胞荧光值)×100％
GAS6-CAR对不同肿瘤细胞系的杀伤作用结果如图3所示。胰腺癌细胞(A)；胶质细胞瘤细胞(B)；非小细胞肺癌细胞(C)；前列腺癌细胞(D)；肝癌细胞(E)； 乳腺瘤细胞(F)；胃癌细胞(G)。结果表明GAS6-CAR对多个肿瘤细胞系均有良好的杀伤效果。
GAS6-CAR对不同胰腺癌细胞系的梯度杀伤结果如图5所示。(A)GAS6-CAR对PANC1的梯度杀伤作用；(B)GAS6-CAR对BXPC3的梯度杀伤作用；(C)GAS6-CAR对ASPC1的梯度杀伤作用。结果表明，GAS6CAR-T细胞对AXL高表达肿瘤细胞的杀伤作用随着效靶比(E:T)升高而逐渐增强。
实施例8：IFNγ细胞因子释放
在本实施例中，检测本发明CAR-T细胞与靶细胞共孵育情况下的细胞因子的释放情况。采用细胞杀伤实验中共孵育的细胞上清进行检测。
方法如下：取实施例7中本发明CAR-T细胞与PANC1、BXPC3、ASPC1、HEK-293T靶细胞(E：T比为4：1)共孵育的细胞上清按照IFN gamma Human ELISA Kit(life technology)检测IFNγ。
用Standard Dilution Buffer溶解标准品，并进行梯度稀释成1000pg/ml、500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.2pg/ml、15.6pg/ml、0pg/ml的标准品。
每孔中加入50ul Incubation buffer、50ul检测样本、50ulIFNγbiotin conjugated solution，混匀后室温静置90分钟。
然后依次按照以下步骤进行操作：
(1)用1*Wash Buffer洗孔4次，每次停留1分钟。
(2)每孔加入100ul 1*Streptavidin-HRP solution，室温静置45分钟。
(3)用1*Wash Buffer洗孔4次，每次停留1分钟。
(4)加入100ul Stabilized chromogen，室温静置30分钟.
(5)每孔加入100ul Stop solution后混匀。
(6)450nm处检测吸光值。
结果如图6所示。GAS6-CAR-T杀伤PANC1和ASPC1后的细胞因子明显增加，BXPC3无明显变化。结果表明，GAS6-CAR-T细胞对肿瘤细胞的杀伤作用伴随着IFNγ释放，提示该杀伤作用与IFNγ释放有关。
实施例9：过表达AXL后对GAS6-CAR-T杀伤作用的影响
根据AXL的CDS区序列，从易锦生物购买AXL过表达质粒(EX-Z7835-Lv105-B，ORF lentiviral expression clone)，瞬时转染AXL过表达质粒。
在转染前一天按照每孔50万靶细胞接种到六孔板内进行培养。转染前，将六孔板每孔换成2ml新鲜培养液(含有血清，不含抗生素)；取两个洁净无菌离心管，分别加入125μl
Figure PCTCN2022130701-appb-000004
Medium，然后于其中一管加入2.5μg质粒DNA，并用枪轻轻 吹打混匀；另一管加入5μl Lipo6000 TM转染试剂，用枪轻轻吹打混匀。室温静置5分钟后，将含有DNA的培养液用枪轻轻加入含Lipo6000 TM转染试剂的培养液中，轻轻颠倒离心管或者用枪轻轻吹打混匀，室温静置5分钟后加入6孔板混匀，于48h后检测过表达效率，并进行GAS6-CAR-T杀伤检测。
将不同胰腺癌细胞系BXPC3-Vector、BXPC3-AXL-luciferase细胞消化计数后调整细胞密度为2×10 4/ml。将100ul luciferase细胞接种于96孔板中，将CAR-T/NT细胞调整细胞密度为1×10 5,按照E:T为4:1接种至黑色96孔板中，每孔接种100ul。将上述靶细胞和T细胞混匀后至于培养箱孵育24小时。
GAS6-CAR-T对胰腺癌细胞系BXPC3过表达AXL后的杀伤结果如图7所示。图7-A为WB检测AXL蛋白表达。图7-B为qPCR检测AXL mRNA水平。图7-C为流式检测AXL表达。图7-D为免疫荧光检测AXL在细胞膜上的表达水平。图7-E为GAS6-CAR对BXPC3过表达AXL后的杀伤作用。图7-F为GAS6-CAR-T对BXPC3过表达AXL后杀伤作用的IFNγ释放。结果显示，相较于对比组BXPC3-Vector，GAS6-CAR-T细胞对过表达AXL的BXPC3-AXL细胞的杀伤率和IFNγ释放量均明显上升。该结果表明GAS6-CAR-T细胞对AXL过表达肿瘤细胞的杀伤作用明显增强。
