CN116178562A - Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1 - Google Patents

Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1 Download PDF

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CN116178562A
CN116178562A CN202111435566.3A CN202111435566A CN116178562A CN 116178562 A CN116178562 A CN 116178562A CN 202111435566 A CN202111435566 A CN 202111435566A CN 116178562 A CN116178562 A CN 116178562A
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cells
car
leu
cell
efna1
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赵旭东
魏文文
张富娟
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West China Hospital of Sichuan University
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West China Hospital of Sichuan University
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Priority to PCT/CN2022/134768 priority patent/WO2023093888A1/en
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Abstract

The invention provides preparation and application of a chimeric antigen receptor immune cell constructed based on EFNA 1. In particular, the invention provides an EFNA1 engineered Chimeric Antigen Receptor (CAR) comprising an extracellular binding domain capable of specifically targeting the EFNA1 receptor. The CAR immune cell has stronger specificity and target affinity, so the killing capacity to the target cell is stronger and the safety is high.

Description

Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1
Technical Field
The invention belongs to the technical field of biological medicines for tumor immunotherapy, relates to a specific chimeric antigen receptor immune cell, and in particular relates to a CAR of a specific targeting EFNA1 receptor, a modified immune response cell thereof, and a preparation method and application thereof.
Background
Cancer is currently considered to be the leading cause of death in various countries around the world and the leading obstacle to extend human life. According to World Health Organization (WHO) statistics in 2019, cancer is the first or second leading cause of death in the 183 out of the people aged 70 years, severely threatening the health of humans. By 2020, there are estimated 1930 cases of new cancer and 1000 cases of cancer patient death worldwide, and by 2040 years according to the current incidence of cancer, 2840 cases of new cancer (including non-melanoma skin cancer other than basal cell carcinoma) will appear worldwide, increasing by 47% than 2020, and overall, the incidence and mortality burden of cancer worldwide is rapidly increasing.
Based on global tumor data statistics 2020, 1900 ten thousand new colorectal cancer patients are expected to die from colorectal cancer in 2020, 935,000 patients; overall, colorectal cancer is the third most frequently seen in morbidity, and mortality is the second most frequently seen, with high morbidity and mortality leading to serious threat to human health. Current treatment for colorectal cancer mainly includes surgery for local treatment, chemotherapy for systemic treatment, targeted therapy, and immunotherapy. With the increasing medical level, significant progress has been made in the systemic treatment of colorectal cancer. Six new chemotherapeutics were introduced to increase the median overall survival of patients with metastatic colorectal cancer from less than 9 months (untreated) to about 24 months. For stage III (lymph node positive) colon cancer patients, the overall survival benefit of fluorouracil-based chemotherapy has been established, and recent data indicate that the incorporation of oxaliplatin into such adjuvant treatment regimens has a higher efficacy, and while the efficacy of the treatment is increasing, there are still parts of the patients who develop malignant progression, and therefore new methods for treating colorectal cancer need to be explored.
Along with the development of medical technology, the life of human beings is prolonged, great progress is made in the treatment of tumors, and the life cycle of cancer patients is prolonged and the life quality is improved greatly by the application of comprehensive treatment means such as surgery, chemotherapy, radiotherapy, biological treatment and the like. In addition to conventional therapies, immunotherapy is also becoming a clinically important means for treating cancer, and more immunotherapeutic drugs are approved for clinical therapies, such as PD1, PDL1 monoclonal antibodies against immune checkpoint inhibitors, and CAR-T cell therapies.
Chimeric antigen-antibody receptor (Chimeric Antigen Receptor-T cell, CAR-T) T cells refer to T cells that, after genetic modification, recognize a specific antigen of interest in an MHC non-limiting manner and continue to activate expansion. The main structure of the polypeptide comprises three types, namely an extracellular ScFv recognition domain, which is used for recognizing and combining targets on tumor cells; the hinge region and transmembrane domain, predominantly derived from CD8 or CD28, anchors the CAR to the cell membrane, and links the extracellular recognition domain with the intracellular signal; intracellular domains, which are activation domains whose number and length differences affect the anti-tumor effect of CAR-T, are currently commonly used, with one activation domain plus one or more co-stimulatory domains, cd3ζ being a common feature of the intracellular portion of CAR that initiates signals to drive T cell killing, whereas co-stimulatory domains are primarily derived from the CD28 receptor family or TNF receptor family such as 4-1bb, ox40 or CD27, also enhancing CAR-T killing by enhancing cytokine secretion or promoting proliferation and persistence.
Normal T cells recognize tumor cells in a manner dependent on the binding of a T cell surface Receptor (TCR) to a major histocompatibility complex (Major Histocompatibility complex.mhc) on the surface of tumor cells, whereas CAR-T cells recognize tumor-associated antigens (Tumor Associated Antigen, TAA) on the surface of tumor cells in a manner independent of MHC, relying only on this structure of CAR, with the specificity of CAR recognition of antigen and the killing properties of T cells.
With the rapid development of gene editing, immunization and other subjects, CAR-T cell therapy has become a new and well-developed therapeutic approach to the treatment of hematological disorders over the past years. CAR-T treatment of malignant tumors remains the current focus of research, from the first treatment of tumors with tumor-infiltrating lymphocytes (TILs) in 1986, to the development of first generation CARs in 1993, until the first approval by the FDA of 2017 for CAR-T for the treatment of relapsed/refractory acute lymphoblastic leukemia, CAR-T treatment has gone through a course of years. Three types of CAR-T are currently used to treat tumors, including Kymriah and yescata approved by the FDA at 8 and 10 months of 2017 for the treatment of relapsed/refractory acute lymphoblastic leukemia and specific types of large B-cell lymphomas, and Tecartus approved at 7 months of 2020 for the treatment of adult mantle cell lymphomas (Mental Cell Lymphoma, MCL). CAR-T treatment has made great progress in hematological disease, and solid tumor treatment has still had certain limitations, mainly due to selection of effective targets and infiltration of CAR-T cells into tumors.
Disclosure of Invention
The invention aims to provide a chimeric antigen receptor immune cell taking an EFNA1 receptor as a target point and a preparation method and an application method thereof.
In a first aspect of the invention there is provided a Chimeric Antigen Receptor (CAR) characterised in that the CAR comprises an extracellular binding domain comprising the structure of EFNA1 or a fragment thereof based on the amino acid sequence shown in SEQ ID No. 1 and in that the extracellular binding domain is capable of specifically binding to the EFNA1 receptor in a ligand receptor manner.
In another preferred embodiment, the EFNA1 receptor is selected from the group consisting of: ephA2.
In another preferred embodiment, the extracellular binding domain has an amino acid sequence derived from EFNA 1.
In another preferred embodiment, the extracellular binding domain comprises an EFNA1 protein or fragment thereof.
In another preferred embodiment, the EFNA1 protein fragment includes the extracellular region of the EFNA1 protein.
In another preferred embodiment, the extracellular binding domain comprises an EFNA1 protein or fragment thereof.
