WO2023093888A1 - Preparation and use of immune cells of chimeric antigen receptor constructed on the basis of efna1 - Google Patents

Preparation and use of immune cells of chimeric antigen receptor constructed on the basis of efna1 Download PDF

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WO2023093888A1
WO2023093888A1 PCT/CN2022/134768 CN2022134768W WO2023093888A1 WO 2023093888 A1 WO2023093888 A1 WO 2023093888A1 CN 2022134768 W CN2022134768 W CN 2022134768W WO 2023093888 A1 WO2023093888 A1 WO 2023093888A1
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cells
car
efna1
chimeric antigen
amino acid
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French (fr)
Chinese (zh)
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赵旭东
魏文文
张富娟
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四川大学华西医院
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Definitions

  • the invention belongs to the technical field of biomedicine for tumor immunotherapy, and relates to specific chimeric antigen receptor immune cells, in particular to a CAR that specifically targets EFNA1 receptors, its modified immune response cells, and its preparation method and application.
  • cancer is considered to be the main cause of death in various countries in the world and the main obstacle to prolonging human life.
  • WHO World Health Organization
  • cancer is the first or second leading cause of death among people before the age of 70 in 183 countries, seriously threatening human health.
  • WHO World Health Organization
  • melanoma skin cancer a 47% increase over 2020, and overall, the burden of cancer incidence and mortality is growing rapidly worldwide.
  • colorectal cancer According to the 2020 global tumor statistics, it is estimated that there will be 19 million new patients with colorectal cancer and 935,000 patients will die of colorectal cancer in 2020; overall, colorectal cancer ranks third in incidence and mortality Ranking second, the high morbidity and mortality make colorectal cancer a serious threat to human health.
  • the treatment methods for colorectal cancer mainly include surgery for local treatment, chemotherapy for systemic treatment, targeted therapy and immunotherapy. With the improvement of medical level, significant progress has been made in systemic treatment of colorectal cancer. Six new chemotherapy drugs were introduced that increased the median overall survival of patients with metastatic colorectal cancer from less than 9 months (untreated) to about 24 months.
  • stage III node-positive colon cancer
  • fluorouracil-based chemotherapy For patients with stage III (node-positive) colon cancer, the overall survival benefit of fluorouracil-based chemotherapy has been established, and recent data suggest that the inclusion of oxaliplatin in this type of adjuvant regimen is more effective, although treatment The effect is continuously improving, but some patients still have malignant progression, so it is necessary to explore new methods for the treatment of colorectal cancer.
  • Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that can recognize specific target antigens in an unrestricted way of MHC after genetic modification, and continuously activate and expand T cells. Its main structure includes three types, namely the extracellular ScFv recognition domain, which is used to recognize and bind the target on tumor cells; the hinge region and transmembrane domain, mainly derived from CD8 or CD28, anchor CAR It is located on the cell membrane and connects the extracellular recognition domain and intracellular signal; the intracellular domain is the activation domain, and the difference in number and length will affect the anti-tumor effect of CAR-T.
  • the extracellular ScFv recognition domain which is used to recognize and bind the target on tumor cells
  • the hinge region and transmembrane domain mainly derived from CD8 or CD28
  • anchor CAR anchor CAR It is located on the cell membrane and connects the extracellular recognition domain and intracellular signal
  • the intracellular domain is the activation domain, and the difference in number and length
  • CD3 ⁇ is a common feature of the intracellular part of CAR, which can initiate signals to drive T cell killing
  • the costimulatory domain is mainly derived from the CD28 receptor family or TNF receptor family such as 4-1BB, OX40 or CD27 , by enhancing the secretion of cytokines or promoting proliferation and persistence can also improve the killing effect of CAR-T.
  • T cell Receptor TCR
  • MHC major histocompatibility complex
  • TAA tumor-associated antigen
  • CAR-T cell therapy has become a new treatment for blood diseases with good curative effect.
  • CAR-T treatment of malignant tumors is still a research hotspot at present.
  • TILs tumor-infiltrating lymphocytes
  • CAR-T therapy has gone through several years.
  • CAR-Ts are currently three CAR-Ts being used to treat tumors, including Kymriah and Yescarta approved by the FDA in August and October 2017 for the treatment of relapsed/refractory acute lymphoblastic leukemia and specific types of large B-cell lymphoma, and Tecartus, approved in July 2020, is used to treat adult mantle cell lymphoma (MCL).
  • CAR-T therapy has made good progress in blood diseases, but there are still some limitations in the treatment of solid tumors, mainly due to the selection of effective targets and the infiltration of CAR-T cells into tumors.
  • the object of the present invention is to provide a chimeric antigen receptor immune cell targeting EFNA1 receptor and its preparation and application method.
  • a chimeric antigen receptor (CAR) is provided, characterized in that, the CAR contains an extracellular binding domain, and the extracellular binding domain comprises a sequence based on SEQ ID NO: 1 The structure of EFNA1 or its fragments of the amino acid sequence shown, and the extracellular binding domain can specifically bind to the EFNA1 receptor in the form of a ligand receptor.
  • the EFNA1 receptor is selected from the following group: EphA2.
  • the extracellular binding domain has an amino acid sequence derived from EFNA1.
  • the extracellular binding domain includes EFNA1 protein or a fragment thereof.
  • the EFNA1 protein fragment includes the extracellular region of the EFNA1 protein.
  • the extracellular binding domain includes EFNA1 protein or a fragment thereof.
  • the extracellular binding domain segment includes the extracellular region of the EFNA1 protein, and the amino acid sequence of the extracellular region corresponds to positions 19-182 of the sequence shown in SEQ ID NO:1.
  • the extracellular domain has the amino acids shown in positions 19-182 of SEQ ID NO:1.
  • the EFNA1 receptor is selected from the following group: EphA2.
  • the extracellular binding domain of the CAR in addition to the first extracellular domain targeting EFNA1 receptor, also includes a second extracellular domain targeting additional targets.
  • the additional target is a tumor-specific target.
  • the EFNA1 protein or its fragments specifically bind to EFNA1 receptors including EphA2.
  • the binding molecule is selected from the following group: EphA2.
  • the EphA2 is EphA2 located on the cell membrane.
  • the EphA2 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 EphA2 is of human or monkey origin.
  • the EphA2 is of human origin.
  • the extracellular domain has the amino acid sequence shown in SEQ ID NO: 1, or has the 1st to 182nd (preferably the 1st) of the sequence shown in SEQ ID NO: 1 19 to 182 amino acid sequence).
  • the EFNA1 protein or its fragment corresponds to the amino acid sequence at positions 19-182 of the sequence shown in SEQ ID NO:1.
  • amino acid sequence of the EFNA1 protein or its fragment 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, EFNA1, 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 as described in 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 CAR 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 CAR as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, and the CAR as described in the third aspect of the present invention.
  • 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 CAR 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 vector as described in the third aspect of the present invention, or a CAR as described in the present invention
  • the use of the host cell according to the fourth aspect, and/or the engineered immune cell according to the fifth aspect of the present invention is used to prepare a drug or preparation for preventing and/or treating diseases related to abnormal expression of EFNA1 receptor.
  • the EFNA1 receptor includes but not limited to EphA2.
  • the abnormal expression of the EFNA1 receptor refers to the overexpression of the EFNA1 receptor.
  • the overexpression of the EFNA1 receptor means that the expression level of the EFNA1 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 EFNA1 receptor include but not limited to tumor, aging, obesity, cardiovascular disease, diabetes, neurodegenerative disease, infectious disease and so on.
  • the diseases related to abnormal expression of EFNA1 receptor include diseases related to abnormal expression of EphA2.
  • the diseases related to the abnormal expression of EphA2 include: tumor, aging, cardiovascular disease, obesity and so on.
  • the disease is a malignant tumor with high expression of EphA2.
  • 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), lymphoma, or a combination thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • lymphoma or a combination thereof.
  • the solid tumor is selected from the group consisting of 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, Pancreatic cancer, prostate cancer, or a combination thereof.
  • the tumor is selected from the group consisting of colorectal cancer, brain tumor, breast cancer, endometrial cancer, bladder cancer, prostate cancer, and pancreatic 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 EFNA1 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 the construction of the EFNA1-CAR vector.
  • A is the sequence diagram of EFNA1, in which 1-18AA is the signal peptide, and 19-182AA is the extracellular domain;
  • B is the structure diagram of the control plasmid MOCK-CAR and EFNA1-CAR, in which the signal peptide, hinge region, transmembrane region All are derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3 ⁇ is derived from human CD3, mKate2 is a fluorescent marker for detecting CAR expression;
  • C is pTomo-EFNA1-CAR carrier HindIII digestion identification.
  • Figure 2 shows the cell expansion fold and total number of EFNA1CAR.
  • Figure 2A T cell proliferation factor 12 days after infection;
  • Figure 2B Statistics of the total number of cells 12 days after infection.
  • Figure 3 shows the efficiency of CAR infection detected by fluorescence microscopy and flow cytometry.
  • A is the result of cell fluorescence expression after 72 hours of MOCK-CAR and EFNA1-CAR infection of T cells, the upper row is the bright field, and the lower row is the fluorescence expression of CAR; B is the result of flow cytometry detection of fluorescence expression.
  • Figure 4 shows the phenotypic detection results of CAR-T cells, and the proportion of CD4 and CD8 positive T cells was detected on d3 and d6 after infection. .
  • Figure 5 shows the killing effect of EFNA1CAR-T on different tumor cell lines (effect-to-target ratio 5:1).
  • Figure 6 shows the results of detection of EphA2 expression in colorectal cancer cell lines DLD1, HCT116 and normal cell lines 293T and COS7.
  • A is the detection of RNA level
  • B is the detection of protein level
  • C is the result of immunofluorescence detection of membrane localization.
  • Figure 7 shows the in vitro detection of EFNA1-CAR on EphA2-positive prostate cancer cell line PC-3; bladder cancer cell line 5637, RT4, J82; colorectal cancer cell line DLD1, HCT116; and EphA2-negative normal cells HEK293T, COS7 The effect-to-target ratio gradient kill assay results.
  • Figure 8 shows the detection results of IFN ⁇ and TNF ⁇ release after killing colorectal cancer cell lines DLD1 and HCT116 at a ratio of 2:1.
  • FIG. 9 shows the efficiency detection results after overexpression of EphA2.
  • A is RNA level; B is protein level; C is immunofluorescence membrane localization detection.
  • Figure 10 shows the killing detection results of EFNA1CAR-T cells on cells overexpressing EphA2. .
  • the inventor After extensive and in-depth research and a large number of screenings, the inventor first developed a chimeric antigen receptor immune cell preparation and application based on EFNA1.
  • the experimental results show that the CAR-T targeting the EFNA1 receptor of the present invention has a significant killing effect on target cells with high expression of EphA2, but has no or almost no killing effect on cells that do not express or low express EphA2, so it has high specificity. sex.
  • the present invention has been accomplished on this basis.
  • EphA2 EphA2
  • EFNA1 EFNA1
  • 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.
  • EphA2 and EphrinA1 EphA2 and EphrinA1
  • Erythropoietin-producing hepatocellular receptors A2 (EphA2) is a member of the receptor tyrosine kinase family. Receptor tyrosine kinase family proteins play an important role in the signaling pathway of tumor cells and participate in the occurrence and development of tumors.
  • Ephrins are ligands of the Eph receptor protein family, which are divided into two subclasses, EphrinA (EphrinA1-6) and EphrinB (EphrinB1-3).
  • EphrinA1 is the most widely studied ligand of EphA2 in tumors, and it is also the most important ligand that binds with high affinity.
  • the protein has a total length of 205 amino acids (amino acid, aa) and binds to the extracellular structure of EphA2. In terms of domain, the interaction between the two is involved in the occurrence and development of various solid tumors.
  • EFNA1 can also bind to EphA1, EphA3 and EphA4.
  • EphrinA1 binds to EphA1 with lower affinity
  • Eph family proteins contains an extracellular conserved N-terminal ligand-binding domain, a cysteine-rich domain containing an epidermal growth factor-like motif, and two fibronectin type III repeats.
  • EphA2 is located on the cell membrane and has 25-35% sequence homology with other Eph receptor family members, and the tyrosine residues in the juxtamembrane domain and the kinase domain are conserved.