实施例10：特异性敲降AXL后对GAS6-CAR-T杀伤作用影响
根据Sigma公司提供的shRNA序列库，选择MISSION shRNA Lentiviral Transduction Particles，选取CDS区Validated过的shRNA，并在NCBI上BLAST选择的每一条shRNA，确保靶点的特异性。
在转染前一天按照每皿100万HEK-293T细胞接种到6cm皿内进行培养。转染前，将6cm皿换成5ml新鲜培养液(含有血清，不含抗生素)；取两个洁净无菌离心管，分别加入250μl
Figure PCTCN2022130701-appb-000005
Medium，然后于其中一管加入5μg质粒DNA，并用枪轻轻吹打混匀；另一管加入10μl Lipo6000 TM转染试剂，用枪轻轻吹打混匀。室温静置5分钟后，将含有DNA的培养液用枪轻轻加入含Lipo6000 TM转染试剂的培养液中，轻轻颠倒离心管或者用枪轻轻吹打混匀，室温静置20分钟后加入6cm皿混匀，分别于48h、72h后收集上清过滤，3000r/min，4℃离心15min，获得含病毒上清液。
在感染前一天按照每孔50万靶细胞接种到六孔板内进行培养。感染前，将六孔板每孔换成1ml新鲜培养液(含有血清，不含抗生素)，再加入1ml病毒上清、2ul polybrane(10mg/ml)；24h后更换完全培养基，72h后检测shRNA敲降效率，并进行GAS6-CAR-T杀伤检测。体外杀伤如前所述，通过荧光值变化检测GAS6-CAR对PANC1及PANC1-shAXL的杀伤。
将PANC1-shCOO2、PANC1-shAXL#1、PANC1-shAXL#2-luciferase细胞消化计数后调整细胞密度为2×10 4/ml。将100ul luciferase细胞接种于96孔板中，将CAR-T/NT细胞调整细胞密度为1×10 5,按照E:T为4:1接种至黑色96孔板中，每孔接种100ul。 将上述靶细胞和T细胞混匀后至于培养箱孵育24小时后检测杀伤效果。
结果如图8所示。图8-A为WB检测AXL蛋白表达。图8-B为qPCR检测AXL mRNA水平。图8-C为流式检测AXL表达。图8-D为免疫荧光检测AXL在细胞膜上的表达水平。图8-E为GAS6-CAR对PANC1敲降AXL后的杀伤作用。图8-F为GAS6-CAR对PANC1沉默AXL后杀伤作用的IFNγ释放。结果显示，相较于对照组PANC1-shCOO2细胞，GAS6-CAR-T细胞对敲低AXL的PANC1-shAXL#1和PANC1-shAXL#1细胞的杀伤率和IFNγ释放量均明显下降。该结果表明，AXL在细胞膜上的敲降降低了GAS6-CAR-T的杀伤作用。
实施例11：GAS6-CAR-T对非肿瘤细胞的杀伤作用
HEK-293T细胞是人胚肾细胞系，按照效靶比0.5:1、1:1、2:1、4:1接种至黑色96孔板中，将GAS6-CAR-T细胞与HEK-293T-luciferase细胞共孵育，通过荧光值变化检测GAS6-CAR-T对HEK-293T细胞的杀伤。
结果如图9所示。图9-A为WB检测AXL蛋白表达。图9-B为qPCR检测AXL mRNA水平。图9-C为流式检测AXL表达。图9-D为免疫荧光检测AXL在细胞膜上的表达水平。图9-E为GAS6-CAR对HEK-293T的杀伤作用。图9-F为GAS6-CAR对HEK-293T杀伤作用的IFNγ释放。
上述结果表明，GAS6-CAR-T对非肿瘤细胞HEK-293T没有显著的杀伤作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种嵌合抗原受体(CAR)，其特征在于，所述的CAR含有一胞外结合域，所述的胞外结合域具有基于如SEQ ID NO:1所示氨基酸序列的GAS6蛋白或其片段的结构，并且所述的胞外结合域能够特异性地结合GAS6受体。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的胞外结合域包含GAS6蛋白或其片段，所述的GAS6蛋白或其片段具有如SEQ ID NO:1所示的氨基酸序列，或具有如SEQ ID NO:1所示序列的第1至678位(较佳地为第31至678位，更佳地第118至678位,更佳地为第261-678位)的氨基酸序列。