In another preferred embodiment, the extracellular binding domain comprises an extracellular region of the EFNA1 protein, the amino acid sequence of which is 19-182 corresponding to the sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the extracellular domain has the amino acids shown in positions 19-182 of SEQ ID NO. 1.
In another preferred embodiment, the EFNA1 receptor is selected from the group consisting of: ephA2.
In another preferred embodiment, the extracellular binding domain of the CAR comprises a second extracellular domain for an additional target in addition to the first extracellular domain for the EFNA1 receptor.
In another preferred embodiment, the additional target is a tumor specific target.
In another preferred embodiment, the EFNA1 protein or fragment thereof specifically binds to EFNA1 receptors, including EphA2.
In another preferred embodiment, the binding molecule is selected from the group consisting of: ephA2.
In another preferred embodiment, said EphA2 is EphA2 located on the cell membrane.
In another preferred embodiment, said EphA2 is derived from a human or non-human mammal.
In another preferred embodiment, the non-human mammal comprises: rodents (e.g., rats, mice), primates (e.g., monkeys); preferably a primate.
In another preferred embodiment, said EphA2 is of human or monkey origin.
In another preferred embodiment, said EphA2 is of human origin.
In another preferred embodiment, the extracellular domain has the amino acid sequence shown in SEQ ID NO. 1, or has the amino acid sequence at positions 1 to 182 (preferably positions 19 to 182) of the sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the EFNA1 protein or fragment thereof corresponds to amino acid sequence from position 19 to 182 of the sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of the EFNA1 protein or fragment thereof is selected from the group consisting of:
(i) A sequence shown in 19 th to 182 th positions of the sequence shown in SEQ ID NO. 1; and
(ii) An amino acid sequence obtained by performing substitution, deletion, alteration or insertion of one or more amino acid residues, or adding 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, to the N-terminus or C-terminus thereof based on the sequence shown at positions 19 to 182 of the sequence shown in SEQ ID NO. 1; and the amino acid sequence obtained has a sequence identity of ≡85% (preferably ≡90%, more preferably ≡95%, for example ≡96%,. Gtoreq.97%,. Gtoreq.98% or ≡99%) with the sequence shown in positions 19 to 182 of the sequence shown in SEQ ID NO. 1; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).
In another preferred embodiment, the CAR has the structure shown in formula I below:
L-EB-H-TM-C-CD3ζ-RP (I)
in the method, in the process of the invention,
each "-" is independently a connecting peptide or peptide bond;
l is an absent or signal peptide sequence;
EB is an extracellular binding domain;
h is a no or hinge region;
TM is a transmembrane domain;
c is an absent or co-stimulatory signaling molecule;
cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;
RP is absent or reporter.
In another preferred embodiment, the reporter protein RP further comprises a self-cleaving recognition site, preferably a T2A sequence, at its N-terminus.
In another preferred embodiment, the reporter protein RP is a fluorescent protein.
In another preferred embodiment, the reporter protein RP is mKate2 red fluorescent protein.
In another preferred example, the amino acid sequence of the mKate2 red fluorescent protein is shown as SEQ ID NO. 2.
In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, EFNA1, GM-CSF, CD4, CD137, or a combination thereof.
In another preferred embodiment, L is a CD8 derived signal peptide.
In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO. 3.
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.
In another preferred embodiment, the H is a CD8 derived hinge region.
In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID NO. 4.
In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
In another preferred embodiment, the TM is a CD 8-derived transmembrane region.
In another preferred embodiment, the amino acid sequence of said TM is shown in SEQ ID NO. 5.
In another preferred embodiment, said C is a costimulatory signaling molecule of a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDS, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), NKG2D, GITR, TLR2, or combinations thereof.
In another preferred embodiment, said C is a costimulatory signaling molecule of 4-1BB origin.
In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID NO. 6.
In another preferred embodiment, the amino acid sequence of the cytoplasmic signaling sequence derived from CD3 zeta is shown in SEQ ID NO. 7.
In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO. 8.
In a second aspect of the invention there is provided a nucleic acid molecule encoding a chimeric antigen receptor according to the first aspect of the invention.
In a third aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.
In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, or combinations thereof.
In another preferred embodiment, the vector is a lentiviral vector.
In another preferred embodiment, the carrier is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM, pHCMV, pLBS.CAG, pHR, pLV, etc.
In another preferred embodiment, the vector is a pTomo lentiviral vector.
In another preferred embodiment, the carrier further comprises a member selected from the group consisting of: promoters, transcription enhancing elements WPRE, long terminal repeat LTR, and the like.
In another preferred embodiment, the vector comprises the nucleotide sequence shown as SEQ ID NO. 9.
In a fourth aspect of the invention there is provided a host cell comprising a vector or chromosome according to the third aspect of the invention incorporating an exogenous nucleic acid molecule according to the second aspect of the invention or expressing a CAR according to the first aspect of the invention.
In a fifth aspect of the invention there is provided an engineered immune cell comprising a vector or chromosome according to the third aspect of the invention incorporating an exogenous nucleic acid molecule according to the second aspect of the invention or expressing a CAR according to the first aspect of the invention.
In another preferred embodiment, the engineered immune cell is selected from the group consisting of: t cells, NK cells, NKT cells, or macrophages.
In another preferred embodiment, the engineered immune cell is a chimeric antigen receptor T cell (CAR-T cell) or a chimeric antigen receptor NK cell (CAR-NK cell).
In another preferred embodiment, the engineered immune cell is a CAR-T cell.
In a sixth aspect of the invention there is provided a method of preparing an engineered immune cell according to the fifth aspect of the invention comprising the steps of: transduction of a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention into an immune cell, thereby obtaining said engineered immune cell.
In another preferred embodiment, the method further comprises the step of performing functional and validity assays on the obtained engineered immune cells.
In a seventh aspect of the invention there is provided a pharmaceutical composition comprising a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and/or an engineered immune cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.
In another preferred embodiment, the formulation is a liquid formulation.
In another preferred embodiment, the formulation is in the form of an injection.
In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1X 10 3 -1×10 8 Individual cells/ml, preferably 1X 10 4 -1×10 7 Individual cells/ml.
In an eighth aspect of the invention there is provided the use of a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, and/or an engineered immune cell according to the fifth aspect of the invention, for the manufacture of a medicament or formulation for the prevention and/or treatment of a disease associated with aberrant expression of the EFNA1 receptor.
In another preferred embodiment, the EFNA1 receptor includes but is not limited to EphA2.
In another preferred embodiment, the abnormal expression of the EFNA1 receptor means that the EFNA1 receptor is overexpressed.
In another preferred embodiment, the over-expression of the EFNA1 receptor means that the expression level of the EFNA1 receptor is more than or equal to 1.5 times, preferably more than or equal to 2 times, more preferably more than or equal to 2.5 times the expression level under normal physiological conditions.
In another preferred embodiment, the diseases associated with abnormal expression of EFNA1 receptor include, but are not limited to, tumor, aging, obesity, cardiovascular disease, diabetes, neurodegenerative disease, infectious disease, etc.
