CN115819613B - Preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor protein - Google Patents

Preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor protein Download PDF

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CN115819613B
CN115819613B CN202210507054.1A CN202210507054A CN115819613B CN 115819613 B CN115819613 B CN 115819613B CN 202210507054 A CN202210507054 A CN 202210507054A CN 115819613 B CN115819613 B CN 115819613B
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car
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leu
cell
msln
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CN115819613A (en
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赵旭东
郭婧
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West China Hospital of Sichuan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Abstract

The invention provides preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor protein. In particular, the invention provides a Chimeric Antigen Receptor (CAR) based on MSLN precursor protein engineering, the CAR comprising an extracellular binding domain capable of specifically targeting MSLN binding proteins including MUC 16. The CAR immune cells have high specificity, high killing capacity and high safety.

Description

Preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor protein
Technical Field
The invention belongs to the field of immune cell therapy, and particularly relates to preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor proteins.
Background
With the improvement of medical level, the prevention and treatment of tumors have been greatly advanced, but there are still great difficulties in diagnosing and treating various tumors, for example, early ovarian cancer is often diagnosed at the later stage of cancer spread (for example, spread to the liver or lung) because the early ovarian cancer usually has no obvious symptoms, the five-year survival rate is low, and the prognosis is extremely poor. How to diagnose early tumors quickly and accurately, and provide new treatment methods for patients lacking surgical treatment indicators has been the search direction for medical workers. With the rapid progress of imaging and molecular biology, new tumor cell specific targets are continuously emerging, and the specific targets are the basis of accurate diagnosis and treatment of tumors.
MUC16 (CA 125) is an important target for tumor treatment, and the expression level of MUC16 is usually low in normal tissues, and abnormally expressed MUC16 is often the causative factor of various diseases. MUC16 has been found to be overexpressed in a variety of tumors, such as ovarian, endometrial, pancreatic, colon, breast, and gastric cancers. In fact, as a class of high molecular weight, highly glycosylated proteins, the expression of MUC16 in normal cells is affected by complex regulation, the expression of which is often limited by the polarity of the epithelial cells. However, in the process of cancer, after cell polarity is lost, MUC16 is expressed on almost all epithelial cell surfaces and interacts with various growth factors, regulating its downstream signaling pathway, inducing the development of cancer.
Recently, the MUC16 mab drug orego has been used for the administration of primary ovarian cancer patients. Second, an anti-MUC 16 antibody drug (JCAR-020), CAR-T, is in a primary clinical trial, also for targeted therapy of ovarian cancer. In addition, a number of drugs targeting MUC16, including bispecific antibodies (BiTE) and antibody conjugated drugs (ADC), are being developed.
As one of the most popular targeted therapies at present, CAR-T cell therapy has achieved more successful clinical outcome in the field of hematologic malignancies. Its function is to redirect T cells to recognize and eliminate cells expressing a particular target antigen. Binding of the CAR to the target antigen expressed on the cell surface is independent of MHC receptors, resulting in potent T cell activation and a strong anti-tumor response. Traditional CARs most commonly consist of a single chain antibody fragment (scFv), a transmembrane region, a cytoplasmic signaling domain (typically derived from CD8, CD28, OX-40 or 4-1 BB). Recent studies have found that constructing chimeric antigen receptor T cells by way of natural selection in vivo, facilitates their differentiation between malignant (tumor) and healthy (non-tumor) cells. By improving the persistence, viability and proliferation of chimeric antigen receptor T cells, better anti-tumor effects are expected. For example, the recognition of endogenous ligand-receptor interactions has been a long-term natural choice, and thus targeting endogenous receptor-ligand recognition regions is also an important choice for CAR construction.