  3. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的GAS6蛋白或其片段的氨基酸序列选自下组：
    (i)如SEQ ID NO:1所示序列的第261至678位所示的序列；和
    (ii)在如SEQ ID NO:1所示序列的第261至678位所示序列的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至30个氨基酸残基，较佳地1至10个氨基酸残基，更佳地1至5个氨基酸残基，从而获得的氨基酸序列；并且所述获得的氨基酸序列与如SEQ ID NO:1所示序列的第261至678位所示序列具有≥85％(优选地≥90％，更优选地≥95％，例如≥96％、≥97％、≥98％或≥99％)的序列同一性；并且所获得的氨基酸序列与(i)所示的序列具有相同或相似的功能。
  4. 如权利要求1-3中任一项所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的结构如下式I所示：
    L-EB-H-TM-C-CD3ζ-RP   (I)
    式中，
    各“-”独立地为连接肽或肽键；
    L是无或信号肽序列；
    EB是胞外结合域；
    H是无或铰链区；
    TM是跨膜结构域；
    C是无或共刺激信号分子；
    CD3ζ是源于CD3ζ的胞浆信号传导序列；
    RP是无或报告蛋白。
  5. 如权利要求4所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体CAR的氨基酸序列如SEQ ID NO:8所示。
  6. 一种核酸分子，其特征在于，所述核酸分子编码如权利要求1所述的嵌合抗原受体。
  7. 一种载体，其特征在于，所述的载体含有如权利要求6所述的核酸分子。
  8. 一种宿主细胞，其特征在于，所述的宿主细胞含有如权利要求7所述的载体或染色体中整合有外源的如权利要求6所述的核酸分子或表达如权利要求1所述的嵌合抗原受体。
  9. 一种工程化免疫细胞，其特征在于，所述的免疫细胞含有如权利要求7所述的载体或染色体中整合有外源的如权利要求6所述的核酸分子或表达如权利要求1所述的嵌合抗原受体。
  10. 一种制备如权利要求9所述的工程化免疫细胞的方法，其特征在于，包括以下步骤：将如权利要求6所述的核酸分子或如权利要求7所述的载体转导入免疫细胞内，从而获得所述工程化免疫细胞。
  11. 一种药物组合物，其特征在于，所述药物组合物含有如权利要求1所述的嵌合抗原受体、如权利要求6所述的核酸分子、如权利要求7所述的载体、如权利要求8所述的宿主细胞，和/或如权利要求9所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  12. 一种如权利要求1所述的嵌合抗原受体、如权利要求6所述的核酸分子、如权利要求7所述的载体、或如权利要求8所述的宿主细胞，和/或如权利要求9所述的工程化免疫细胞的用途，其特征在于，用于制备预防和/或治疗GAS6受体异常表达相关疾病的药物或制剂。
  13. 如权利要求12所述的用途，其特征在于，所述的GAS6受体异常表达相关的疾病包括：受体酪氨酸激酶AXL异常表达相关的疾病的药物或制剂。
  14. 如权利要求13所述的用途，其特征在于，所述的AXL异常表达相关的疾病包括：肿瘤、静脉血栓栓塞性疾病，系统性红斑狼疮，慢性肾功能衰竭和先兆子痫等。
  15. 如权利要求14所述的用途，其特征在于，所述的肿瘤选自下组：胰腺癌、肝癌、胶质细胞瘤、胃癌、前列腺癌。
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