  • EphA2 The expression of EphA2 is involved in the occurrence and development of various tumors such as colorectal cancer. Colon cancer clinical specimens showed significantly higher EphA2 expression levels compared with paired normal tissues. Furthermore, high EphA2 mRNA and protein expression in stage II/III colorectal cancer tissues was found to be associated with poorer overall survival in both univariate and multivariate analyses. Data have shown that EphA2 is highly expressed in KRAS-mutated colorectal cancer cells, and the level of EphA2 is regulated by KRAS-driven MAPK and RalGDS-RalA pathways. EpHA2 has a poor prognosis in stage II/III CRC, which may be due to its ability to promote cell migration and invasion. Therefore, EphA2 can be used as a very valuable therapeutic target for CAR-T design, and the response characteristics and capabilities for the treatment of colorectal cancer have not yet been reported.
  • the current inhibitors against EphA2 are mainly antibodies.
  • Coffman et al. screened two antibodies, EA2 and B233, which promoted the phosphorylation and degradation of EphA2 in tumor cells, and inhibited the growth of lung and breast cancer tumors in vivo; a new EphA2 humanized monoclonal antibody DS-8895a was released in 2016 Purified in 2018, Burvenich et al.
  • the CAR-T cells targeting EphA2 were used to treat glioma by Beijing Xuanwu Hospital in 2018 Initiate clinical research; there are other EphA2-targeted CAR-Ts in the preclinical research stage for the treatment of lung cancer and esophageal cancer, etc.
  • the existing CAR-Ts targeting EphA2 are all designed based on EphA2 antibodies. However, if the antibody affinity is too low, the ability to target and bind tumor cells is poor, and if the antibody affinity is too high, excessive immune reactions will easily occur. Patient tolerance is poor.
  • the present invention integrates EFNA1 fragments into CAR vectors for the first time through genetic engineering, and modifies related immune cells, so as to achieve specific killing of EFNA1 receptor-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 an EFNA1 protein or a fragment thereof that can specifically bind the EphA2 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 19 to 182 of the sequence shown in SEQ ID NO:1.
  • 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 intracellular domain of a combination of the CD8 hinge and transmembrane regions, the 4-1BB co-stimulatory domain and the CD3 ⁇ signaling domain.
  • the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which refer to at most 10 compared with the amino acid sequence at positions 19 to 182 of SEQ ID NO: 1, preferably At most 8, more preferably at most 5, and most preferably at most 3 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 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.
  • 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 EphA2.
  • a chimeric antigen receptor immune cell which comprises the chimeric antigen receptor specifically targeting EFNA1 receptor (preferably EphA2) 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 EFNA1 receptors (preferably EphA2).
  • 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.
  • 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 especially 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 is contemplated for introducing 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 (eg, T cells) transduced with a lentiviral vector (LV) encoding an expression cassette of the invention.
  • the transduced T cells can target EphA2, a marker of tumor cells, and activate T cells cooperatively, causing an immune response from immune cells, thereby significantly improving their killing efficiency against tumor cells.
  • the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the step of: administering the CAR-cell 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-T 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.
  • EphA2 CAR-T cells elicit a specific immune response against EphA2 cells.
  • the data disclosed herein specifically discloses lentiviral vectors comprising the EFNA1 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 EFNA1 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.
  • Cancers include non-solid tumors such as hematological tumors, such as leukemias and lymphomas.
  • Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemia 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
  • 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.
  • a composition 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 a pharmaceutical composition comprising T cells as described herein may be dosed at a dose of 10 4 to 10 9 cells/kg body weight, preferably at a dose of 10 5 to 10 6 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.
  • the 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.
  • the 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.
  • EphA2 is basically not expressed on the cell membrane of normal cells, but its expression will be up-regulated under pressure stress conditions (such as tumors), so the CAR of the present invention only targets malignant cells with high expression of EphA2 on the cell membrane. cells, but basically no killing effect on normal cells.
  • the present invention utilizes ligand-receptor binding mode instead of scfv in the traditional sense.
  • EphA2 ligand EphrinA1 (EFNA1) (NM_004428.3), human CD8 ⁇ hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3 ⁇ intracellular region gene sequence information.
  • the corresponding nucleotide sequence is obtained by artificial synthesis method or PCR method.
  • the CD8 signal peptide and EphrinA1 (EFNA1) extracellular region were synthesized at BGI Corporation, and the nucleotide sequence of the CAR molecule was double-digested with XbaI (Thermo) and NheI (Thermo), and inserted by T4 DNA ligase (NEB) CD8TM, 4-1BB, and CD3 ⁇ have been inserted into the lentiviral vector pTomo.
  • the schematic diagram of the extracellular domain of EFNA1 is shown in Figure 1A
  • the schematic diagram of the full length of CAR is shown in Figure 1B, where MOCK is the control group.
  • All plasmids were extracted with QIAGEN’s endotoxin-free large extraction kit, and the purified plasmids were transfected into 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 of OPTI-MEM dissolved plasmid mixture (core plasmid 20ug, pCMV ⁇ R8.9 10ug, PMD2.G 4ug); in another centrifuge tube 2ml OPTIMEM and 68ul lipo 6000. 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 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.
  • CD3+ T cells were isolated from human peripheral blood with Ficool separation medium, and purified CD3+ T cells were obtained from RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). For T cells
  • CD3/CD28 magnetic beads were activated (Life technology), and then 200U/ml IL2 (PeproTech) was added to stimulate the culture for 48 hours before virus infection.
  • lentiboost lentivirus was used to infect T cells at an MOI of 20 to prepare CAR-T cells, and the culture medium was changed one day after infection.
  • Example 4 Positive rate of infected CAR-T cells detected by flow cytometry
  • the CAR-T cells, Mock control group, and NTR cell control group were collected by centrifugation 72 hours after virus infection, washed once with PBS, discarded the supernatant, and resuspended the cells in PBS containing 2% FBS, and the positive rate was detected by flow cytometry.
  • NTR refers to uninfected T cells
  • MOCK refers to T cells infected with a control virus without CD7 extracellular binding domain.
  • Results The results of transfection efficiency are shown in Figure 3, in which Figure 3A represents the results observed under a fluorescent microscope. The results show that because of the co-expressed CAR-mKate2 fusion protein in CAR cells, the expressed fusion protein is cleaved by T2A to form a mKate2 protein exhibits red fluorescence in cells.
  • Figure 3B represents the results of flow cytometry, and the results show that the positive rate of CAR-T expressing the CAR of the present invention is about 80%.
  • Example 5 The killing effect of EFNA1CAR-T on various tumor cell lines
  • the killing effect of the CAR-T cells of the present invention on various tumor cell lines was detected with an effect-to-target ratio of 5:1.
  • EFNA1CAR-T was effective against bladder cancer cell lines (RT4), prostate cancer cell lines (PC3), glioma cell lines (U87), breast cancer cell lines (MCF7), pancreatic cancer cell lines (BXPC3 , PANC1) and colorectal cancer cell lines (DLD1, HCT116) these tumor cells have significant killing effect.
  • RNA level and protein level and immunofluorescence detection of EphA2 expression in colorectal cancer tumor cells DLD1, HCT116 and normal cells 293T and COS7 wherein, the RNA expression level was detected by RT-PCR (Fig. 6A), and the protein level was detected by Western Blot ( Figure 6B), molecular localization by immunofluorescence ( Figure 6C), COS7 cells served as a negative control.
  • Embodiment 7 Target cell construction carrying luciferase
  • the luciferase fragment was amplified by PCR from the pGL3-luciferase plasmid, and then ligated into the pTomo vector by XbaI and BamHI to construct the pTomo-EGFP-T2A-lucferase plasmid.
  • IRES and puromycin fragments were amplified from pTomo and PLkO.1 plasmids respectively.
  • the pTomo-EGFP-T2A-luciferase-IRES-Puro plasmid was successfully constructed by ligation of three fragments.
  • the cell line expressing luciferase was used in the cell killing assay in Example 8.
  • Example 8 Gradient killing effect of CAR-T cells on tumor cells
  • the target cells used include: target cells expressing EphA2: prostate cancer cell PC-3; bladder cancer tumor cells 5637, RT4, J82; colorectal cancer cells DLD1, HCT116; target cells that do not express or substantially do not express EphA2: 293T ,COS7.
  • Cytotoxic killer cells% (1-target cell fluorescence value with effector cells/target cell fluorescence value without effector cells)*100%
  • the method is as follows: take the CAR-T cells of the present invention and colorectal cancer in Example 7
  • the cell supernatants co-incubated with DLD1 and HCT116 target cells were used to detect IFN ⁇ according to the IFN gamma Human ELISA Kit (life technology).
  • Example 10 Effect of overexpression of EphA2 on tumor killing effect of EFNA1-CAR-T
  • 293T and COS7 are EphA2-negative normal cell lines, and EFNA1-CAR-T has no killing effect on 293T and COS7 that do not express EphA2.
  • the EphA2 coding region was synthesized in vitro and pTomo-CMV-EphA2-luciferase-IRES-EGFP was constructed by restriction enzyme digestion. Lentivirus was packaged in vitro and infected 293T cells and COS7 cells.
  • the 293T cell line stably overexpressing EphA2 (called 293T-EphA2over) and the COS7 cell line stably overexpressing EphA2 (called COS7-EphA2over) were obtained.
  • the CAR-T cells of the present invention have a significant killing effect, but the CAR-T cells of the present invention have no killing effect on normal 293T and COS7 cells (Figure 10A).
  • the IFN ⁇ secretion of CAR-T cells of the present invention was significantly up-regulated, and it was basically the same as that of the control group for normal cells ( FIG. 10B ).
  • the above results all suggest that the CAR-T cells of the present invention have a specific killing effect on EphA2 overexpressing cells, but basically have no killing effect on cells that do not express EphA2, and have good safety.
  • EphA1, EphA2, EphA3, and EphA4 are expressed on a variety of cells, and the Eph receptor protein family has a variety of ligands (including at least EphrinA1-6 and EphrinB1-3, etc. 9 ligands).
  • EphA2 plays an important role in the formation of tumors. Compared with normal tissues, its expression is significantly upregulated in various tumor tissues, making it an ideal target for the treatment of tumors with high EphA2 expression.
  • EFNA1 ligands bind to four Eph receptor family members to varying degrees
  • the inventors have found through research that CAR immune cells constructed based on EFNA1 have significantly high specificity for tumors with high EphA2 expression. and high killing activity, but it does not show obvious killing activity on normal cells that do not express EphA2, so it has high specificity and safety, and is suitable for targeting EphA2-positive tumors.
  • EphA2 EphA2
  • EFNA1 EFNA1
  • our chimeric antigen receptor immune cells based on EFNA1 can recognize EFNA1 receptors well, and have very high specificity and killing activity against tumor cells with high expression of EphA2, but have no effect on normal cells that do not express EphA2.
  • the killing effect can be used to treat tumors with high expression of EphA2.

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Abstract

Provided are preparation and a use of immune cells of a chimeric antigen receptor (CAR) constructed on the basis of EFNA1. Specifically, provided is a CAR modified on the basis of EFNA1, the CAR comprises an extracellular binding domain, and the extracellular binding domain is capable of specifically targeting the EFNA1 receptor. The CAR immune cells have strong specificity and target affinity, so that the CAR immune cells are relatively strong in killing capability for target cells and high in safety.

Description

基于EFNA1构建的嵌合抗原受体免疫细胞制备及其应用Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1 技术领域technical field
本发明属于肿瘤免疫治疗生物医药技术领域，涉及特异性嵌合抗原受体免疫细胞，具体涉及一种特异性靶向EFNA1受体的一种CAR，其修饰的免疫应答细胞，及其制备方法和应用。The invention belongs to the technical field of biomedicine for tumor immunotherapy, and relates to specific chimeric antigen receptor immune cells, in particular to a CAR that specifically targets EFNA1 receptors, its modified immune response cells, and its preparation method and application.