In another preferred embodiment, the disorder associated with aberrant expression of EFNA1 receptor comprises a disorder associated with aberrant expression of EphA 2.
In another preferred embodiment, the EphA2 expression-related disorder comprises: tumors, aging, cardiovascular diseases, obesity, etc.
In another preferred embodiment, the disease is a malignancy in which EphA2 is highly expressed.
In another preferred embodiment, the tumor includes a hematological tumor and a solid tumor.
In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), lymphoma, or a combination thereof.
In another preferred embodiment, the solid tumor is selected from the group consisting of: breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian and esophageal cancer, glioma, lung cancer, pancreatic cancer, prostate cancer, or a combination thereof.
In another preferred embodiment, the tumor is selected from the group consisting of: colorectal cancer, brain tumor, breast cancer, endometrial cancer, bladder cancer, prostate cancer, pancreatic cancer.
In a ninth aspect of the invention there is provided the use of an engineered immune cell as described in the fifth aspect of the invention, or a pharmaceutical composition as described in the seventh aspect of the invention, for the prevention and/or treatment of cancer or tumour.
In a tenth aspect of the invention there is provided a method of treating a disease comprising administering to a subject in need of treatment an effective amount of an engineered immune cell according to the fifth aspect of the invention, or a pharmaceutical composition according to the seventh aspect of the invention.
In another preferred embodiment, the disease is a disease associated with aberrant expression of the EFNA1 receptor.
In another preferred embodiment, the disease is cancer or tumor.
In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells).
In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).
In another preferred embodiment, the methods described can be used in combination with other therapeutic methods.
In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, and the like.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
FIG. 1 shows a schematic diagram of EFNA1-CAR vector construction.
Wherein A is an EFNA1 sequence diagram, wherein 1-18AA is a signal peptide, and 19-182AA is an extracellular domain; b is a schematic diagram of the structures of plasmids MOCK-CAR and EFNA1-CAR of a control group, wherein the signal peptide, the hinge region and the transmembrane region are all derived from human CD8 molecules, 4-1BB is derived from human CD137, CD3 zeta is derived from human CD3, mKate2 is a fluorescent label and is used for detecting CAR expression; c is the HindIII cleavage identification of pTomo-EFNA1-CAR vector.
Figure 2 shows cell expansion fold and total number of EFNA1 CAR. FIG. 2A fold proliferation 12 days post T cell infection; FIG. 2B statistics of total number of cells 12 days after infection.
Figure 3 shows fluorescence microscopy and flow cytometry to detect CAR infection efficiency.
Wherein A is the result of cell fluorescence expression after T cells are infected by MOCK-CAR and EFNA1-CAR for 72 hours, wherein the upper row is bright field, and the lower row is CAR fluorescence expression; b is the result of flow detection fluorescence expression.
Figure 4 shows the phenotypic assay results of CAR-T cells, detecting the proportion of d3, d6 detected CD4, CD8 positive T cells after infection. .
Figure 5 shows the killing effect of EFNA1 CAR-T on different tumor cell lines (effective target ratio 5:1).
Wherein, A. bladder cancer cell line; B. a prostate cancer cell line; C. glioma cell lines; D. a breast cancer cell line; e, f, pancreatic cancer cell lines; g, h colorectal cancer cell line.
FIG. 6 shows the results of the detection of the expression of EphA2 by colorectal cancer tumor cell lines DLD1, HCT116 and normal cell lines 293T and COS 7.
Wherein A is RNA level detection; b is protein level detection; c is the positioning result of the immunofluorescence detection membrane.
FIG. 7 shows in vitro detection of the prostate cancer cell line PC-3 positive for EphA2 by EFNA 1-CAR; bladder cancer cell line 5637, rt4, j82; colorectal cancer tumor cell line DLD1, HCT116; and EphA2 negative normal cells HEK293T, COS7, results of the target specific gradient killing assay.
Fig. 8 shows the following 2:1 results of ifnγ and tnfα release assays after killing of colorectal cancer cell line DLD1, HCT 116.
FIG. 9 shows the results of efficiency measurements after over-expression of EphA 2.
Wherein a is RNA level; b is protein level; c is immunofluorescent membrane localization detection.
FIG. 10 shows the results of detection of killing of cells overexpressing EphA2 by EFNA1 CAR-T cells. .
Detailed Description
Through extensive and intensive studies, the inventors of the present invention have developed, for the first time, a preparation method and an application of a chimeric antigen receptor immune cell constructed based on EFNA1 through a large number of screening. Experimental results show that the CAR-T targeting the EFNA1 receptor has remarkable killing effect on the target cells with high expression of the EphA2, and has no or basically no killing effect on the cells with no or low expression of the EphA2, thus having higher specificity. The present invention has been completed on the basis of this finding.
Experiments of the invention show that although different erythropoietin hepatocyte receptors (such as EphA1, ephA2, ephA3 and EphA 4) are expressed on various cells, and the Eph receptor protein family has various ligands (at least 9 ligands including EphrinA1-6, ephrinB1-3 and the like) and certain cross reactivity exists between the Eph receptor protein and the ligand, the chimeric antigen receptor immune cell has obvious high specificity and high killing activity on tumors with high expression of EphA2, but does not show obvious killing activity on normal cells without expressing EphA2, so the chimeric antigen receptor immune cell has high specificity and safety and is suitable for targeting tumors with positive EphA 2.
In addition, in tumor cells, there may be cases of abnormally high expression of several Eph receptors other than EphA2, which exceeds the physiological expression level. Therefore, based on the characteristic that EFNA1 can bind the Eph receptors, the anti-tumor peptide also has the potential of preventing immune escape phenomenon caused by target loss in treatment and preventing tumor recurrence.
Terminology
In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other 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. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology 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, as the scope of the present invention will be limited only by the appended claims.
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, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur.
As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …" or "consisting of …".
"transduction," "transfection," "transformation," or the terms used herein refer to the process of transferring an exogenous polynucleotide into a host cell, and transcription and translation to produce a polypeptide product, including the use of plasmid molecules to introduce the exogenous polynucleotide into the host cell (e.g., E.coli).
"Gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce an RNA or protein product of the gene.
"Polynucleotide" refers to polymeric forms of nucleotides of any length, including Deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. The term polynucleotide as used herein refers to single-and double-stranded molecules that are interchangeable. Unless otherwise indicated, polynucleotides in any of the embodiments described herein include a double stranded form and two complementary single strands that are known or predicted to constitute the double stranded form.
Conservative amino acid substitutions are known in the art. In some embodiments, the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine. Furthermore, the invention provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.
The meaning of all parameters, dimensions, materials and configurations described herein will be readily understood by those skilled in the art. The actual parameters, dimensions, materials, and/or configurations may depend upon the specific application for which the invention is used. It will be appreciated by those skilled in the art that the examples or claims are given by way of example only and that the scope of the invention which can be covered by the embodiments of the invention is not limited to the specifically described and claimed scope within the scope of the equivalents or claims.