In the tumor formation process, MSLN is combined with ovarian cancer antigen (MUC 16) to promote adhesion of cancer cells, thereby promoting thoracic peritoneal implantation and metastasis diffusion of tumors. Whereas anti-MSLN antibodies recognize the CA125 binding domain and block mesothelin-MUC 16 dependent cell attachment on cancer cells. Furthermore, the study demonstrated that the region of cell surface mesothelin consisting of 64 amino acids at the N-terminus (residues 296-359) is the smallest fragment with complete binding activity to MUC16 (CA 125).
Thus, there is an urgent need in the art to develop CAR-T therapies that target MUC16 (CA 125). Whether chimeric antigen receptors constructed based on the MUC16 ligand MSLN precursor protein have the ability to recognize MUC16 and activate immune effector cell activity would be of interest.
Disclosure of Invention
The invention aims to provide a chimeric antigen receptor immune cell constructed based on MSLN precursor protein and taking MSLN binding protein including MUC16 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), said CAR comprising an extracellular binding domain, and said extracellular binding domain comprising the structure of a MSLN precursor protein or a fragment thereof based on the amino acid sequence shown in SEQ ID NO. 1,
And, the extracellular binding domain is capable of specifically binding to the MSLN-binding protein.
In another preferred embodiment, the binding is ligand receptor binding.
In another preferred embodiment, the MSLN-binding protein comprises MUC16.
In another preferred embodiment, the MSLN-binding protein comprises MUC16 located on the cell membrane.
In another preferred embodiment, the extracellular binding domain has an amino acid sequence derived from a MSLN precursor protein.
In another preferred embodiment, the extracellular binding domain comprises a MSLN precursor protein or fragment thereof.
In another preferred embodiment, the extracellular binding domain comprises a fragment of a membrane-bound mature MSLN of the MSLN precursor protein.
In another preferred embodiment, the amino acid sequence of the membrane-bound mature MSLN fragment is shown at positions 296 to 598 (preferably positions 296 to 362) of the sequence of SEQ ID NO: 1.
In another preferred embodiment, the extracellular binding domain further comprises an N-terminal extension fragment of a membrane-bound mature MSLN of the MSLN precursor protein.
In another preferred embodiment, the amino acid sequence of the N-terminal extension fragment is shown in positions 287 to 295 (preferably position 290 and 295) of the sequence SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of the extracellular binding domain is shown at positions 290 to 362 of the sequence of SEQ ID NO. 1.
In another preferred embodiment, the MSLN-binding protein 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, 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 MSLN binding protein.
In another preferred embodiment, the additional target is a tumor specific target.
In another preferred embodiment, the extracellular binding domain comprises a MSLN precursor protein or fragment thereof having the amino acid sequence shown in SEQ ID NO. 1, or having the amino acid sequence at positions 290 to 362 of the sequence shown in SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of the extracellular binding domain is selected from the group consisting of:
(i) A sequence shown in positions 290 to 362 of the sequence shown in SEQ ID NO. 1; and
(ii) Amino acid sequence obtained by substitution, deletion, alteration or insertion of one or more amino acid residues, or addition of 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues at the N-terminus or C-terminus thereof, based on the sequence shown at positions 290 to 362 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 at positions 290 to 362 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 that specifically binds to MSLN binding protein;
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 (e.g., green fluorescent protein, yellow fluorescent protein, red 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, 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, CD3 epsilon, 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 28-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 another preferred embodiment, the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO. 9.
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 a T cell, NK cell, NKT cell, macrophage or a combination thereof.
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, 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 preparation of a medicament or formulation for the prevention and/or treatment of a disease in which MSLN-binding protein is highly expressed.
In another preferred embodiment, the MSLN-binding protein comprises MUC16.
In another preferred embodiment, the disease associated with high expression of MSLN-binding protein includes, but is not limited to, tumor, aging, obesity, cardiovascular disease, diabetes, neurodegenerative disease, infectious disease, etc.
In another preferred embodiment, the disease associated with high expression of MSLN-binding protein comprises: tumors, aging, cardiovascular diseases, obesity, etc.