背景技术Background technique
目前癌症被认为是世界上各个国家的主要死亡原因及延长人类寿命的主要障碍。根据世界卫生组织(WHO)2019年的统计，在183个国家中年龄为70岁之前的人群中，癌症是第一或第二大死亡原因，严重威胁着人类的健康。到2020年，全球估计有1930万新发病例和1000万例癌症患者死亡，根据目前癌症的发病率估计到2040年，全球将会出现2840万例癌症新发病例(包括基底细胞癌除外的非黑色素瘤皮肤癌)，比2020年增加47％，总体而言，世界范围内癌症的发病率和死亡率负担正在迅速增长。At present, cancer is considered to be the main cause of death in various countries in the world and the main obstacle to prolonging human life. According to the statistics of the World Health Organization (WHO) in 2019, cancer is the first or second leading cause of death among people before the age of 70 in 183 countries, seriously threatening human health. By 2020, it is estimated that there will be 19.3 million new cases and 10 million deaths of cancer patients worldwide. According to the current incidence of cancer, it is estimated that by 2040, there will be 28.4 million new cases of cancer worldwide (including non-basal cell carcinomas except basal cell carcinoma). melanoma skin cancer), a 47% increase over 2020, and overall, the burden of cancer incidence and mortality is growing rapidly worldwide.
根据2020全球肿瘤数据统计，预计2020年会有1900万新发结直肠癌患者，935，000例患者死于结直肠癌；总体来说，在发病率上结直肠癌占第三位，死亡率居第二位，居高的发病率和死亡率使结直肠癌严重威胁着人类的健康。目前结直肠癌的治疗手段主要包括局部治疗的手术，***治疗的化疗，靶向治疗以及免疫治疗。随着医疗水平的提高，***治疗结直肠癌方面已经取得了重大进展。引入了六种新的化疗药物，使转移性结直肠癌患者的中位总生存期从不到9个月(未经治疗)增加到大约24个月。对于III期(***阳性)结肠癌患者，已经确立了以氟尿嘧啶为基础的化疗的总体生存获益，最新的数据表明，将奥沙利铂纳入此类辅助治疗方案具有更高的疗效，虽然治疗效果不断提高，但仍有部分患者出现恶性进展，因此需要探索新的方法治疗结直肠癌。According to the 2020 global tumor statistics, it is estimated that there will be 19 million new patients with colorectal cancer and 935,000 patients will die of colorectal cancer in 2020; overall, colorectal cancer ranks third in incidence and mortality Ranking second, the high morbidity and mortality make colorectal cancer a serious threat to human health. Currently, the treatment methods for colorectal cancer mainly include surgery for local treatment, chemotherapy for systemic treatment, targeted therapy and immunotherapy. With the improvement of medical level, significant progress has been made in systemic treatment of colorectal cancer. Six new chemotherapy drugs were introduced that increased the median overall survival of patients with metastatic colorectal cancer from less than 9 months (untreated) to about 24 months. For patients with stage III (node-positive) colon cancer, the overall survival benefit of fluorouracil-based chemotherapy has been established, and recent data suggest that the inclusion of oxaliplatin in this type of adjuvant regimen is more effective, although treatment The effect is continuously improving, but some patients still have malignant progression, so it is necessary to explore new methods for the treatment of colorectal cancer.
随着医学技术的发展，人类的寿命不断延长，在肿瘤的治疗上取得了很大的进展，手术、化疗、放疗和生物治疗等综合治疗手段的应用，使得癌症患者的生存期不断延长，生存质量也取得了很大的提升。除了常规治疗手段，免疫治疗也成为了治疗癌症的临床重要手段，越来越多的免疫治疗药物被批准用于临床治疗，如针对免疫检查点抑制剂的PD1,PDL1单克隆抗体以及CAR-T细胞治疗。With the development of medical technology, the life span of human beings has been continuously extended, and great progress has been made in the treatment of tumors. The application of comprehensive treatment methods such as surgery, chemotherapy, radiotherapy and biological therapy has continuously extended the survival period of cancer patients. Quality has also been greatly improved. In addition to conventional treatment methods, immunotherapy has also become an important clinical method for the treatment of cancer. More and more immunotherapy drugs have been approved for clinical treatment, such as PD1, PDL1 monoclonal antibody and CAR-T for immune checkpoint inhibitors. cell therapy.
嵌合抗原抗体受体(Chimeric Antigen Receptor-T cell,CAR-T)T细胞是指经基因修饰后，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。其主要结构包括三类，即胞外的ScFv识别结构域，此区域用于对肿瘤细胞上的靶点进行识别结合；铰链区和跨膜结构域，主要来源于CD8或CD28，将CAR锚定于细胞膜，且连接胞外识别域与胞内信号；胞内域，是激活域，其数量和长度 差异会影响CAR-T的抗肿瘤效果，目前常用的是一个激活域加一个或多个共刺激结构域，CD3ζ是CAR胞内部分的常见特征，其能启动信号以驱动T细胞的杀伤作用，而共刺激域主要来源于CD28受体家族或者TNF受体家族如4-1BB,OX40或CD27，通过增强细胞因子分泌或者促进增殖及持久性也提高CAR-T的杀伤效果。Chimeric Antigen Receptor-T cell (CAR-T) T cells refer to T cells that can recognize specific target antigens in an unrestricted way of MHC after genetic modification, and continuously activate and expand T cells. Its main structure includes three types, namely the extracellular ScFv recognition domain, which is used to recognize and bind the target on tumor cells; the hinge region and transmembrane domain, mainly derived from CD8 or CD28, anchor CAR It is located on the cell membrane and connects the extracellular recognition domain and intracellular signal; the intracellular domain is the activation domain, and the difference in number and length will affect the anti-tumor effect of CAR-T. Currently, one activation domain plus one or more common Stimulatory domain, CD3ζ is a common feature of the intracellular part of CAR, which can initiate signals to drive T cell killing, while the costimulatory domain is mainly derived from the CD28 receptor family or TNF receptor family such as 4-1BB, OX40 or CD27 , by enhancing the secretion of cytokines or promoting proliferation and persistence can also improve the killing effect of CAR-T.
正常T细胞依赖于T细胞表面受体(T cell Receptor,TCR)及肿瘤细胞表面的主要组织相容复合物(Major Histocompatibility complex.MHC)结合的方式识别肿瘤细胞,而CAR-T细胞以不依赖于MHC的方式只依赖于CAR这个结构来识别肿瘤细胞表面的肿瘤相关抗原(Tumor Associated Antigen，TAA)，具有CAR对抗原识别的特异性及T细胞的杀伤特性。Normal T cells rely on the combination of T cell receptor (T cell Receptor, TCR) and the major histocompatibility complex (MHC) on the surface of tumor cells to recognize tumor cells, while CAR-T cells recognize tumor cells independently. The method based on MHC only relies on the structure of CAR to recognize tumor-associated antigen (Tumor Associated Antigen, TAA) on the surface of tumor cells, which has the specificity of CAR for antigen recognition and the killing characteristics of T cells.
过去这些年来，随着基因编辑，免疫及其他学科的综合快速发展，CAR-T细胞治疗成为治疗血液病一个新的并取得不错疗效的治疗手段。CAR-T治疗恶性肿瘤仍然是目前的研究热点，自1986年首次用肿瘤浸润淋巴细胞(TILs)***，到1993年第一代CAR的研发，直至2017年FDA首次批准CAR-T用于治疗复发/难治性急性淋巴细胞白血病，CAR-T治疗已经走过数年的历程。目前有三种CAR-T被用于***，包括2017年8月和10月被FDA批准的Kymriah和Yescarta被用于治疗复发/难治的急性淋巴细胞白血病及特定类型大B细胞淋巴瘤，及2020年7月批准的Tecartus被用于治疗成人套细胞淋巴瘤(Mental Cell Lymphoma,MCL)。CAR-T治疗在血液病方面取得了很好的进展，实体瘤的治疗仍然存在一定的局限性，主要原因是有效靶点的选择以及CAR-T细胞对肿瘤的浸润。In the past few years, with the comprehensive and rapid development of gene editing, immunization and other disciplines, CAR-T cell therapy has become a new treatment for blood diseases with good curative effect. CAR-T treatment of malignant tumors is still a research hotspot at present. Since 1986, tumor-infiltrating lymphocytes (TILs) were first used to treat tumors, to the development of the first-generation CAR in 1993, until 2017, when the FDA first approved CAR-T for treatment. For relapsed/refractory acute lymphoblastic leukemia, CAR-T therapy has gone through several years. There are currently three CAR-Ts being used to treat tumors, including Kymriah and Yescarta approved by the FDA in August and October 2017 for the treatment of relapsed/refractory acute lymphoblastic leukemia and specific types of large B-cell lymphoma, and Tecartus, approved in July 2020, is used to treat adult mantle cell lymphoma (MCL). CAR-T therapy has made good progress in blood diseases, but there are still some limitations in the treatment of solid tumors, mainly due to the selection of effective targets and the infiltration of CAR-T cells into tumors.
发明内容Contents of the invention
本发明的目的就是提供一种以EFNA1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。The object of the present invention is to provide a chimeric antigen receptor immune cell targeting EFNA1 receptor and its preparation and application method.
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，所述的胞外结合域包括基于SEQ ID NO:1所示氨基酸序列的EFNA1或其片段的结构，并且所述的胞外结合域能够特异性地与EFNA1受体以配体受体方式结合。In a first aspect of the present invention, a chimeric antigen receptor (CAR) is provided, characterized in that, the CAR contains an extracellular binding domain, and the extracellular binding domain comprises a sequence based on SEQ ID NO: 1 The structure of EFNA1 or its fragments of the amino acid sequence shown, and the extracellular binding domain can specifically bind to the EFNA1 receptor in the form of a ligand receptor.
在另一优选例中，所述的EFNA1受体选自下组：EphA2。In another preferred example, the EFNA1 receptor is selected from the following group: EphA2.
在另一优选例中，所述的胞外结合域具有来源于EFNA1的氨基酸序列。In another preferred example, the extracellular binding domain has an amino acid sequence derived from EFNA1.
在另一优选例中，所述的胞外结合域包括EFNA1蛋白或其片段。In another preferred example, the extracellular binding domain includes EFNA1 protein or a fragment thereof.
在另一优选例中，所述的EFNA1蛋白片段包括EFNA1蛋白的胞外区。In another preferred example, the EFNA1 protein fragment includes the extracellular region of the EFNA1 protein.
在另一优选例中，所述的胞外结合域包括EFNA1蛋白或其片段。In another preferred example, the extracellular binding domain includes EFNA1 protein or a fragment thereof.
在另一优选例中，所述的胞外结合域段包括EFNA1蛋白的胞外区，所述胞外区的氨基酸序列为对应于SEQ ID NO:1所示序列的19-182位。In another preferred example, the extracellular binding domain segment includes the extracellular region of the EFNA1 protein, and the amino acid sequence of the extracellular region corresponds to positions 19-182 of the sequence shown in SEQ ID NO:1.
在另一优选例中，所述胞外结构域具有如SEQ ID NO:1的19-182位所示的氨基酸。In another preferred example, the extracellular domain has the amino acids shown in positions 19-182 of SEQ ID NO:1.
在另一优选例中，所述的EFNA1受体选自下组：EphA2。In another preferred example, the EFNA1 receptor is selected from the following group: EphA2.
在另一优选例中，所述的CAR的胞外结合域除了含有针对EFNA1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。In another preferred example, in addition to the first extracellular domain targeting EFNA1 receptor, the extracellular binding domain of the CAR also includes a second extracellular domain targeting additional targets.
在另一优选例中，所述的额外靶点为肿瘤特异性靶点。In another preferred example, the additional target is a tumor-specific target.
在另一优选例中，所述的EFNA1蛋白或其片段与包括EphA2在内的EFNA1受体具有特异性结合。In another preferred example, the EFNA1 protein or its fragments specifically bind to EFNA1 receptors including EphA2.
在另一优选例中，所述的结合分子选自下组：EphA2。In another preferred example, the binding molecule is selected from the following group: EphA2.
在另一优选例中，所述的EphA2是位于细胞膜上EphA2。In another preferred embodiment, the EphA2 is EphA2 located on the cell membrane.
在另一优选例中，所述的EphA2来源于人或非人哺乳动物。In another preferred example, the EphA2 is derived from human or non-human mammal.
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。In another preferred example, the non-human mammals include: rodents (such as rats, mice), primates (such as monkeys); preferably primates.
