WO2023093888A1 - Préparation et utilisation de cellules immunitaires d'un récepteur antigénique chimérique construit sur la base de efna1 - Google Patents

Préparation et utilisation de cellules immunitaires d'un récepteur antigénique chimérique construit sur la base de 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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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

L'invention concerne la préparation et l'utilisation de cellules immunitaires d'un récepteur antigénique chimérique (CAR) construit sur la base de EFNA1. Spécifiquement, l'invention concerne un CAR modifié sur la base de EFNA1, le CAR comprenant un domaine de liaison extracellulaire, et le domaine de liaison extracellulaire étant capable de cibler spécifiquement le récepteur EFNA1. Les cellules immunitaires CAR présentent une forte spécificité et une forte affinité cible, de sorte que les cellules immunitaires CAR présentent une capacité de destruction relativement forte des cellules cibles et une forte innocuité.
PCT/CN2022/134768 2021-11-29 2022-11-28 Préparation et utilisation de cellules immunitaires d'un récepteur antigénique chimérique construit sur la base de efna1 WO2023093888A1 (fr)

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