In another preferred embodiment, the disease is a malignancy in which MSLN-binding protein is highly expressed.
In another preferred embodiment, the high expression of MSLN binding protein refers to the ratio of the expression level (F1) of MSLN binding protein to the expression level (F0) under normal physiological conditions (i.e.F1/F0) is not less than 1.5, preferably not less than 2, more preferably not less than 2.5.
In another preferred embodiment, the tumor comprises a solid tumor or a hematological tumor.
In another preferred embodiment, the solid tumor is selected from the group consisting of: pancreatic cancer, breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian and esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal cancer, or combinations thereof.
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), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.
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 disorder is a disorder in which MSLN binding protein is highly expressed.
In another preferred embodiment, the high expression of the MSLN binding protein means that the expression level of the MSLN binding protein 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 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 of MSLN-CAR vector construction.
Wherein A is a schematic diagram of MSLN precursor protein sequence, wherein 1-34AA is signal peptide, 34-596AA is extracellular domain, 34-286AA is megakaryocyte enhancement factor domain, 296-598AA is membrane-bound mature MSLN; b is a schematic diagram of the structures of plasmids MOCK-CAR and MSLN-CAR of a control group (wherein the antigen binding domain of the MSLN-CAR is A diagram 290-362 AA), 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, and mKate2 is a fluorescent label for detecting CAR expression; c is the HindIII digestion identification of pTomo-MSLN-CAR vector.
Figure 2 shows the results of CAR transfection efficiency assays.
Wherein A is the result of cell fluorescence expression after T cells are infected by MOCK-CAR and MSLN-CAR for 72 hours, BF is bright field, mKate2 is CAR fluorescence expression; b is the result of flow detection fluorescence expression.
Figure 3 shows the killing effect of MSLN-CAR-T on different tumor cell lines.
Wherein, A is ovarian cancer. And B, cervical cancer. C, breast cancer.
Fig. 4 shows the results of the detection of MUC16 expression by different ovarian cancer cell lines.
Wherein A is WB to detect the expression of MUC16 protein in different ovarian cancer cell lines. B is qPCR to detect MUC16 mRNA levels. C is immunofluorescence to detect the expression level of MUC16 on cell membrane.
Figure 5 shows the gradient killing results of MSLN-CAR-T against different ovarian cancer cell lines.
Figure 6 shows IFN- γ release results after killing of different ovarian cancer cell lines by MSLN-CAR-T.
Figure 7 shows that overexpression of MUC16 in ovarian cancer cell line SKOV3 enhances MSLN-CAR killing.
Wherein A is WB to detect MUC16 protein expression. B is qPCR to detect MUC16 mRNA levels. C is immunofluorescence to detect the expression level of MUC16 on cell membrane. D is the detection of killing of SKOV3-MUC16 by MSLN-CAR. E is the cytokine IFN-gamma release assay result.
FIG. 8 shows the killing results of MSLN-CAR-T on the normal cell line HEK-293T.
Detailed Description
Through extensive and intensive studies, the inventors of the present invention have developed, for the first time, a chimeric antigen receptor immune cell constructed based on a MSLN precursor protein through a large number of screens. The single chain antibody scFv or endogenous receptor/ligand can serve as the target recognition region of the CAR, but CAR recognition targets and activation intracellular signaling are affected by a number of factors, and much research is required to determine whether the resulting CAR works. The inventor discovers through tests that, by taking a specific fragment (namely 290-362 amino acid sequences) of the mesothelin precursor protein as an extracellular binding domain of the CAR, the constructed CAR-T cell can specifically bind to MUC16 positive target cells (such as tumor cells), and has strong killing capacity and high safety. The present invention has been completed on the basis of this finding.
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 the present application, each of the following terms shall have the meanings given below, unless explicitly 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 application 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.