在另一优选例中，所述的EphA2是人源的或猴源的。In another preferred example, the EphA2 is of human or monkey origin.
在另一优选例中，所述的EphA2是人源的。In another preferred example, the EphA2 is of human origin.
在另一优选例中，所述的胞外结构域具有如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182位(较佳地为第19至182位)的氨基酸序列。In another preferred example, the extracellular domain has the amino acid sequence shown in SEQ ID NO: 1, or has the 1st to 182nd (preferably the 1st) of the sequence shown in SEQ ID NO: 1 19 to 182 amino acid sequence).
在另一优选例中，所述的EFNA1蛋白或其片段对应于SEQ ID NO:1所示序列的19-182位的氨基酸序列。In another preferred example, the EFNA1 protein or its fragment corresponds to the amino acid sequence at positions 19-182 of the sequence shown in SEQ ID NO:1.
在另一优选例中，所述的EFNA1蛋白或其片段的氨基酸序列选自下组：In another preferred example, the amino acid sequence of the EFNA1 protein or its fragment is selected from the following group:
(i)如SEQ ID NO:1所示序列的第19至182位所示的序列；和(i) the sequence shown in the 19th to 182nd positions of the sequence shown in SEQ ID NO: 1; and
(ii)在如SEQ ID NO:1所示序列的第19至182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***，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19至182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。(ii) On the basis of the sequence shown in the 19th to 182nd positions of the sequence shown in SEQ ID NO: 1, one or more amino acid residues are replaced, deleted, changed or inserted, or at the N-terminal or C-terminal 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are added to the end, thereby obtaining an amino acid sequence; and the amino acid sequence obtained is the same as SEQ ID NO : The sequence shown in the 19th to 182nd positions of the sequence shown in 1 has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) Sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).
在另一优选例中，所述CAR的结构如下式I所示：In another preferred example, the structure of the CAR is shown in the following formula I:
L-EB-H-TM-C-CD3ζ-RP   (I)L-EB-H-TM-C-CD3ζ-RP (I)
式中，In the formula,
各“-”独立地为连接肽或肽键；Each "-" is independently a connecting peptide or a peptide bond;
L是无或信号肽序列；L is nothing or a signal peptide sequence;
EB是胞外结合域；EB is the extracellular binding domain;
H是无或铰链区；H is none or hinge region;
TM是跨膜结构域；TM is the transmembrane domain;
C是无或共刺激信号分子；C is none or a co-stimulatory signal molecule;
CD3ζ是源于CD3ζ的胞浆信号传导序列；CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;
RP是无或报告蛋白。RP is none or a reporter protein.
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。In another preferred example, the reporter protein RP also includes a self-cleavage recognition site at its N-terminus, preferably a T2A sequence.
在另一优选例中，所述的报告蛋白RP为荧光蛋白。In another preferred embodiment, the reporter protein RP is a fluorescent protein.
在另一优选例中，所述的报告蛋白RP为mKate2红色荧光蛋白。In another preferred embodiment, the reporter protein RP is mKate2 red fluorescent protein.
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。In another preferred example, the amino acid sequence of the mKate2 red fluorescent protein is shown in SEQ ID NO:2.
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、EFNA1、GM-CSF、CD4、CD137、或其组合。In another preferred embodiment, said L is a signal peptide of a protein selected from the following group: CD8, CD28, EFNA1, GM-CSF, CD4, CD137, or a combination thereof.
在另一优选例中，所述的L是CD8来源的信号肽。In another preferred example, said L is a signal peptide derived from CD8.
在另一优选例中，所述L的氨基酸序列如SEQ ID NO:3所示。In another preferred example, the amino acid sequence of said L is shown in SEQ ID NO:3.
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。In another preferred embodiment, said H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.
在另一优选例中，所述的H是CD8来源的铰链区。In another preferred example, said H is a hinge region derived from CD8.
在另一优选例中，所述H的氨基酸序列如SEQ ID NO:4所示。In another preferred example, the amino acid sequence of the H is shown in SEQ ID NO:4.
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。In another preferred example, 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.
在另一优选例中，所述的TM是CD8来源的跨膜区。In another preferred example, the TM is a transmembrane region derived from CD8.
在另一优选例中，所述TM的氨基酸序列如SEQ ID NO:5所示。In another preferred example, the amino acid sequence of the TM is shown in SEQ ID NO:5.
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。In another preferred example, 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.
在另一优选例中，所述的C是4-1BB来源的共刺激信号分子。In another preferred example, said C is a co-stimulatory signal molecule derived from 4-1BB.
在另一优选例中，所述C的氨基酸序列如SEQ ID NO:6所示。In another preferred example, the amino acid sequence of C is shown in SEQ ID NO:6.
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。In another preferred example, the amino acid sequence of the cytoplasmic signaling sequence derived from CD3ζ is shown in SEQ ID NO:7.
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。In another preferred example, the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO:8.
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。In the second aspect of the present invention, a nucleic acid molecule encoding the chimeric antigen receptor as described in the first aspect of the present invention is provided.
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。In the third aspect of the present invention, a vector is provided, characterized in that the vector contains the nucleic acid molecule as described in the second aspect of the present invention.
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。In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
在另一优选例中，所述载体为慢病毒载体。In another preferred example, the vector is a lentiviral vector.
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。In another preferred embodiment, the vector is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV and the like.
在另一优选例中，所述的载体是pTomo慢病毒载体。In another preferred example, the vector is pTomo lentiviral vector.
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。In another preferred example, the vector further includes a promoter, a transcriptional enhancer element WPRE, a long terminal repeat sequence LTR, etc. selected from the group.
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。In another preference, the vector comprises the nucleotide sequence shown in SEQ ID NO:9.
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CAR。In the fourth aspect of the present invention, a host cell is provided, the 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 CAR as described in the first aspect of the present invention.
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CAR。In the fifth aspect of the present invention, there is provided 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. A nucleic acid molecule or expression of the CAR as described in the first aspect of the present invention.
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。In another preferred example, the engineered immune cells are selected from the group consisting of T cells, NK cells, NKT cells, or macrophages.
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)。In another preferred example, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).
在另一优选例中，所述的工程化免疫细胞是CAR-T细胞。In another preferred example, the engineered immune cells are CAR-T cells.
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。In 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.
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。In another preferred example, the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
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CAR、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。In the seventh aspect of the present invention, a pharmaceutical composition is provided, which contains the CAR as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, and the CAR as described in the third aspect of the present invention. The carrier according to the aspect, 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.
在另一优选例中，所述制剂为液态制剂。In another preferred example, the formulation is a liquid formulation.
在另一优选例中，所述制剂的剂型为注射剂。In another preferred example, the dosage form of the preparation is injection.
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。 In another preferred example, 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.
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CAR、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EFNA1受体异常表达相关的疾病的药物或制剂。In the eighth aspect of the present invention, there is provided a CAR 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 vector as described in the third aspect of the present invention, or a CAR as described in the present invention The use of the host cell according to the fourth aspect, and/or the engineered immune cell according to the fifth aspect of the present invention, is used to prepare a drug or preparation for preventing and/or treating diseases related to abnormal expression of EFNA1 receptor.
在另一优选例中，所述的EFNA1受体包括但不限于EphA2。In another preferred example, the EFNA1 receptor includes but not limited to EphA2.
在另一优选例中，所述的EFNA1受体异常表达是指所述EFNA1受体过度表达。In another preferred example, the abnormal expression of the EFNA1 receptor refers to the overexpression of the EFNA1 receptor.
在另一优选例中，所述EFNA1受体过度表达是指所述EFNA1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。In another preferred example, the overexpression of the EFNA1 receptor means that the expression level of the EFNA1 receptor is ≥ 1.5 times, preferably ≥ 2 times, more preferably ≥ 2.5 times the expression level under normal physiological conditions.
在另一优选例中，所述的EFNA1受体异常表达相关的疾病包括但不限于肿瘤、衰老、肥胖、心血管疾病、糖尿病、神经退行性疾病、感染性疾病等。In another preferred example, the diseases related to the abnormal expression of EFNA1 receptor include but not limited to tumor, aging, obesity, cardiovascular disease, diabetes, neurodegenerative disease, infectious disease and so on.
在另一优选例中，所述的EFNA1受体异常表达相关的疾病包括EphA2异常表达相关的疾病。In another preferred example, the diseases related to abnormal expression of EFNA1 receptor include diseases related to abnormal expression of EphA2.
在另一优选例中，所述的EphA2异常表达相关的疾病包括：肿瘤、衰老、心血管疾病、肥胖等。In another preferred example, the diseases related to the abnormal expression of EphA2 include: tumor, aging, cardiovascular disease, obesity and so on.
在另一优选例中，所述的疾病是EphA2高表达的恶性肿瘤。In another preferred example, the disease is a malignant tumor with high expression of EphA2.
在另一优选例中，所述肿瘤包括血液肿瘤和实体瘤。In another preferred example, the tumor includes blood tumors and solid tumors.
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)、淋巴瘤，或其组合。In another preferred example, 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), lymphoma, or a combination thereof.
在另一优选例中，所述的实体瘤选自下组：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、胰腺癌、***癌，或其组合。In another preferred example, the solid tumor is selected from the group consisting of 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, Pancreatic cancer, prostate cancer, or a combination thereof.
在另一优选例中，所述的肿瘤选自下组：结直肠癌、脑肿瘤、乳腺癌、子宫内膜癌、膀胱癌、***癌、胰腺癌。In another preferred example, the tumor is selected from the group consisting of colorectal cancer, brain tumor, breast cancer, endometrial cancer, bladder cancer, prostate cancer, and pancreatic cancer.
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。In 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.
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。In the tenth aspect of the present invention, there is provided 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.
在另一优选例中，所述疾病为EFNA1受体异常表达相关的疾病。In another preferred example, the disease is a disease related to abnormal expression of EFNA1 receptor.
在另一优选例中，所述疾病为癌症或肿瘤。In another preferred example, the disease is cancer or tumor.
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)。In another preferred example, the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from the subject (autologous cells).
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)。In another preferred example, the engineered immune cells or the CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).
在另一优选例中，所述的方法可与其他治疗方法联合使用。In another preferred example, the above method can be used in combination with other treatment methods.
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。In another preferred example, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and other methods.
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。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.
附图说明Description of drawings
图1显示了EFNA1-CAR载体构建示意图。Figure 1 shows a schematic diagram of the construction of the EFNA1-CAR vector.
其中，A为EFNA1序列示意图，其中1-18AA为信号肽、19-182AA为胞外结构域；B为对照组质粒MOCK-CAR及EFNA1-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-EFNA1-CAR载体HindIII酶切鉴定。Among them, A is the sequence diagram of EFNA1, in which 1-18AA is the signal peptide, and 19-182AA is the extracellular domain; B is the structure diagram of the control plasmid MOCK-CAR and EFNA1-CAR, in which the signal peptide, hinge region, transmembrane region All are derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3ζ is derived from human CD3, mKate2 is a fluorescent marker for detecting CAR expression; C is pTomo-EFNA1-CAR carrier HindIII digestion identification.
图2显示了EFNA1CAR的细胞扩增倍数及总数。图2A.T细胞感染后12天后增殖倍数；图2B.感染12天后细胞总数统计。Figure 2 shows the cell expansion fold and total number of EFNA1CAR. Figure 2A. T cell proliferation factor 12 days after infection; Figure 2B. Statistics of the total number of cells 12 days after infection.
图3显示了荧光显微镜及流式细胞术检测CAR感染效率。Figure 3 shows the efficiency of CAR infection detected by fluorescence microscopy and flow cytometry.
其中，A为MOCK-CAR及EFNA1-CAR感染T细胞72小时后细胞荧光表达结果，其中上排为明场，下排为CAR荧光表达；B为流式检测荧光表达结果。Among them, A is the result of cell fluorescence expression after 72 hours of MOCK-CAR and EFNA1-CAR infection of T cells, the upper row is the bright field, and the lower row is the fluorescence expression of CAR; B is the result of flow cytometry detection of fluorescence expression.
图4显示了CAR-T细胞的表型检测结果，检测了感染后d3,d6检测CD4,CD8阳性T细胞的比例。。Figure 4 shows the phenotypic detection results of CAR-T cells, and the proportion of CD4 and CD8 positive T cells was detected on d3 and d6 after infection. .