All definitions and uses herein should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.
All references, patents and patent applications cited herein are incorporated by reference with respect to the subject matter in which they are cited, and in some cases may contain the entire document.
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 embodiments.
In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" may refer to a value or composition that is within an acceptable error of 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 measured.
The term "administering" refers to physically introducing a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, e.g., by injection or infusion.
EphA2 and EphrinA1
Erythropoietin hepatocyte receptor A2 (erythropoetin-producing hepatocellular receptors A, ephA 2) is one of the members of the receptor tyrosine kinase family. The receptor tyrosine kinase family proteins play an important role in the signal path of tumor cells and participate in the development and development process of tumors.
Ephrins (EFNs) are ligands of the Eph receptor protein family, and are divided into two subclasses, ephrinA (EphrinA 1-6) and EphrinB (EphrinB 1-3).
EphrinA1 (EFNA 1) is the most widely studied ligand of EphA2 in tumors, and is also the most dominant ligand for binding with high affinity, and the total length of the protein is 205 amino acids (aa), and the interaction between the two is bound on the extracellular domain of EphA2, and the occurrence and development processes of various solid tumors are involved. In addition to EphA2, EFNA1 can bind to EphA1, ephA3, and EphA 4. EphrinA1 binds to EphA1 with lower affinity, the most predominant high affinity ligands for EphA3 are EphrinA2 (kd=1 nM) and EphrinA3 (kd=5 nM), and the most predominant high affinity ligands for EphA4 are EphrinA2 (kd=10 nM) and EphrinA4 (kd=5 nM).
The structure of Eph family proteins comprises 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 localized on the cell membrane, has 25-35% sequence homology with other Eph receptor family members, and is conserved at tyrosine residues in the membrane-proximal domain and the kinase domain.
EphA2 expression is involved in the development and progression of various tumors such as colorectal cancer. Clinical specimens of intestinal cancer showed significantly higher EphA2 expression levels compared to paired normal tissues. Furthermore, in univariate and multivariate analyses, high EphA2 mRNA and protein expression in stage II/III colorectal cancer tissues was found to be associated with poor overall survival. There are data showing that EphA2 is highly expressed in KRAS mutated colorectal cancer cells and that levels of EphA2 are regulated by KRAS-driven MAPK and RalGDS-RalA pathways. EpHA2 is poorly predicted in phase II/III CRC, possibly due to its ability to promote cell migration and invasion. EphA2 can therefore be a very valuable therapeutic target for CAR-T design, and the response characteristics and capabilities for treating colorectal cancer have not been reported.
As tumor antigen targets, inhibitors against EphA2 are currently predominantly antibodies. Coffman et al screened two antibodies EA2 and B233, promoted phosphorylation and degradation of EphA2 in tumor cells, and inhibited growth of lung and breast cancer tumors in vivo; novel EphA2 humanized monoclonal antibody DS-8895a was purified in 2016 and Burvenich et al demonstrated the ability to inhibit breast and intestinal tumor growth in vivo; using single-chain antibody of EphA2 as CAR sequence, CAR-T cells targeting EphA2 for treatment of glioma initiated clinical study at 2018 by beijing Xuan Wu hospital; there are other EphA 2-targeting CAR-T in preclinical research stages for the treatment of lung and esophageal cancer, and the like. In general, existing EphA 2-targeting CAR-T were designed based on EphA2 antibodies, however, the ability of the antibodies to bind tumor cells was poor with too low an affinity, excessive immune responses were likely to occur with too high an affinity, and patient tolerance was poor.
Therefore, a receptor/ligand which is naturally combined with the target molecule is selected, the characteristic advantage of the combination conservation which is evolved under natural conditions of the receptor/ligand and the ligand is utilized to design a CAR sequence, and the affinity of the CAR sequence is more suitable, so that the problem of unsuitable affinity of an artificially designed antibody can be better overcome; meanwhile, the studies of the present invention demonstrate that the use of the natural ligand of EphA2 as an extracellular recognition domain does not affect CAR-T cell proliferation and CD4/CD8 expression.
Based on the method, the EFNA1 segment is integrated into the CAR carrier in a genetic engineering mode for the first time, and related immune cells are modified, so that the positive cell specific killing of the EFNA1 receptor is realized, and the method can be used for treating related diseases.
Chimeric Antigen Receptor (CAR) of the invention
Chimeric immune antigen receptor (Chimeric antigen receptor, CAR) consists of extracellular antigen recognition region, transmembrane region and intracellular co-stimulatory signaling region.
The design of the CAR goes through the following process: the first generation of CARs had only one intracellular signaling component, cd3ζ or fcγri molecule, which, due to the presence of only one activation domain within the cell, only caused transient T cell proliferation and less cytokine secretion, and did not provide long-term T cell proliferation signaling and sustained in vivo anti-tumor effects, and therefore did not achieve good clinical efficacy. The second generation CAR introduces a co-stimulatory molecule such as CD28, 4-1BB, OX40 and ICOS based on the original structure, and has greatly improved function compared with the first generation CAR, and further enhances the persistence of CAR-T cells and the killing ability to tumor cells. Some new immune co-stimulatory molecules such as CD27, CD134 are concatenated on the basis of the second generation CARs, developing into third and fourth generation CARs.
The extracellular segment of the CAR recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity, and secretion of cytokines, thereby clearing the target cell. Patient autologous cells (or heterologous donors) are first isolated, CAR-producing immune cells are activated and genetically engineered, and then injected into the same patient. This way the probability of graft versus host disease is very 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 hematological malignancy therapy, which is not achieved by any conventional therapeutic means, and has triggered a hot tide of clinical research worldwide.
In particular, the Chimeric Antigen Receptor (CAR) of the invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
The extracellular domain includes a target-specific binding member. The extracellular domain may be ScFv of an antibody based on specific binding of an antigen-antibody, or may be a native sequence or a derivative thereof based on specific binding of a ligand-receptor.
In the present invention, the extracellular domain of the chimeric antigen receptor is an EFNA1 protein or fragment thereof that specifically binds to the EphA2 target of the CAR of the 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 as SEQ ID NO. 1.
The 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 connect a transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the CD8 hinge region, the transmembrane region, the 4-1BB costimulatory domain, and the CD3ζ signaling domain.
In the present invention, the extracellular binding domain of the CAR of the invention also includes sequence-based conservative variants, meaning that up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence at positions 19 to 182 of SEQ ID NO. 1 to form a polypeptide.
In the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids of the original amino acid sequence.
In the present invention, the number of the added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, most preferably 1.
For hinge and transmembrane regions (transmembrane domains), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.
The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands. The intracellular domains in the CARs of the invention include a 4-1BB costimulatory domain and a signaling domain of cd3ζ.
In one embodiment of the invention, the CAR is one that can specifically target EphA 2.