MSLN binding protein MUC16 (carbohydrate related antigen CA 125)
As used herein, the term "MSLN-binding protein" refers to a protein capable of binding MSLN, including but not limited to MUC16.
MUC16 (also called CA 125) is a highly glycosylated type I transmembrane protein found in 1981 by the monoclonal antibody OC125 produced by Bast et al in mice immunized with human ovarian cancer cells. In 2001, the first cDNA clone was reported to have an average molecular weight between 250 and 500 kilodaltons and also to be highly glycosylated with O-linked and N-linked oligosaccharides. The peptide backbone of MUC16 consists of an N-terminal region, a Ser/Thr/Pro-rich Tandem Repeat (TR) (156 amino acids each with both N-and O-gly-cosystems) and a C-terminal region with a short cytoplasmic tail. SEA domains with high levels of O-glycosylation in the TR repeat may bind MSLN.
MUC16 is a tumor-specific antigen overexpressed in ovarian cancer, and is currently the most widely used clinically, important serum biomarker for diagnosing ovarian cancer. Serum MUC16 from 90% of ovarian cancer patients is associated with the progression of the disease, and is therefore also commonly used as a marker to monitor disease progression and recurrence. CA125 (MUC 16) can inhibit the cytolytic response of ovarian cancer natural killer cells and can inhibit the immune response against ovarian cancer cells.
MUC16 is also over-expressed in other tumors than ovarian cancer, including cervical, fallopian, pancreatic, colon, peritoneal, nasopharyngeal, lung, breast, and gastric cancers, among others. Therefore, can be used as a target for treating tumors, especially various solid tumors.
Mesothelin (MSLN) and MSLN precursor protein
The MSLN gene is located on chromosome 1p13.3, full length 8kD. The gene comprises an open reading frame of 1884bp, and encodes 17 exons and 628 amino acids. The precursor protein of MSLN is a glycoprotein with a length of about 69kD anchored to the cell membrane with glycosyl phosphotidylinositol, which can be hydrolyzed by proteolytic enzymes into 2 parts, wherein the N-terminal is a 31kD soluble protein with megakaryocyte stimulating activity, called megakaryocyte potentiator (megakaryocyte-potentiating factor MPF); whereas membrane-bound proteins with a C-terminus of about 40kD have cell adhesion properties, called MSLNs, the N-terminus (residues 296-359) of which binds CA 125. MUC16-MSLN interactions play a role in cancer cell adhesion, and anti-MSLN antibodies can eliminate MSLN binding to MUC 16-expressing positive cells and block MUC 16/mesothelin dependent cell adhesion.
Based on the method, the MSLN precursor protein fragment is integrated into the CAR carrier in a genetic engineering mode for the first time, and related immune cells are modified, so that the specific killing of MUC16 positive cells is realized, and the method can be used for treating related diseases. The invention constructs CARs using the N-terminal fragment of MSLN (SEQ ID NO. 1AA296-362) and the fragment of 6 amino acids linked to the N-terminal of the MSLN precursor protein. The CARs of the invention are constructed based on fragments of MSLN precursor proteins, capable of binding to MSLN receptors, including MUC 16.
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 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 comprises an extracellular domain, a transmembrane domain, and an intracellular domain.
The extracellular domain includes a target-specific binding member. The extracellular domain may be an scFv of an antibody based on specific binding of an antigen-antibody, or may be a native sequence or derivative thereof based on specific binding of a ligand-receptor.
In the present invention, the extracellular domain of the chimeric antigen receptor is a MSLN precursor protein or fragment thereof that specifically binds to the MUC16 target of the CAR of the present invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence at positions 290 to 362 of the sequence shown in SEQ ID NO. 1.
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 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 CD28 signaling 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 290 to 362 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 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 a CAR that can specifically target MUC 16.
Chimeric antigen receptor immune cells (CAR-immune cells)
In the present invention, there is provided a chimeric antigen receptor immune cell comprising the chimeric antigen receptor of the present invention having a specific target MUC 16.