图5显示了EFNA1CAR-T对不同肿瘤细胞系的杀伤作用(效靶比5：1)。Figure 5 shows the killing effect of EFNA1CAR-T on different tumor cell lines (effect-to-target ratio 5:1).
其中，A.膀胱癌细胞系；B.***癌细胞系；C.胶质瘤细胞系；D.乳腺癌细胞系；E,F.胰腺癌细胞系；G,H.结直肠癌细胞系。Among them, A. bladder cancer cell line; B. prostate cancer cell line; C. glioma cell line; D. breast cancer cell line; E, F. pancreatic cancer cell line; G, H. colorectal cancer cell line.
图6显示了结直肠癌肿瘤细胞系DLD1,HCT116及正常细胞系293T及COS7的EphA2表达检测的结果。Figure 6 shows the results of detection of EphA2 expression in colorectal cancer cell lines DLD1, HCT116 and normal cell lines 293T and COS7.
其中，A为RNA水平检测；B为蛋白水平检测；C为免疫荧光检测膜定位结果。Among them, A is the detection of RNA level; B is the detection of protein level; C is the result of immunofluorescence detection of membrane localization.
图7显示了体外检测EFNA1-CAR对EphA2阳性的***癌细胞系PC-3；膀胱癌 细胞系5637，RT4,J82；结直肠癌肿瘤细胞系DLD1,HCT116；以及EphA2阴性的正常细胞HEK293T,COS7的效靶比梯度杀伤检测结果。Figure 7 shows the in vitro detection of EFNA1-CAR on EphA2-positive prostate cancer cell line PC-3; bladder cancer cell line 5637, RT4, J82; colorectal cancer cell line DLD1, HCT116; and EphA2-negative normal cells HEK293T, COS7 The effect-to-target ratio gradient kill assay results.
图8显示了以2：1对结直肠癌细胞系DLD1,HCT116杀伤后IFNγ和TNFα释放检测结果。Figure 8 shows the detection results of IFNγ and TNFα release after killing colorectal cancer cell lines DLD1 and HCT116 at a ratio of 2:1.
图9显示了过表达EphA2后的效率检测结果。Figure 9 shows the efficiency detection results after overexpression of EphA2.
其中，A是RNA水平；B是蛋白水平；C是免疫荧光膜定位检测。Among them, A is RNA level; B is protein level; C is immunofluorescence membrane localization detection.
图10显示了EFNA1CAR-T细胞对过表达EphA2的细胞的杀伤检测结果。。Figure 10 shows the killing detection results of EFNA1CAR-T cells on cells overexpressing EphA2. .
具体实施方式Detailed ways
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EFNA1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EFNA1受体的CAR-T具有对EphA2高表达靶细胞的显著杀伤效果，而对于不表达或低表达EphA2的细胞没有或基本没有杀伤效果，因此具有较高特异性。在此基础上完成了本发明。After extensive and in-depth research and a large number of screenings, the inventor first developed a chimeric antigen receptor immune cell preparation and application based on EFNA1. The experimental results show that the CAR-T targeting the EFNA1 receptor of the present invention has a significant killing effect on target cells with high expression of EphA2, but has no or almost no killing effect on cells that do not express or low express EphA2, so it has high specificity. sex. The present invention has been accomplished on this basis.
本发明的实验表明，虽然不同的***肝细胞受体(如EphA1、EphA2、EphA3和EphA4)在多种细胞上有表达，并且Eph受体蛋白家族存在多种配体(至少包括EphrinA1-6和EphrinB1-3等9种配体)，并且Eph受体蛋白与配体之间存在一定的交叉反应性，但本发明的嵌合抗原受体免疫细胞对EphA2高表达的肿瘤具有明显的高特异性和高杀伤活性，但对不表达EphA2的正常细胞则不表现出明显的杀伤活性，因此具有高特异性和安全性，适合靶向EphA2阳性的肿瘤。Experiments of the present invention show that although different erythropoietin hepatocyte receptors (such as EphA1, EphA2, EphA3 and EphA4) are expressed on various cells, and there are multiple ligands in the Eph receptor protein family (including at least EphrinA1 -6 and 9 ligands such as EphrinB1-3), and there is a certain cross-reactivity between the Eph receptor protein and the ligand, but the chimeric antigen receptor immune cells of the present invention have obvious effects on tumors with high expression of EphA2 It has high specificity and high killing activity, but it does not show obvious killing activity on normal cells that do not express EphA2, so it has high specificity and safety, and is suitable for targeting EphA2-positive tumors.
另外，在肿瘤细胞中，除EphA2外的其他几个Eph受体也可能存在超出生理表达量的异常高表达情况。因此基于EFNA1能够结合这些Eph受体的特点，也使其具备防止由于靶点在治疗中丢失引起的免疫逃逸现象、预防肿瘤的复发的潜力。In addition, in tumor cells, several other Eph receptors besides EphA2 may also have abnormally high expression beyond the physiological expression. Therefore, based on the characteristics that EFNA1 can bind to these Eph receptors, it also has the potential to prevent immune escape caused by the loss of the target during treatment and prevent tumor recurrence.
术语the term
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。For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Before the present invention is described, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, the scope of the present invention being limited only by the appended claims.
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)。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
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。As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance can but does not have to occur.
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”。As used herein, 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".
“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)。"Transduction", "transfection", "transformation" or the terms used herein refer 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).
“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。"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.
“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。"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.
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。Conservative amino acid substitutions are known in the art. In some embodiments, potential substituting 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 . In addition, the present invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.
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。A person skilled in the art will readily understand the meaning of all parameters, dimensions, materials and configurations described herein. Actual parameters, dimensions, materials and/or configurations will depend upon the particular application being described using the invention. Those skilled in the art can understand that the embodiments or claims are given by way of example only, and within the scope of equivalents or claims, the scope covered by the embodiments of the present invention is not limited to what is specifically described and claimed scope.
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。Definitions herein and all definitions used should be read over dictionary definitions or definitions in documents incorporated by reference.
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。All references, patents and patent applications cited herein are incorporated by reference with respect to their cited subject matter, and in some cases may include the entire document.
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。It should be understood that for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these examples.
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。In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.
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。The term "about" can refer to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
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***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。The term "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.
EphA2和EphrinA1EphA2 and EphrinA1
***肝细胞受体A2(erythropoietin-producing hepatocellular receptors A2,EphA2)是受体酪氨酸激酶家族的成员之一。受体酪氨酸激酶家族蛋白在肿瘤细胞的信号通路中起到重要作用，参与肿瘤的发生发展过程。Erythropoietin-producing hepatocellular receptors A2 (EphA2) is a member of the receptor tyrosine kinase family. Receptor tyrosine kinase family proteins play an important role in the signaling pathway of tumor cells and participate in the occurrence and development of tumors.
Ephrins(EFN)是Eph受体蛋白家族的配体，分为EphrinA(EphrinA1-6)和EphrinB(EphrinB1-3)两个亚类。Ephrins (EFN) are ligands of the Eph receptor protein family, which are divided into two subclasses, EphrinA (EphrinA1-6) and EphrinB (EphrinB1-3).
EphrinA1(EFNA1)是EphA2在肿瘤中研究最广泛的配体，也是以高亲和力结合的最主要的配体，该蛋白总长度为205个氨基酸(amino acid,aa)，结合在EphA2的细胞外结构域上，其二者间的相互作用，参与了多种实体肿瘤的发生与发展过程。除EphA2之外，EFNA1还能与EphA1、EphA3和EphA4结合。EphrinA1以较低亲和力与EphA1结合，EphA3的最主要高亲和力配体是EphrinA2(Kd＝1nM)和EphrinA3(Kd＝5nM)，EphA4的最主要高亲和力配体是EphrinA2(Kd＝10nM)和EphrinA4(Kd＝5nM)。EphrinA1 (EFNA1) is the most widely studied ligand of EphA2 in tumors, and it is also the most important ligand that binds with high affinity. The protein has a total length of 205 amino acids (amino acid, aa) and binds to the extracellular structure of EphA2. In terms of domain, the interaction between the two is involved in the occurrence and development of various solid tumors. In addition to EphA2, EFNA1 can also bind to EphA1, EphA3 and EphA4. EphrinA1 binds to EphA1 with lower affinity, the most important high-affinity ligands of EphA3 are EphrinA2 (Kd=1nM) and EphrinA3 (Kd=5nM), the most important high-affinity ligands of EphA4 are EphrinA2 (Kd=10nM) and EphrinA4 ( Kd = 5nM).
Eph家族蛋白的结构包含细胞外的保守N端配体结合结构域、含有表皮生长因子样基序的富含半胱氨酸结构域和两个纤连蛋白III型重复序列。The structure of Eph family proteins contains an extracellular conserved N-terminal ligand-binding domain, a cysteine-rich domain containing an epidermal growth factor-like motif, and two fibronectin type III repeats.
EphA2定位在细胞膜上，与其他Eph受体家族成员具有25-35％的序列同源性，在近膜结构域和激酶结构域中的酪氨酸残基是保守的。EphA2 is located on the cell membrane and has 25-35% sequence homology with other Eph receptor family members, and the tyrosine residues in the juxtamembrane domain and the kinase domain are conserved.
EphA2的表达参与结直肠癌等多种肿瘤的发生发展。与配对的正常组织相比，肠癌临床标本显示出明显更高的EphA2表达水平。此外，在单变量和多变量分析中，发现II/III期结直肠癌组织中高EphA2mRNA和蛋白表达与较差的总体存活率有关。有数据表明EphA2在KRAS突变的结直肠癌细胞中高表达，并且EphA2的水平受KRAS驱动的MAPK和RalGDS-RalA途径的调节。EpHA2在II/III期CRC中预后不良，这可能是由于其促进细胞迁移和侵袭的能力。因此EphA2可作为CAR-T设计的非常有价值的治疗靶标，且用于治疗结直肠癌的应答特点与能力尚未报道。The expression of EphA2 is involved in the occurrence and development of various tumors such as colorectal cancer. Colon cancer clinical specimens showed significantly higher EphA2 expression levels compared with paired normal tissues. Furthermore, high EphA2 mRNA and protein expression in stage II/III colorectal cancer tissues was found to be associated with poorer overall survival in both univariate and multivariate analyses. Data have shown that EphA2 is highly expressed in KRAS-mutated colorectal cancer cells, and the level of EphA2 is regulated by KRAS-driven MAPK and RalGDS-RalA pathways. EpHA2 has a poor prognosis in stage II/III CRC, which may be due to its ability to promote cell migration and invasion. Therefore, EphA2 can be used as a very valuable therapeutic target for CAR-T design, and the response characteristics and capabilities for the treatment of colorectal cancer have not yet been reported.
作为肿瘤抗原靶标，目前针对EphA2的抑制剂主要为抗体。Coffman等筛选了两种抗体EA2和B233，在肿瘤细胞中促进了EphA2的磷酸化和降解，在体内抑制肺癌和乳腺癌肿瘤的生长；一种新型的EphA2人源化单抗DS-8895a于2016年纯化出，Burvenich等证明在体内能够抑制乳腺和肠道肿瘤的生长；以EphA2的单链抗体为CAR序列，靶向EphA2的CAR-T细胞用于治疗神经胶质瘤由北京 宣武医院于2018发起临床研究；还有其他正在临床前研究阶段的靶向EphA2的CAR-T，用于治疗肺癌和食道癌等。总的来说，现有的靶向EphA2的CAR-T均是基于EphA2的抗体设计的，然而，抗体亲和力过低靶向结合肿瘤细胞的能力差，抗体亲和力过高易发生过度的免疫反应，病人耐受性差。As a tumor antigen target, the current inhibitors against EphA2 are mainly antibodies. Coffman et al. screened two antibodies, EA2 and B233, which promoted the phosphorylation and degradation of EphA2 in tumor cells, and inhibited the growth of lung and breast cancer tumors in vivo; a new EphA2 humanized monoclonal antibody DS-8895a was released in 2016 Purified in 2018, Burvenich et al. proved that it can inhibit the growth of breast and intestinal tumors in vivo; using the single-chain antibody of EphA2 as the CAR sequence, the CAR-T cells targeting EphA2 were used to treat glioma by Beijing Xuanwu Hospital in 2018 Initiate clinical research; there are other EphA2-targeted CAR-Ts in the preclinical research stage for the treatment of lung cancer and esophageal cancer, etc. In general, the existing CAR-Ts targeting EphA2 are all designed based on EphA2 antibodies. However, if the antibody affinity is too low, the ability to target and bind tumor cells is poor, and if the antibody affinity is too high, excessive immune reactions will easily occur. Patient tolerance is poor.