Chimeric antigen receptor immune cells (CAR-immune cells)
In the present invention, there is provided a chimeric antigen receptor immune cell comprising a chimeric antigen receptor of the present invention having a specific targeting EFNA1 receptor (preferably EphA 2).
The chimeric antigen receptor immune cells of the invention can be CAR-T cells, also can be CAR-NK cells and CAR-macrophages. Preferably, the chimeric antigen receptor immune cells of the invention are CAR-T cells.
As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to CAR-T cells according to the fifth aspect of the invention.
CAR-T cells have the following advantages over other T cell-based therapies: (1) the course of action of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor markers, CAR gene construction for a certain tumor marker can be widely utilized once completed; (3) The CAR can utilize not only tumor protein markers but also glycolipid non-protein markers, so that the target range of the tumor markers is enlarged; (4) The use of autologous patient cells reduces the risk of rejection; (5) The CAR-T cells have an immunological memory function and can survive in vivo for a long time.
As used herein, the terms "CAR-NK cells", "CAR-NK cells of the invention" all refer to CAR-NK cells of the fifth aspect of the invention. The CAR-NK cells of the invention are useful for tumors that are highly expressed by the EFNA1 receptor, preferably EphA 2.
Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion by non-antigen specific pathways. New functions may be obtained by engineered (genetically modified) NK cells, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.
CAR-NK cells also have advantages over CAR-T cells, such as: (1) The perforin and the granzyme are released to directly kill tumor cells, and the perforin and granzyme have no killing effect on normal cells of the organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storms; (3) Is easy to expand and develop into a ready-made product in vitro. In addition, similar to CAR-T cell therapy.
Carrier body
Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.
The invention also provides vectors comprising the nucleic acid molecules of the invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
In brief summary, the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.
The expression constructs of the invention may 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, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.
The nucleic acid may be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.
Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).
Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.
Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act cooperatively or independently to initiate transcription.
One 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 levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses 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 invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may 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.
The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. Typically, the reporter gene is the following gene: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al 2000FEBS Letters479:79-82). In one embodiment of the invention, the reporter gene is a gene encoding a mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, constructs with a minimum of 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to regulate promoter-driven transcription.
Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell 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, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). A preferred method of introducing the polynucleotide into a host cell is calcium phosphate transfection.
Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.
Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery tool is a liposome (e.g., an artificial membrane vesicle).
In the case of non-viral delivery systems, an exemplary delivery means is a liposome. Lipid formulations are 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. The nucleic acid associated with the lipid may be encapsulated into the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. 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 be present 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 naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
In a preferred embodiment of the invention, the vector is a lentiviral vector.
Formulations
The present invention provides a pharmaceutical composition comprising a chimeric antigen receptor CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention or an engineered immune cell according to the fifth aspect of the inventionAcceptable carriers, diluents or excipients. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said CAR-T cells in said formulation is 1 x 10 3 -1×10 8 Individual cells/ml, more preferably 1X 10 4 -1×10 7 Individual cells/ml.
In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.
Therapeutic applications
The invention includes therapeutic applications with cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding the expression cassettes of the invention. The transduced T cells can target a tumor cell marker EphA2, and synergistically activate the T cells to cause immune cell immune response, so that the killing efficiency of the transduced T cells on the tumor cells is remarkably improved.
Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering the CAR-cells of the invention to a mammal.
In one embodiment, the invention includes a class of cell therapies in which autologous T cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This way the probability of graft versus host disease is very low and the antigen is recognized by T cells in a non-MHC restricted manner. Furthermore, a CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to persistent tumor control.
In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-modified T cells induce an immune response specific for an antigen binding domain in the CAR. For example, CAR-T cells of EphA2 elicit a specific immune response against EphA2 cells.
Although the data disclosed herein specifically disclose lentiviral vectors comprising the EFNA1 protein or fragment thereof, hinge and transmembrane regions, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.
Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Cancers include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) types of cancers treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, e.g., sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
Hematological cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematogenic) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous (myelogenous) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, hodgkin's disease, non-hodgkin's lymphomas (indolent and high grade forms), multiple myelomas, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
The CAR-modified T cells of the invention can also be used as a vaccine type for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.
For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) Expanding the cells, ii) introducing nucleic acid encoding the CAR into the cells, and/or iii) cryopreserving the cells.
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 (i.e., transduced or transfected in vitro) with vectors expressing the CARs disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.
In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the 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; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.
The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.
When "effective amount" is indicated, "When an immunologically effective amount "," an anti-tumor effective amount "," a tumor-inhibiting effective amount "or" a therapeutic amount "is used, the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences in the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be administered at 10 4 To 10 9 A dose of individual cells/kg body weight, preferably 10 5 To 10 6 Individual cells/kg body weight doses (including all integer values within those ranges) are administered. T cell compositions may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.
Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intradesmally, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are 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. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.
In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapies for specific tumor patients. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.
The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, 1X 10 will be administered per treatment or per course of treatment 6 Up to 1X 10 10 The CAR-T cells of the invention are administered to a patient by, for example, intravenous infusion.
The main advantages of the invention include:
(a) Target specificity: in the case of EphA2, ephA2 is not substantially expressed on the cell membrane of normal cells, but expression is upregulated under stress conditions (e.g., tumors), so that the inventive CAR is directed only to malignant cells whose cell membrane highly expresses EphA2, and has substantially no killing effect on normal cells.
(b) The present invention utilizes the mode of action of ligand binding to receptor, rather than scfv in the traditional sense.
(c) The CAR of the invention based on a specific segment of a natural ligand receptor, safety tests in animals, in particular primates, are of more reference value for clinical applications.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. Table a summary of amino acid sequences to which the invention relates
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Example 1: preparation of EFNA1-CAR vectors
Based on the nucleotide sequence (NM_ 004428.3) of EphA2 ligand EphrinA1 (EFNA 1), human CD8 alpha hinge region, human CD8 transmembrane region, human 4-1BB intracellular region and human CD3 zeta intracellular region gene sequence information, the corresponding nucleotide sequence is obtained by artificial synthesis method or PCR method. CD8 signal peptide and EphrinA1 (EFNA 1) extracellular domain were synthesized in Huada gene, and the nucleotide sequence of the CAR molecule was digested with XbaI (Thermo) and NheI (Thermo) and inserted into lentiviral vector pTomo into which CD8TM, 4-1BB and CD3 zeta had been inserted via T4 DNA ligase (NEB) ligation. The extracellular domain of EFNA1 is schematically shown in FIG. 1A, and the full length of CAR is schematically shown in FIG. 1B, wherein MOCK is the control group.
The recombinant plasmid was sequenced and the sequencing results were aligned to confirm whether the plasmid was correct. The results indicate that the coding sequence of the CAR was correctly inserted into the predetermined position of the plasmid (fig. 1C).
All plasmids were extracted with QIAGEN endotoxinfree megapump kit and purified plasmids were lentivirally packaged with Biyundian lipo6000 transfected 293T cells.