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 cell can be used for tumors with high MUC16 expression.
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 invention provides an engineered immune cell 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 a fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. 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 marker MUC16 of tumor cells, and synergistically activate the T cells to cause immune response of immune cells, 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 MUC16 elicit a cell-specific immune response against MUC 16.
Although the data disclosed herein specifically disclose lentiviral vectors comprising a MSLN precursor 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 to 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) and solid tumors. Types of cancers treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemia or lymphoid malignancies, benign and malignant tumors, such as 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 referring to "effective amount", "immunologically effective amount", "antineoplastic effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", 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 of 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: MUC16 is not substantially expressed on the cell membrane of normal cells, but is highly expressed on part of the tumor cell membrane. The CAR immune cells of the invention are only aimed at malignant cells with high expression of MUC16 in cell membranes, and have small effect on other cells which do not express or low expression of MUC 16.
(b) The present invention utilizes the mode of action of ligand binding to receptor, rather than scfv in the traditional sense. The selectivity and affinity of receptor-ligand interactions are naturally selected over a long period of time, and conservation of receptor-ligand interactions determines that safety assays in animals, particularly primates, are more responsive to their safety in humans.
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.
Reagents, plasmids, and cells in the examples of the present application are commercially available unless otherwise indicated. Table 1 summarizes the sequences of the present application.
TABLE 1 summary of the sequences to which the application relates
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Table 2 shows the cell lines used in the examples.
TABLE 2 cell lines
Cell lines Type(s)
OVCAR3 Ovarian cancer cells
SKOV3 Ovarian cancer cells
Hela Cervical cancer cell
MDA-MB468 Breast cancer cells
MDA-MB231 Breast cancer cells
Example 1: preparation of MSLN-CAR vector
Based on the MSLN nucleotide sequence (NC_ 000016), human CD8 signal peptide, 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. The CD8 signal peptide and the MSLN extracellular region were synthesized and the nucleotide sequence of the CAR molecule was double digested by agoi (Thermo) and NheI (Thermo) and inserted into the lentiviral vector pTomo, into which the CD8 transmembrane region, 4-1BB costimulatory domain, CD3 zeta signaling region had been inserted, via a T4 DNA ligase (NEB) ligation. Competent E.coli (Stbl 3) was transformed.
Sequencing the recombinant plasmid, and comparing the sequencing results to confirm whether the plasmid is correct, wherein the sequencing primer is a universal sequencing primer. Both sequencing and restriction characterization showed 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 HEK-293T cells.
Example 2: virus package
HEK-293T cells were cultured in 15cm dishes for virus packaging. 2ml of OPTIMEM-dissolved plasmid mixture (core plasmid 20. Mu.g, pCMV. DELTA.R 8.9. Mu.g, PMD2.G 4. Mu.g) was prepared after transfection at about 80% -90% confluence of HEK-293T cells; in another centrifuge tube 2ml OPTI MEM and 68. Mu.l lipo6000. 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. The virus supernatants were collected for 48 hours and 72 hours, centrifuged at 3000rpm for 20 minutes at 4℃and filtered through a 0.45 μm filter, and centrifuged at 25000rpm for 2.5 hours at 4℃for virus concentration. After the concentrated virus was solubilized with 30. Mu.l of the virus lysate overnight, the virus titer was detected by Q PCR. The results show that the virus titer meets the requirements.
Example 3: CAR-T cell preparation
Mononuclear cells were isolated from human peripheral blood using Ficol separation, and purified from RosetteSep Human T Cell Enrichment Cocktail (Stemc Cell technologies) to obtain purified CD3 + T cells. T cells were activated with CD3/CD28 magnetic beads (Life technology), and IL2 (PeproTech) was added at a final concentration of 200U/ml, followed by 48 hours of stimulated culture for virus infection. Lentiviruses infected T cells in the presence of lentiboost to prepare CAR-T cells at moi=20. The medium was changed one day after infection.