因此选择与靶标分子天然结合的受体/配体，利用二者在自然条件下进化出的结合保守性的特点优势设计CAR序列，其亲和力较适宜能够更好地克服人工设计的抗体亲和力不适宜问题；同时，本发明的研究证明使用EphA2的天然配体作为胞外识别域并不影响CAR-T细胞的增殖及CD4/CD8表达。Therefore, choose the receptor/ligand that naturally binds to the target molecule, and design the CAR sequence by taking advantage of the characteristics of binding conservation evolved under natural conditions. The more appropriate affinity can better overcome the unsuitable affinity of artificially designed antibodies. Questions; at the same time, the research of the present invention proves that using the natural ligand of EphA2 as the extracellular recognition domain does not affect the proliferation and CD4/CD8 expression of CAR-T cells.
基于此，本发明首次将EFNA1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EFNA1受体阳性的细胞特异杀伤，可用于相关疾病的治疗。Based on this, the present invention integrates EFNA1 fragments into CAR vectors for the first time through genetic engineering, and modifies related immune cells, so as to achieve specific killing of EFNA1 receptor-positive cells, which can be used for the treatment of related diseases.
本发明嵌合抗原受体(CAR)Chimeric antigen receptor (CAR) of the present invention
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。Chimeric antigen receptor (CAR) is composed of extracellular antigen recognition region, transmembrane region and intracellular co-stimulatory signal region.
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。The design of CAR has gone through the following process: the first-generation CAR has only one intracellular signaling component CD3ζ or FcγRI 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. On the basis of the second-generation CAR, 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.
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。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. First, 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-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。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.
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。Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
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。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.
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EphA2靶点的EFNA1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19至182位的氨基酸序列。In the present invention, the extracellular domain of the chimeric antigen receptor is an EFNA1 protein or a fragment thereof that can specifically bind the EphA2 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 19 to 182 of the sequence shown in SEQ ID NO:1.
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。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. As used herein, 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.
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铰链区及跨膜区、4-1BB共刺激结构域和CD3ζ信号结构域组合的细胞内结构域融合。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. Preferably, the antigen binding domain is fused to the intracellular domain of a combination of the CD8 hinge and transmembrane regions, the 4-1BB co-stimulatory domain and the CD3ζ signaling domain.
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19至182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，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。In the present invention, the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which refer to at most 10 compared with the amino acid sequence at positions 19 to 182 of SEQ ID NO: 1, preferably At most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
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％。In the present invention, 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%.
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。In the present invention, 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.
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。For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to include the transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some instances, 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.
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。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。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. The intracellular domain in the CAR of the present invention includes a 4-1BB co-stimulatory domain and a CD3ζ signaling domain.
在本发明的一个实施方式中，所述的CAR是可以特异性靶向EphA2的CAR。In one embodiment of the present invention, the CAR is a CAR that can specifically target EphA2.
嵌合抗原受体免疫细胞(CAR-免疫细胞)Chimeric Antigen Receptor Immune Cells (CAR-Immune Cells)
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EFNA1受体(优选地为EphA2)的嵌合抗原受体。In the present invention, a chimeric antigen receptor immune cell is provided, which comprises the chimeric antigen receptor specifically targeting EFNA1 receptor (preferably EphA2) of the present invention.
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。The chimeric antigen receptor immune cells of the present invention may be CAR-T cells, or CAR-NK cells, or CAR-macrophages. Preferably, the chimeric antigen receptor immune cells of the present invention are CAR-T cells.
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。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。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.
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EFNA1受体(优选地为EphA2)高表达的肿瘤。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 EFNA1 receptors (preferably EphA2).
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。Natural killer (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.
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。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.
载体carrier
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。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. Alternatively, the gene of interest can be produced synthetically.
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。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.
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。In brief overview, 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.
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。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. In another embodiment, the present invention provides gene therapy vectors.
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。The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Particular vectors of interest include expression vectors, replication vectors, probe production vectors and sequencing vectors.
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)。Furthermore, 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. Generally, 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).
已经开发许多基于病毒的***，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***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***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***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。A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, 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. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.
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。Additional promoter elements, such as enhancers, 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.
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。An example of 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). However, other 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. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of 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. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
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。In order to assess the expression of the CAR polypeptide or a part thereof, 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. In other aspects, 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.
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***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。Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Typically, 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, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82). In one embodiment of the present invention, 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. Typically, 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.
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。Methods of introducing genes into cells and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.
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。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.
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***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。Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, especially 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.
将多核苷酸引入宿主细胞的化学手段包括胶体分散***，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的***，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***为脂质体(例如，人造膜囊)。Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipid-based systems. plastid. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle).
在使用非病毒传递***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。Where a non-viral delivery system is used, an exemplary delivery vehicle is liposomes. The use of lipid formulations is contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, 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 To liposomes, 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. For example, they may exist in a bilayer structure, as micelles or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, 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.
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。In a preferred embodiment of the present invention, the vector is a lentiviral vector.
制剂preparation
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。 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. In one embodiment, the formulation is a liquid formulation. Preferably, the preparation is an injection. Preferably, 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.
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。In one embodiment, 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. The formulations of the invention are preferably formulated for intravenous administration.
治疗性应用therapeutic application
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EphA2，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。The invention includes therapeutic applications of cells (eg, T cells) transduced with a lentiviral vector (LV) encoding an expression cassette of the invention. The transduced T cells can target EphA2, a marker of tumor cells, and activate T cells cooperatively, causing an immune response from immune cells, thereby significantly improving their killing efficiency against tumor cells.
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-细胞。Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the step of: administering the CAR-cell of the present invention to the mammal.
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-T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。In one embodiment, 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. In this way, the probability of suffering from graft-versus-host disease is extremely low, and the antigen is recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T can treat all cancers that express that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
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，EphA2的CAR-T细胞引起抗EphA2细胞的特异性免疫应答。In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time. Additionally, 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. For example, EphA2 CAR-T cells elicit a specific immune response against EphA2 cells.
尽管本文公开的数据具体公开了包括EFNA1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。Although the data disclosed herein specifically discloses lentiviral vectors comprising the EFNA1 protein or fragments thereof, the hinge and transmembrane regions, and the 4-1BB and CD3ζ signaling domains, the invention should be construed as including the inclusion of the EFNA1 protein as part of the construct. Any number of variations of each.
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。癌症包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)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。Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors. Cancers include non-solid tumors such as hematological tumors, such as leukemias and lymphomas. Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemia or lymphoid malignancies, benign and malignant Tumors, and malignancies such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and childhood tumors/cancers.
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。Hematological cancers are cancers of the blood or bone marrow. Examples of 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.
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。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. Preferably, the mammal is a human.
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。For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into the mammal: i) expanding the cells, ii) introducing a CAR-encoding nucleic acid into the cells, and/or iii) cryopreserving the cells.
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。Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, 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. Alternatively, the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.
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。In addition to the use of cell-based vaccines for ex vivo immunization, the invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
本发明提供了***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。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.
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。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. Briefly, 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. Such 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. The compositions of the invention are preferably formulated for intravenous administration.
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。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.
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。 When an "effective amount", "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount" is indicated, the precise amount of a composition 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 a pharmaceutical composition comprising T cells as described herein may be dosed at a dose of 10 4 to 10 9 cells/kg body weight, preferably at a dose of 10 5 to 10 6 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.
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，***或感染位置。Administration of the compositions to a subject may be by any convenient means, including by spraying, injection, swallowing, infusion, implantation or implantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, 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.
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。In certain embodiments of the invention, 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. In a further embodiment, the 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. In a further embodiment, 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. For example, in one embodiment, a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, following transplantation, the subject receives an infusion of expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered before or after surgery.
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。 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.
本发明的主要优点包括：The main advantages of the present invention include:
(a)靶点特异：以EphA2为例，EphA2在正常细胞的细胞膜上基本不表达，但在压力应激情况下(如肿瘤)表达会上调，从而本发明CAR仅针对细胞膜高表达EphA2的恶性细胞，而对正常细胞基本上无杀伤作用。(a) Target specificity: Taking EphA2 as an example, EphA2 is basically not expressed on the cell membrane of normal cells, but its expression will be up-regulated under pressure stress conditions (such as tumors), so the CAR of the present invention only targets malignant cells with high expression of EphA2 on the cell membrane. cells, but basically no killing effect on normal cells.
(b)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。(b) The present invention utilizes ligand-receptor binding mode instead of scfv in the traditional sense.
(c)本发明的基于天然配体受体特定区段的CAR，在动物特别是灵长类动物中的安全性测试对临床应用更具有参考价值。(c) The safety test of the CAR based on the specific segment of the natural ligand receptor of the present invention in animals, especially primates, has more reference value for clinical application.
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。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacture conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
表A本发明涉及的氨基酸序列总结Table A summary of the amino acid sequences involved in the present invention
Figure PCTCN2022134768-appb-000001
Figure PCTCN2022134768-appb-000001
Figure PCTCN2022134768-appb-000002
Figure PCTCN2022134768-appb-000002
Figure PCTCN2022134768-appb-000003
Figure PCTCN2022134768-appb-000003
Figure PCTCN2022134768-appb-000004
Figure PCTCN2022134768-appb-000004
实施例1：制备EFNA1-CAR载体Example 1: Preparation of EFNA1-CAR vector
基于EphA2配体EphrinA1(EFNA1)的核苷酸序列(NM_004428.3)、人的CD8α铰链区、人的CD8跨膜区、人的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。在华大基因公司合成CD8信号肽及EphrinA1(EFNA1)胞外区域，并通过XbaI(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***已将CD8TM、4-1BB、CD3ζ***的慢病毒载体pTomo中。EFNA1胞外结构域示意图如图1A，CAR全长示意图如图1B，其中MOCK为对照组。Based on the nucleotide sequence of EphA2 ligand EphrinA1 (EFNA1) (NM_004428.3), human CD8α hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3ζ intracellular region gene sequence information, The corresponding nucleotide sequence is obtained by artificial synthesis method or PCR method. The CD8 signal peptide and EphrinA1 (EFNA1) extracellular region were synthesized at BGI Corporation, and the nucleotide sequence of the CAR molecule was double-digested with XbaI (Thermo) and NheI (Thermo), and inserted by T4 DNA ligase (NEB) CD8TM, 4-1BB, and CD3ζ have been inserted into the lentiviral vector pTomo. The schematic diagram of the extracellular domain of EFNA1 is shown in Figure 1A, and the schematic diagram of the full length of CAR is shown in Figure 1B, where MOCK is the control group.
将重组质粒进行测序，比对测序结果以确认质粒是否正确。结果表明，CAR的编码序列正确地***了质粒的预定位置(图1C)。Sequence the recombinant plasmid, and compare the sequencing results to confirm whether the plasmid is correct. The results showed that the coding sequence of CAR was correctly inserted into the predetermined position of the plasmid (Fig. 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293T细胞进行慢病毒包装。All plasmids were extracted with QIAGEN’s endotoxin-free large extraction kit, and the purified plasmids were transfected into 293T cells with Biyuntian lipo6000 for lentiviral packaging.
实施例2：病毒包装Example 2: Viral Packaging
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 OPTI-MEM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lipo 6000。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。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 of OPTI-MEM dissolved plasmid mixture (core plasmid 20ug, pCMVΔR8.9 10ug, PMD2.G 4ug); in another centrifuge tube 2ml OPTIMEM and 68ul lipo 6000. 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 293T cells, incubated at 37°C for 6 hours, and then the medium was removed. Refill with pre-warmed complete medium. 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.