Example 2: virus package
HEK-293T cells were cultured in 15cm dishes for virus packaging. Preparing 2ml of OPTI-MEM dissolved plasmid mixture (core plasmid 20ug, pCMV delta R8.9 ug, PMD2.G 4 ug) after transfection of HEK-293T cells with confluence of about 80% -90%; in another centrifuge tube 2ml OPTIMEM and 68ul lipo 6000. After standing at room temperature for 5min, the plasmid complex was added to the liposome complex, and standing at room temperature for 20min. The mixture was added dropwise to 293T cells, and the medium was removed after incubation at 37℃for 6 hours. The preheated complete medium was re-added. After collecting the virus supernatant for 48 hours and 72 hours, it was centrifuged at 3000rpm at 4℃for 20 minutes. After filtration through a 0.45um filter, the virus was concentrated by centrifugation at 25000rpm for 2.5 hours at 4 ℃. After the concentrated virus was solubilized with 30ul of virus lysate overnight, the virus titer was detected by QPCR. The results show that the virus titer meets the requirements.
Example 3: CAR-T cell preparation
Monocytes were isolated from human peripheral blood using Ficol isolation and purified CD3+ T cells were obtained from RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3/CD28 magnetic beads (Life technology), 200U/ml IL2 (PeproTech) was added, and after 48 hours of stimulated incubation, virus infection was performed. Lentiviruses infected T cells in the presence of lentiboost at moi=20 to prepare CAR-T cells, and the medium was changed one day after infection.
The resulting fold of CAR-T cell proliferation (fig. 2A) and total number results (fig. 2B) indicate that constructing EphA 2-targeted CARs using EFNA1 does not affect CAR-T cell proliferation.
Example 4: detection of positive rate of infected CAR-T cells by flow cytometry
CAR-T cells, mock control and NTR cell control after 72 hours of virus infection were collected separately by centrifugation, the supernatant was washed once with PBS, the cells were resuspended in PBS containing 2% fbs, and the positive rate was detected by flow.
The results of transfection efficiency are shown in FIG. 3. Where NTR is an uninfected T cell and MOCK is a T cell infected with a control virus without the CD7 extracellular binding domain.
Results: the results of transfection efficiency are shown in FIG. 3, wherein FIG. 3A represents the results of observation under a fluorescence microscope, and the results indicate that the CAR cells, because of the co-expressed CAR-mKate2 fusion protein, the expressed fusion protein is cleaved by T2A, and the formed mKate2 protein shows red fluorescence in cells. Fig. 3B represents the results of a flow assay showing that CAR-T positive rates expressing CARs of the invention are seen to be around 80%.
At the same time, on day 3 and day 6 after infection, the T cells were subjected to CD4/CD8 flow assay. The results are shown in FIG. 4: EFNA1 as a target recognition domain did not affect CAR-T phenotype, i.e. did not affect the proportion of CD4, CD8 positive T cells.
Example 5: killing effect of EFNA1 CAR-T on various tumor cell lines
And 5:1 to detect killing of various tumor cell lines by CAR-T cells of the invention.
The results are shown in FIG. 5: EFNA1 CAR-T has a remarkable killing effect on tumor cells of bladder cancer cell line (RT 4), prostate cancer cell line (PC 3), glioma cell line (U87), breast cancer cell line (MCF 7), pancreatic cancer cell line (BXPC 3, PANC 1) and colorectal cancer cell line (DLD 1, HCT 116).
Example 6: detection of EphA2 expression in colorectal cancer cells and normal cells
RNA levels and protein levels and immunofluorescence detection of EphA2 expression in colorectal tumor cells DLD1, HCT116 and normal cells 293T and COS7, wherein RNA expression levels were detected by RT-PCR (fig. 6A), protein levels were detected by Western Blot (fig. 6B), molecular localization was performed by immunofluorescence (fig. 6C), and COS7 cells served as negative controls.
The results are shown in FIG. 6, which shows that colorectal cancer cell lines DLD1 and HCT116 both expressed EphA2 and were localized on the cell membrane, with higher levels of HCT116 expression. Whereas normal cells 293T and COS7 do not express EphA2.
Example 7: construction of target cells carrying luciferases
The luciferase fragment was PCR amplified from pGL3-luciferase plasmid, and then ligated into pTomo vector by XbaI and BamHI to construct pTomo-EGFP-T2A-luciferase 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 three fragment ligation. Lentiviral packaging and titre assays human PC-3,5637, rt4, j82, dld1 and HCT116 cell lines were infected with moi=100, respectively, and after 48 hours, cell lines stably expressing luciferases were screened with puromycin (1 ug/ml) for 1 week and used in the cell killing assay of example 8.
Example 8: efficient target specific gradient killing of CAR-T cells on tumor cells
In this example, the killing ability of CAR-T cells of the invention against different target cells was tested. The target cells used include: target cells expressing EphA 2: prostate cancer cells 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 EphA 2: 293T, COS7.
The cell density was adjusted to 2 x 10 x 4/ml after digestion and counting of the cells carrying luciferases. 100ul of cells carrying luciferases were seeded in black 96-well plates, CAR-T/NT cells were adjusted to a cell density of 1 x 10≡5, and 0.5:1, 1:1, 2:1, 4:1, 8:1 were seeded in black 96-well plates at E:T, 100ul per well. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours. Cell supernatants were collected and frozen at-80℃for detection of IFNγ release. Cell killing was detected with promega fluorescence detection kit, first cells were treated with 20ul of 1 x plb lysate for 20 min, and immediately after addition of 100ul of substrate per well were detected with BioTek microplate reader.
Cytotoxic killer cell% = (1-target cell fluorescence value with effector cells/target cell fluorescence value with null effector cells) ×100%
The results are shown in FIG. 7. The results show that the killing effect of EFNA1-CAR-T cells on tumor cells gradually increases with the increase of the effective target ratio (E: T).
Example 9: IFNgamma cytokine release
In this example, cytokine release was detected in the case of co-incubation of CAR-T cells of the invention with target cells. Cell supernatants co-incubated in cell killing experiments were used for detection.
Taking ifnγ as an example, the method is as follows: ifnγ was detected according to IFN gamma Human ELISA Kit (life technology) from cell supernatants of example 7, in which CAR-T cells of the invention were incubated with colorectal cancer DLD1, HCT116 target cells (ET ratio 2:1).
The standard was dissolved with Standard Dilution Buffer and diluted in a gradient to 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0 pg/ml.
50ul Incubation buffer, 50ul of detection sample and 50ul of IFN gamma biotin conjugated solution are added into each hole, and the mixture is stirred uniformly and then kept stand for 90 minutes at room temperature.
Then sequentially operating according to the following steps:
(1) Wells were washed 4 times with 1 XWash Buffer, each for 1 min.
(2) 100ul 1*Streptavidin-HRP solution was added to each well and allowed to stand at room temperature for 45 minutes.
(3) Wells were washed 4 times with 1 XWash Buffer, each for 1 min.
(4) 100ul Stabilized chromogen was added thereto and allowed to stand at room temperature for 30 minutes.
(5) 100ul of Stop solution was added to each well and mixed well.