实施例3：CAR-T细胞制备Example 3: Preparation of CAR-T cells
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Mononuclear cells were isolated from human peripheral blood with Ficool separation medium, and purified CD3+ T cells were obtained from RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). For T cells
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＝20感染T细胞制备CAR-T细胞，感染一天后更换培养基。CD3/CD28 magnetic beads were activated (Life technology), and then 200U/ml IL2 (PeproTech) was added to stimulate the culture for 48 hours before virus infection. In the presence of lentiboost, lentivirus was used to infect T cells at an MOI of 20 to prepare CAR-T cells, and the culture medium was changed one day after infection.
得到的CAR-T细胞增殖倍数(图2A)及总数结果(图2B)所示,结果表明，使用EFNA1构建靶向EphA2的CAR不会影响CAR-T细胞的增殖。The obtained CAR-T cell proliferation fold (Figure 2A) and total number results (Figure 2B) are shown, the results show that the use of EFNA1 to construct a CAR targeting EphA2 will not affect the proliferation of CAR-T cells.
实施例4：流式细胞仪检测感染CAR-T细胞的阳性率Example 4: Positive rate of infected CAR-T cells detected by flow cytometry
分别离心收集病毒感染72小时后的CAR-T细胞、Mock对照组和NTR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。The CAR-T cells, Mock control group, and NTR cell control group were collected by centrifugation 72 hours after virus infection, washed once with PBS, discarded the supernatant, and resuspended the cells in PBS containing 2% FBS, and the positive rate was detected by flow cytometry.
转染效率的结果如图3所示。其中，NTR为未感染的T细胞，MOCK为感染了无CD7胞外结合域的对照病毒的T细胞.The results of transfection efficiency are shown in FIG. 3 . Among them, NTR refers to uninfected T cells, and MOCK refers to T cells infected with a control virus without CD7 extracellular binding domain.
结果：转染效率的结果如图3所示，其中图3A代表荧光显微镜下观察结果，结果表明，CAR细胞因为共表达的CAR-mKate2融合蛋白，因此表达后的融合蛋白经T2A切割，形成的mKate2蛋白在胞内表现出红色荧光。图3B代表流式检测结果，结果显示可见表达本发明CAR的CAR-T阳性率在80％左右。Results: The results of transfection efficiency are shown in Figure 3, in which Figure 3A represents the results observed under a fluorescent microscope. The results show that because of the co-expressed CAR-mKate2 fusion protein in CAR cells, the expressed fusion protein is cleaved by T2A to form a mKate2 protein exhibits red fluorescence in cells. Figure 3B represents the results of flow cytometry, and the results show that the positive rate of CAR-T expressing the CAR of the present invention is about 80%.
同时对感染后第3天，第6天的T细胞进行CD4/CD8流式检测。结果如图4所示：EFNA1作为靶点识别域不影响CAR-T表型，即不影响CD4,CD8阳性T细胞的比例。At the same time, CD4/CD8 flow cytometry was performed on the T cells on the 3rd day and the 6th day after infection. The results are shown in Figure 4: EFNA1 as the target recognition domain does not affect the CAR-T phenotype, that is, it does not affect the proportion of CD4, CD8 positive T cells.
实施例5：EFNA1CAR-T对各种肿瘤细胞系的杀伤作用Example 5: The killing effect of EFNA1CAR-T on various tumor cell lines
以5：1的效靶比检测本发明的CAR-T细胞对各种肿瘤细胞系的杀伤作用。The killing effect of the CAR-T cells of the present invention on various tumor cell lines was detected with an effect-to-target ratio of 5:1.
结果如图5所示：EFNA1CAR-T对膀胱癌细胞系(RT4)、***癌细胞系(PC3)、胶质瘤细胞系(U87)、乳腺癌细胞系(MCF7)、胰腺癌细胞系(BXPC3、PANC1)和结直肠癌细胞系(DLD1、HCT116)这些肿瘤细胞均有显著的杀伤作用。The results are shown in Figure 5: EFNA1CAR-T was effective against bladder cancer cell lines (RT4), prostate cancer cell lines (PC3), glioma cell lines (U87), breast cancer cell lines (MCF7), pancreatic cancer cell lines (BXPC3 , PANC1) and colorectal cancer cell lines (DLD1, HCT116) these tumor cells have significant killing effect.
实施例6：结直肠癌细胞及正常细胞中EphA2的表达检测Example 6: Detection of EphA2 expression in colorectal cancer cells and normal cells
RNA水平和蛋白水平以及免疫荧光检测结直肠癌肿瘤细胞DLD1,HCT116及正常细胞293T和COS7中EphA2的表达，其中，通过RT-PCR检测RNA表达水平(图6A),通过Western Blot检测蛋白水平(图6B),通过免疫荧光进行分子定位(图6C)，COS7细胞作为阴性对照。RNA level and protein level and immunofluorescence detection of EphA2 expression in colorectal cancer tumor cells DLD1, HCT116 and normal cells 293T and COS7, wherein, the RNA expression level was detected by RT-PCR (Fig. 6A), and the protein level was detected by Western Blot ( Figure 6B), molecular localization by immunofluorescence (Figure 6C), COS7 cells served as a negative control.
结果见图6，结果表明，结直肠癌细胞系DLD1和HCT116均表达EphA2且定位于细胞膜上，其中HCT116表达水平更高。而正常细胞293T及COS7不表达 EphA2。The results are shown in Figure 6. The results showed that both colorectal cancer cell lines DLD1 and HCT116 expressed EphA2 and localized on the cell membrane, and the expression level of HCT116 was higher. The normal cells 293T and COS7 did not express EphA2.
实施例7：携带luciferase的靶细胞构建Embodiment 7: Target cell construction carrying luciferase
从pGL3-luciferase质粒中PCR扩增出luciferase片段，然后通过XbaI和BamHI连接进入pTomo载体构建pTomo-EGFP-T2A-lucferase质粒。分别从pTomo及PLkO.1质粒中分别扩增IRES及puromycin片段。通过三片段连接成功构建pTomo-EGFP-T2A-luciferase-IRES-Puro质粒。慢病毒包装及滴度测定如前所述，按照MOI＝100分别感染人PC-3,5637,RT4,J82,DLD1及HCT116细胞系，48小时后用puromycin(1ug/ml)筛选1周得到稳定表达luciferase的细胞系，用于实施例8的细胞杀伤检测实验中。The luciferase fragment was amplified by PCR from the pGL3-luciferase plasmid, and then ligated into the pTomo vector by XbaI and BamHI to construct the pTomo-EGFP-T2A-lucferase plasmid. IRES and puromycin fragments were amplified from pTomo and PLkO.1 plasmids respectively. The pTomo-EGFP-T2A-luciferase-IRES-Puro plasmid was successfully constructed by ligation of three fragments. Lentivirus packaging and titer determination were as described above, respectively infecting human PC-3, 5637, RT4, J82, DLD1 and HCT116 cell lines according to MOI=100, and after 48 hours, they were screened with puromycin (1ug/ml) for 1 week to obtain stability The cell line expressing luciferase was used in the cell killing assay in Example 8.
实施例8：CAR-T细胞对肿瘤细胞的效靶比梯度杀伤Example 8: Gradient killing effect of CAR-T cells on tumor cells
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：表达EphA2的靶细胞：***癌细胞PC-3；膀胱癌肿瘤细胞5637、RT4、J82；结直肠癌细胞DLD1、HCT116；不表达或基本上不表达EphA2的靶细胞：293T,COS7。In this example, the killing ability of the CAR-T cells of the present invention on different target cells was tested. The target cells used include: target cells expressing EphA2: prostate cancer cell PC-3; bladder cancer tumor cells 5637, RT4, J82; colorectal cancer cells DLD1, HCT116; target cells that do not express or substantially do not express EphA2: 293T ,COS7.
将携带luciferase的上述细胞消化计数后调整细胞密度为2*10^4/ml。将100ul携带luciferase的细胞接种于黑色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0.5:1、1:1、2:1、4:1、8:1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。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。After digesting and counting the above cells carrying luciferase, adjust the cell density to 2*10^4/ml. Inoculate 100ul of luciferase-carrying cells in a black 96-well plate, adjust the cell density of CAR-T/NT cells to 1*10^5, according to E:T: 0.5:1, 1:1, 2:1, 4: 1. Inoculate 8:1 into a black 96-well plate, inoculate 100ul per well. Mix the above-mentioned target cells and T cells and incubate in an incubator for 24 hours. The cell supernatant was collected and frozen at -80°C to detect the release of IFNγ. Cell killing was detected with a promega fluorescence detection kit. First, 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.
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％Cytotoxic killer cells%=(1-target cell fluorescence value with effector cells/target cell fluorescence value without effector cells)*100%
结果如图7所示。结果表明，EFNA1-CAR-T细胞对肿瘤细胞的杀伤作用随着效靶比(E:T)升高而逐渐增强。The result is shown in Figure 7. The results showed that the killing effect of EFNA1-CAR-T cells on tumor cells was gradually enhanced as the effector-to-target ratio (E:T) increased.
实施例9：IFNγ细胞因子释放Example 9: IFNγ Cytokine Release
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。In this embodiment, the release of cytokines in the case of co-incubation of CAR-T cells of the present invention and target cells was detected. Cell supernatants co-incubated in cell killing assays were used for detection.
以IFNγ为例，方法如下：取实施例7中本发明CAR-T细胞与结直肠癌Taking IFNγ as an example, the method is as follows: take the CAR-T cells of the present invention and colorectal cancer in Example 7
DLD1,HCT116靶细胞(ET比为2：1)共孵育的细胞上清，按照IFN gamma Human ELISA Kit(life technology)检测IFNγ。The cell supernatants co-incubated with DLD1 and HCT116 target cells (ET ratio 2:1) were used to detect IFNγ according to the IFN gamma Human ELISA Kit (life technology).
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。Dissolve the standard in Standard Dilution Buffer, and perform gradient dilution to produce 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0pg/ml standard .
每孔中加入50ul Incubation buffer、50ul检测样本、50ulIFNγbiotin conjugatedAdd 50ul Incubation buffer, 50ul detection sample, 50ulIFNγbiotin conjugated to each well
solution，混匀后室温静置90分钟。solution, mix well and let stand at room temperature for 90 minutes.
然后依次按照以下步骤进行操作：Then follow the steps below in order:
(1)用1*Wash Buffer洗孔4次，每次停留1分钟。(1) Wash the wells 4 times with 1*Wash Buffer, and stay for 1 minute each time.
(2)每孔加入100ul 1*Streptavidin-HRP solution，室温静置45分钟。(2) Add 100ul 1*Streptavidin-HRP solution to each well and let stand at room temperature for 45 minutes.
(3)用1*Wash Buffer洗孔4次，每次停留1分钟。(3) Wash the wells 4 times with 1*Wash Buffer, and stay for 1 minute each time.
(4)加入100ul Stabilized chromogen，室温静置30分钟.(4) Add 100ul Stabilized chromogen and let stand at room temperature for 30 minutes.
(5)每孔加入100ul Stop solution后混匀。(5) Add 100ul Stop solution to each well and mix well.
(6)450nm处检测吸光值。(6) Detect the absorbance at 450nm.
类似地，TNFα的检测用Human TNFαELISA Kit(BD Bioscience)试剂盒进行。Similarly, the detection of TNFα was carried out with the Human TNFα ELISA Kit (BD Bioscience) kit.
结果如图8A、B所示。在与结直肠癌肿瘤细胞共孵育时，本发明的CAR-T细胞分泌的细胞因子IFNγ及TNFα量均显著上升。该结果提示EFNA1-CAR-T细胞对结直肠癌肿瘤细胞的杀伤作用与IFNγ及TNFα释放有关。The results are shown in Figure 8A,B. When co-incubating with colorectal cancer tumor cells, the amounts of cytokines IFNγ and TNFα secreted by the CAR-T cells of the present invention are significantly increased. The results suggest that the killing effect of EFNA1-CAR-T cells on colorectal cancer cells is related to the release of IFNγ and TNFα.
实施例10：过表达EphA2后对EFNA1-CAR-T肿瘤杀伤作用影响Example 10: Effect of overexpression of EphA2 on tumor killing effect of EFNA1-CAR-T
293T和COS7是EphA2阴性的正常细胞系，EFNA1-CAR-T对不表达EphA2的293T和COS7无杀伤作用。在本实施例中，体外合成EphA2编码区并通过酶切连接构建pTomo-CMV-EphA2-luciferase-IRES-EGFP。体外包装慢病毒并感染293T细胞和COS7细胞。获得稳定过表达EphA2的293T细胞系(称为293T-EphA2over)和稳定过表达EphA2的COS7细胞系(称为COS7-EphA2over)。293T and COS7 are EphA2-negative normal cell lines, and EFNA1-CAR-T has no killing effect on 293T and COS7 that do not express EphA2. In this example, the EphA2 coding region was synthesized in vitro and pTomo-CMV-EphA2-luciferase-IRES-EGFP was constructed by restriction enzyme digestion. Lentivirus was packaged in vitro and infected 293T cells and COS7 cells. The 293T cell line stably overexpressing EphA2 (called 293T-EphA2over) and the COS7 cell line stably overexpressing EphA2 (called COS7-EphA2over) were obtained.
结果如图9所示，293T-EphA2over和COS7-EphA2over细胞系均可稳定过表达EphA2。The results are shown in Figure 9, both 293T-EphA2over and COS7-EphA2over cell lines can stably overexpress EphA2.
按实施例8的方法进行体外杀伤实验，通过luciferase荧光检测EFNA1-CAR-T对293T-EphA2over和COS7-EphA2over细胞的杀伤作用In vitro killing experiments were carried out according to the method of Example 8, and the killing effect of EFNA1-CAR-T on 293T-EphA2over and COS7-EphA2over cells was detected by luciferase fluorescence
结果如图10所示。结果表明，对于EphA2在细胞膜上过表达的细胞系The results are shown in Figure 10. The results showed that for cell lines overexpressing EphA2 on the cell membrane
293T-EphA2over和COS7-EphA2over，本发明的CAR-T细胞具有显著的杀伤作用，而对正常293T及COS7细胞，本发明CAR－T细胞无杀伤作用(图10A)。并且，在与EphA2过表达的细胞系共孵育时，本发明的CAR-T细胞IFNγ分泌量明显上调，对正常细胞则与对照组基本相同(图10B)。上述结果均提示，本发明的CAR-T细胞对EphA2过表达细胞有着特异性杀伤作用，而对不表达EphA2的细胞基本无杀伤力，具有良好的安全性。293T-EphA2over and COS7-EphA2over, the CAR-T cells of the present invention have a significant killing effect, but the CAR-T cells of the present invention have no killing effect on normal 293T and COS7 cells (Figure 10A). Moreover, when co-incubating with EphA2 overexpressed cell lines, the IFNγ secretion of CAR-T cells of the present invention was significantly up-regulated, and it was basically the same as that of the control group for normal cells ( FIG. 10B ). The above results all suggest that the CAR-T cells of the present invention have a specific killing effect on EphA2 overexpressing cells, but basically have no killing effect on cells that do not express EphA2, and have good safety.
讨论discuss
不同的***肝细胞受体(如EphA1、EphA2、EphA3和EphA4)在 多种细胞上有表达，并且Eph受体蛋白家族存在多种配体(至少包括EphrinA1-6和EphrinB1-3等9种配体)。Different erythropoietin hepatocyte receptors (such as EphA1, EphA2, EphA3, and EphA4) are expressed on a variety of cells, and the Eph receptor protein family has a variety of ligands (including at least EphrinA1-6 and EphrinB1-3, etc. 9 ligands).
EphA2分子在肿瘤形成过程中发挥重要的作用，相比于正常组织，其在各种肿瘤组织中表达明显上调，使其成为一个用于治疗EphA2高表达肿瘤的理想靶点。此外，虽然既往研究表明EFNA1配体与四种Eph受体家族成员均有不同程度的结合，但本发明人经过研究发现，基于EFNA1构建的CAR免疫细胞对EphA2高表达的肿瘤具有明显的高特异性和高杀伤活性，但对不表达EphA2的正常细胞则不表现出明显的杀伤活性，因此具有高特异性和安全性，适合靶向EphA2阳性的肿瘤。The EphA2 molecule plays an important role in the formation of tumors. Compared with normal tissues, its expression is significantly upregulated in various tumor tissues, making it an ideal target for the treatment of tumors with high EphA2 expression. In addition, although previous studies have shown that EFNA1 ligands bind to four Eph receptor family members to varying degrees, the inventors have found through research that CAR immune cells constructed based on EFNA1 have significantly high specificity for tumors with high EphA2 expression. and high killing activity, but it does not show obvious killing activity on normal cells that do not express EphA2, so it has high specificity and safety, and is suitable for targeting EphA2-positive tumors.
另外，在肿瘤细胞中，除EphA2外的其他几个Eph受体也可能存在超出生理表达量的异常高表达情况。因此基于EFNA1能够结合这些Eph受体的特点，也使其具备防止由于靶点在治疗中丢失引起的免疫逃逸现象、预防肿瘤的复发的潜力。In addition, in tumor cells, several other Eph receptors besides EphA2 may also have abnormally high expression beyond the physiological expression. Therefore, based on the characteristics that EFNA1 can bind to these Eph receptors, it also has the potential to prevent immune escape caused by the loss of the target during treatment and prevent tumor recurrence.
因此，我们基于EFNA1构建的嵌合体抗原受体免疫细胞，能很好的识别EFNA1受体，对EphA2高表达的肿瘤细胞具有非常高的特异性和杀伤活性，而对不表达EphA2的正常细胞无杀伤作用，可以用于治疗EphA2高表达的肿瘤。Therefore, our chimeric antigen receptor immune cells based on EFNA1 can recognize EFNA1 receptors well, and have very high specificity and killing activity against tumor cells with high expression of EphA2, but have no effect on normal cells that do not express EphA2. The killing effect can be used to treat tumors with high expression of EphA2.
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。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (15)

  1. 一种嵌合抗原受体(CAR)，其特征在于，所述的嵌合抗原受体含有一胞外结合域，所述的胞外结合域包括基于SEQ ID NO:1所示氨基酸序列的EFNA1或其片段的结构，并且所述的胞外结合域能够特异性地与EFNA1受体以配体受体方式结合。A chimeric antigen receptor (CAR), characterized in that the chimeric antigen receptor contains an extracellular binding domain, and the extracellular binding domain includes EFNA1 based on the amino acid sequence shown in SEQ ID NO:1 or the structure of its fragment, and the extracellular binding domain can specifically bind to the EFNA1 receptor in the form of a ligand receptor.
  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括EFNA1蛋白或其片段，所述的EFNA1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182位(较佳地为19至182位)的氨基酸序列。The chimeric antigen receptor according to claim 1, wherein the extracellular binding domain comprises EFNA1 protein or a fragment thereof, and the EFNA1 protein or a fragment thereof has amino acids as shown in SEQ ID NO:1 sequence, or have the amino acid sequence of positions 1 to 182 (preferably positions 19 to 182) of the sequence shown in SEQ ID NO:1.
  3. 如权利要求2所述的嵌合抗原受体，其特征在于，所述的EFNA1蛋白或其片段的氨基酸序列选自下组：The chimeric antigen receptor according to claim 2, wherein the amino acid sequence of the EFNA1 protein or a fragment thereof is selected from the group consisting of:
    (i)如SEQ ID NO:1所示序列的第19至182位所示的序列；和(i) the sequence shown in the 19th to 182nd positions of the sequence shown in SEQ ID NO: 1; and
    (ii)在如SEQ ID NO:1所示序列的第19至182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***，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19至182位所示序列具有≥85％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。(ii) On the basis of the sequence shown in the 19th to 182nd positions of the sequence shown in SEQ ID NO: 1, one or more amino acid residues are replaced, deleted, changed or inserted, or at the N-terminal or C-terminal 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are added to the end, thereby obtaining an amino acid sequence; and the amino acid sequence obtained is the same as SEQ ID NO 1. The sequences shown in positions 19 to 182 of the sequence shown in 1 have a sequence identity of ≥85%; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).
  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：The chimeric antigen receptor according to any one of claims 1-3, wherein the structure of the chimeric antigen receptor is shown in formula I below:
    L-EB-H-TM-C-CD3ζ-RP  (I)L-EB-H-TM-C-CD3ζ-RP (I)
    式中，In the formula,
    各“-”独立地为连接肽或肽键；Each "-" is independently a connecting peptide or a peptide bond;
    L是无或信号肽序列；L is nothing or a signal peptide sequence;
    EB是胞外结合域；EB is the extracellular binding domain;
    H是无或铰链区；H is none or hinge region;
    TM是跨膜结构域；TM is the transmembrane domain;
    C是无或共刺激信号分子；C is none or a co-stimulatory signal molecule;
    CD3ζ是源于CD3ζ的胞浆信号传导序列；CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;
    RP是无或报告蛋白。RP is none or a reporter protein.
  5. 如权利要求4所述的CAR，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。The CAR according to claim 4, wherein the amino acid sequence of the chimeric antigen receptor CAR is as shown in SEQ ID NO:8.
  6. 如权利要求1所述的CAR，其特征在于，所述的EFNA1受体选自下组：EphA2。The CAR according to claim 1, wherein the EFNA1 receptor is selected from the group consisting of EphA2.
  7. 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。A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the chimeric antigen receptor as claimed in claim 1.
  8. 一种载体，其特征在于，所述的载体含有如权利要求7所述的核酸分子。A carrier, characterized in that said carrier contains the nucleic acid molecule as claimed in claim 7.
  9. 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8所述的载体或染色体中整合有外源的如权利要求7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。A host cell, characterized in that, the host cell contains the carrier as claimed in claim 8 or the chromosome is integrated with an exogenous nucleic acid molecule as claimed in claim 7 or expresses the chimera molecule as claimed in claim 1 Synthetic antigen receptors.
  10. 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8所述的载体或染色体中整合有外源的如权利要求7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。An engineered immune cell, characterized in that, the immune cell contains the carrier as claimed in claim 8 or the exogenous nucleic acid molecule as claimed in claim 7 is integrated in the chromosome or expresses the nucleic acid molecule as claimed in claim 1 chimeric antigen receptors.
  11. 如权利要求10所述的工程化免疫细胞，其特征在于，所述的工程化免疫细胞是CAR-T细胞。The engineered immune cell according to claim 10, wherein the engineered immune cell is a CAR-T cell.
  12. 一种制备如权利要求10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7所述的核酸分子或如权利要求8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。A method for preparing the engineered immune cell according to claim 10, characterized in that it comprises the following steps: transducing the nucleic acid molecule according to claim 7 or the vector according to claim 8 into the immune cell, Thus, the engineered immune cells are obtained.
  13. 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7所述的核酸分子、如权利要求8所述的载体、如权利要求9所述的宿主细胞，和/或如权利要求10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。A pharmaceutical composition, characterized in that the pharmaceutical composition contains the chimeric antigen receptor as claimed in claim 1, the nucleic acid molecule as claimed in claim 7, the carrier as claimed in claim 8, the nucleic acid molecule as claimed in claim 8, the The host cell according to claim 9, and/or the engineered immune cell according to claim 10, and a pharmaceutically acceptable carrier, diluent or excipient.
  14. 一种如权利要求1所述的嵌合抗原受体、如权利要求7所述的核酸分子、如权利要求8所述的载体、或如权利要求9所述的宿主细胞，和/或如权利要求10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EFNA1受体异常表达相关疾病的药物或制剂。A chimeric antigen receptor as claimed in claim 1, a nucleic acid molecule as claimed in claim 7, a vector as claimed in claim 8, or a host cell as claimed in claim 9, and/or as claimed in claim The use of the engineered immune cell according to claim 10 is characterized in that it is used for the preparation of drugs or preparations for preventing and/or treating diseases related to abnormal expression of EFNA1 receptor.
  15. 如权利要求14所述的用途，其特征在于，所述EFNA1受体异常表达相关疾病为肿瘤，优选地所述肿瘤选自下组：结直肠癌、脑肿瘤、乳腺癌、子宫内膜癌、膀胱癌、***癌、和胰腺癌。The use according to claim 14, characterized in that the disease related to the abnormal expression of EFNA1 receptor is a tumor, preferably the tumor is selected from the group consisting of colorectal cancer, brain tumor, breast cancer, endometrial cancer, Bladder cancer, prostate cancer, and pancreatic cancer.
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