(6) Absorbance was measured at 450 nm.
Similarly, detection of tnfα was performed using the Human tnfα ELISA Kit (BD Bioscience) Kit.
The results are shown in FIG. 8, A, B. The amounts of cytokines ifnγ and tnfα secreted by CAR-T cells of the invention both increased significantly upon co-incubation with colorectal cancer tumor cells. This result suggests that the killing effect of EFNA1-CAR-T cells on colorectal cancer tumor cells is related to ifnγ and tnfα release.
Example 10: effect on EFNA1-CAR-T tumor killing after EphA2 overexpression
293T and COS7 are normal cell lines negative for EphA2, and EFNA1-CAR-T has no killing effect on 293T and COS7 that do not express EphA 2. In this example, the EphA2 coding region was synthesized in vitro and pTomo-CMV-EphA2-luciferase-IRES-EGFP was constructed by cleavage ligation. Lentiviruses were packaged in vitro and infected with 293T cells and COS7 cells. A293T cell line (referred to as 293T-EphA2 over) stably overexpressing EphA2 and a COS7 cell line (referred to as COS7-EphA2 over) stably overexpressing EphA2 were obtained.
As a result, as shown in FIG. 9, both 293T-EphA2 over and COS7-EphA2 over cell lines stably overexpressed EphA2.
In vitro killing experiments were performed as described in example 8 and the killing of EFNA1-CAR-T on 293T-EphA2 over and COS7-EphA2 over cells was detected by luciferase fluorescence
The results are shown in FIG. 10. The results indicate that the CAR-T cells of the invention have significant killing effects on cell lines 293T-EphA2 over and COS7-EphA2 over, where EphA2 is overexpressed on the cell membrane, whereas the CAR-T cells of the invention have no killing effects on normal 293T and COS7 cells (fig. 10A). Furthermore, the amount of ifnγ secretion by CAR-T cells of the invention was significantly up-regulated when incubated with EphA2 overexpressing cell lines, and was essentially the same for normal cells as for the control group (fig. 10B). The results show that the CAR-T cell has specific killing effect on the EphA2 over-expression cell, has no killing effect on the cell which does not express the EphA2, and has good safety.
Discussion of the invention
Different erythropoietin hepatocyte receptors (e.g., ephA1, ephA2, ephA3, and EphA 4) are expressed on a variety of cells, and there are a variety of ligands for the Eph receptor protein family (including at least 9 ligands for EphrinA1-6 and EphrinB 1-3).
EphA2 molecules play an important role in the tumor formation process, and compared with normal tissues, the expression of the EphA2 molecules in various tumor tissues is obviously up-regulated, so that the EphA2 molecules become an ideal target for treating EphA2 high-expression tumors. In addition, although previous studies indicate that EFNA1 ligand binds to four Eph receptor family members to different degrees, the inventors have found that CAR immune cells constructed based on EFNA1 have significantly high specificity and high killing activity against EphA 2-highly expressed tumors, but do not exhibit significant killing activity against normal cells that do not express EphA2, and thus have high specificity and safety, and are suitable for targeting EphA 2-positive tumors.
In addition, in tumor cells, there may be cases of abnormally high expression of several Eph receptors other than EphA2, which exceeds the physiological expression level. Therefore, based on the characteristic that EFNA1 can bind the Eph receptors, the anti-tumor peptide also has the potential of preventing immune escape phenomenon caused by target loss in treatment and preventing tumor recurrence.
Therefore, the chimeric antigen receptor immune cells constructed based on the EFNA1 can well recognize the EFNA1 receptor, have very high specificity and killing activity on tumor cells with high EphA2 expression, have no killing effect on normal cells without EphA2 expression, and can be used for treating tumors with high EphA2 expression.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.
SEQUENCE LISTING
<110> Huaxi Hospital at university of Sichuan
<120> preparation of chimeric antigen receptor immune cell constructed based on EFNA1 and application thereof
<130> P2021-1799
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<170> PatentIn version 3.5
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atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccggatcgcc acaccgtctt ctggaacagt tcaaatccca agttccggaa tgaggactac 120
accatacatg tgcagctgaa tgactacgtg gacatcatct gtccgcacta tgaagatcac 180
tctgtggcag acgctgccat ggagcagtac atactgtacc tggtggagca tgaggagtac 240
cagctgtgcc agccccagtc caaggaccaa gtccgctggc agtgcaaccg gcccagtgcc 300
aagcatggcc cggagaagct gtctgagaag ttccagcgct tcacaccttt caccctgggc 360
aaggagttca aagaaggaca cagctactac tacatctcca aacccatcca ccagcatgaa 420
gaccgctgct tgaggttgaa ggtgactgtc agtggcaaaa tcactcacag tcctcaggcc 480
catgacaatc cacaggagaa gagacttgca gcagatgacc cagaggtgcg ggttctacat 540
agcatcggtc acagtacgcc agcgccgcga ccaccaacac cggcgcccac catcgctagc 600
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 660
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 720
gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 780
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 840
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 900
agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 960
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1020
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1080
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1140
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1200
caggccctgc cccctcgc 1218
<210> 10
<211> 660
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Asp Arg His Thr Val Phe Trp Asn Ser Ser Asn
20 25 30
Pro Lys Phe Arg Asn Glu Asp Tyr Thr Ile His Val Gln Leu Asn Asp
35 40 45
Tyr Val Asp Ile Ile Cys Pro His Tyr Glu Asp His Ser Val Ala Asp
50 55 60
Ala Ala Met Glu Gln Tyr Ile Leu Tyr Leu Val Glu His Glu Glu Tyr
65 70 75 80
Gln Leu Cys Gln Pro Gln Ser Lys Asp Gln Val Arg Trp Gln Cys Asn
85 90 95
Arg Pro Ser Ala Lys His Gly Pro Glu Lys Leu Ser Glu Lys Phe Gln
100 105 110
Arg Phe Thr Pro Phe Thr Leu Gly Lys Glu Phe Lys Glu Gly His Ser
115 120 125
Tyr Tyr Tyr Ile Ser Lys Pro Ile His Gln His Glu Asp Arg Cys Leu
130 135 140
Arg Leu Lys Val Thr Val Ser Gly Lys Ile Thr His Ser Pro Gln Ala
145 150 155 160
His Asp Asn Pro Gln Glu Lys Arg Leu Ala Ala Asp Asp Pro Glu Val
165 170 175
Arg Val Leu His Ser Ile Gly His Ser Thr Pro Ala Pro Arg Pro Pro
180 185 190
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
195 200 205
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
210 215 220
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
225 230 235 240
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
245 250 255
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
260 265 270
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
275 280 285
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
290 295 300
Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
305 310 315 320
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
325 330 335
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
340 345 350
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
355 360 365
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
370 375 380
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
385 390 395 400
Gln Ala Leu Pro Pro Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu
405 410 415
Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ser Glu Leu Ile
420 425 430
Lys Glu Asn Met His Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn
435 440 445
His His Phe Lys Cys Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu Gly
450 455 460
Thr Gln Thr Met Arg Ile Lys Ala Val Glu Gly Gly Pro Leu Pro Phe
465 470 475 480
Ala Phe Asp Ile Leu Ala Thr Ser Phe Met Tyr Gly Ser Lys Thr Phe
485 490 495
Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro
500 505 510
Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val
515 520 525
Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr
530 535 540
Asn Val Lys Ile Arg Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met
545 550 555 560
Gln Lys Lys Thr Leu Gly Trp Glu Ala Ser Thr Glu Thr Leu Tyr Pro
565 570 575
Ala Asp Gly Gly Leu Glu Gly Arg Ala Asp Met Ala Leu Lys Leu Val
580 585 590
Gly Gly Gly His Leu Ile Cys Asn Leu Lys Thr Thr Tyr Arg Ser Lys
595 600 605
Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val Tyr Tyr Val Asp Arg
610 615 620
Arg Leu Glu Arg Ile Lys Glu Ala Asp Lys Glu Thr Tyr Val Glu Gln
625 630 635 640
His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly
645 650 655
His Lys Leu Asn
660
<210> 11
<211> 1983
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccggatcgcc acaccgtctt ctggaacagt tcaaatccca agttccggaa tgaggactac 120
accatacatg tgcagctgaa tgactacgtg gacatcatct gtccgcacta tgaagatcac 180
tctgtggcag acgctgccat ggagcagtac atactgtacc tggtggagca tgaggagtac 240
cagctgtgcc agccccagtc caaggaccaa gtccgctggc agtgcaaccg gcccagtgcc 300
aagcatggcc cggagaagct gtctgagaag ttccagcgct tcacaccttt caccctgggc 360
aaggagttca aagaaggaca cagctactac tacatctcca aacccatcca ccagcatgaa 420
gaccgctgct tgaggttgaa ggtgactgtc agtggcaaaa tcactcacag tcctcaggcc 480
catgacaatc cacaggagaa gagacttgca gcagatgacc cagaggtgcg ggttctacat 540
agcatcggtc acagtacgcc agcgccgcga ccaccaacac cggcgcccac catcgctagc 600
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 660
agggggctgg acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 720
gtccttctcc tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg 780
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 840
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 900
agcgcagacg cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta 960
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1020
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1080
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1140
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1200
caggccctgc cccctcgcgg cagtggagag ggcagaggaa gtctgctaac atgcggtgac 1260
gtcgaggaga atcctggccc aatgagcgag ctgattaagg agaacatgca catgaagctg 1320
tacatggagg gcaccgtgaa caaccaccac ttcaagtgca catccgaggg cgaaggcaag 1380
ccctacgagg gcacccagac catgagaatc aaggcggtcg agggcggccc tctccccttc 1440
gccttcgaca tcctggctac cagcttcatg tacggcagca aaaccttcat caaccacacc 1500
cagggcatcc ccgacttctt taagcagtcc ttccccgagg gcttcacatg ggagagagtc 1560
accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc 1620
tgcctcatct acaacgtcaa gatcagaggg gtgaacttcc catccaacgg ccctgtgatg 1680
cagaagaaaa cactcggctg ggaggcctcc accgagaccc tgtaccccgc tgacggcggc 1740
ctggaaggca gagccgacat ggccctgaag ctcgtgggcg ggggccacct gatctgcaac 1800
ttgaagacca catacagatc caagaaaccc gctaagaacc tcaagatgcc cggcgtctac 1860
tatgtggaca gaagactgga aagaatcaag gaggccgaca aagagaccta cgtcgagcag 1920
cacgaggtgg ctgtggccag atactgcgac ctccctagca aactggggca caagcttaat 1980
tag 1983

Claims (10)

1. A Chimeric Antigen Receptor (CAR), comprising an extracellular binding domain comprising the structure of EFNA1 or a fragment thereof based on the amino acid sequence set forth in SEQ ID No. 1, wherein said extracellular binding domain is capable of specifically binding to the EFNA1 receptor in a ligand receptor manner.
2. The chimeric antigen receptor according to claim 1, wherein the extracellular binding domain comprises an EFNA1 protein or a fragment thereof, said EFNA1 protein or fragment thereof having the amino acid sequence shown in SEQ ID No. 1 or having the amino acid sequence from position 1 to 182 (preferably from position 19 to 182) of the sequence shown in SEQ ID No. 1.
3. The chimeric antigen receptor according to claim 1 or 2, wherein the chimeric antigen receptor has the structure according to formula I:
L-EB-H-TM-C-CD3ζ-RP(I)
in the method, in the process of the invention,
each "-" is independently a connecting peptide or peptide bond;
l is an absent or signal peptide sequence;
EB is an extracellular binding domain;
h is a no or hinge region;
TM is a transmembrane domain;
c is an absent or co-stimulatory signaling molecule;
cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;
RP is absent or reporter.
4. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.
5. A vector comprising the nucleic acid molecule of claim 4.
6. A host cell comprising the vector or chromosome of claim 5 integrated with an exogenous nucleic acid molecule of claim 4 or expressing the chimeric antigen receptor of claim 1.
7. An engineered immune cell comprising the vector of claim 5 or the nucleic acid molecule of claim 4 or expressing the chimeric antigen receptor of claim 1 integrated exogenously into a chromosome.
8. A method of preparing the engineered immune cell of claim 7, comprising the steps of: transduction of the nucleic acid molecule according to claim 4 or the vector according to claim 5 into an immune cell, thereby obtaining said engineered immune cell.
9. A pharmaceutical composition comprising the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, and/or the engineered immune cell of claim 7, and a pharmaceutically acceptable carrier, diluent or excipient.
10. Use of a chimeric antigen receptor according to claim 1, a nucleic acid molecule according to claim 4, a vector according to claim 5, or a host cell according to claim 6, and/or an engineered immune cell according to claim 7, for the preparation of a medicament or formulation for the prevention and/or treatment of diseases associated with aberrant expression of the EFNA1 receptor.
CN202111435566.3A 2021-11-29 2021-11-29 Preparation and application of chimeric antigen receptor immune cells constructed based on EFNA1 Pending CN116178562A (en)

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CN101123983A (en) * 2004-10-27 2008-02-13 米迪缪尼股份有限公司 Modulation of antibody specificity by tailoring the affinity to cognate antigens
EP3575403A1 (en) * 2015-08-05 2019-12-04 Yoo Young Pharm Co., Ltd. Chimeric antigen receptor, and t cells in which chimeric antigen receptor is expressed
JP2021500894A (en) * 2017-10-25 2021-01-14 ノバルティス アーゲー Method for producing chimeric antigen receptor-expressing cells
AU2019331496A1 (en) * 2018-08-31 2021-03-18 Novartis Ag Methods of making chimeric antigen receptor-expressing cells
KR20210073520A (en) * 2018-09-05 2021-06-21 포세이다 테라퓨틱스, 인크. Allogeneic cell compositions and methods of use thereof
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