Example 4: detection of positive rate of infected CAR-T cells by flow cytometry
CAR-T cells and NT cells (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.
Results: the results of transfection efficiency are shown in FIG. 2.
As shown in fig. 2A, CAR-T2A-mKate2 fusion proteins expressed by CAR-T cells, after cleavage, formed mKate2 proteins exhibited red fluorescence in cells.
As shown in fig. 2B, detection using flow cytometry indicated a positive expression rate of CAR or mKate2 CAR-T of about 60%.
Example 5: construction of target cells carrying luciferases
The pTomo-CMV-Luciferase-IRES-Puro lentivirus packaging procedure was the same as in example 2.
OVCAR3, SKOV3, hela, MDA-MB-468, MDA-MB-231, HEK-293T cells were successfully obtained by screening with Puromycin (1. Mu.g/ml) for 2 weeks after virus infection of OVCAR3, SKOV3, hela, MDA-MB-468, MDA-MB-231, HEK-293T-luciferase cells.
Example 6: CAR-T cell killing
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 highly expressing MUC 16: OVCAR3; target cells that do not express or underexpress MUC 16: SKOV3, hela, MDA-MB-468, MDA-MB-231.
The cell densities were adjusted to 2.5X10 after the OVCAR3, SKOV3, hela, MDA-MB-468, MDA-MB-231, HEK-293T-luciferase cell digestions were counted 4 /ml. Mu.l of luciferase cells were seeded in 96-well plates and the CAR-T and NT cells were adjusted to cell densities1×10 5 Per ml, in a 4:1 effective target ratio E: T, into a black 96-well plate, 100 μl per well. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours.
Cell densities were adjusted to 2.5X10 after OVCAR3, SKOV3-luciferase cell digestions were counted 4 /ml. Mu.l of OVCAR3, SKOV3-luciferase cells were seeded in 96-well plates and the CAR-T and NT cells were adjusted to a cell density of 2.5X10 4 、5×10 4 、1×10 5 、2×10 5 Per ml, 1:1, 2:1, 4:1, 8:1 E:T was plated into black 96-well plates, 100 μl per well. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours.
The cell supernatants were collected and stored at-80℃for detection of IFN-gamma release (see example 8). Cell killing was detected with promega fluorescence detection kit, cells were first treated with 30 μl of 1 x plb lysate for 20 min, and immediately after addition of 30 μl of substrate per well were detected with BioTek microplate reader.
Cytotoxic killer cell% = (1-target cell fluorescence value at effector cell-containing/target cell fluorescence value at effector cell-null) ×100%
Results: the results of killing of different tumor cell lines by MSLN-CAR-T are shown in FIG. 3. The results of ovarian cancer cells (A), cervical cancer cells (B) and breast cancer cells (C) show that MSLN-CAR-T has good killing effect on various tumor cell lines.
Example 7: expression of MUC16 by ovarian cancer cells and CAR-T cytotoxicity experiments
(1) Cellular immunofluorescence: target cells were plated on discs of 24-well plates, after 24 hours, cells were fixed with 4% Paraformaldehyde (PFA) for 20 minutes, washed three times with PBST for 5 minutes each; blocking with 10% goat serum was performed for 1 hour at room temperature and incubated overnight with antibodies specifically recognizing NUC 16. The next day, wash with PBST three times, five minutes each. Secondary antibodies specifically recognizing the primary antibody were labeled with CY3 and incubated at room temperature for 1 hour. After three PBST washes, DAPI stained nuclei. Confocal microscopy imaging.
(2) Immunoblotting: collecting 6cm culture dish cells, centrifuging at 4deg.C for 5min at 5500 r/min; removing supernatant, adding RIPA cell lysate containing protease inhibitor PMSF according to cell number, performing on-ice lysis for 20min, centrifuging at 4deg.C for 30min, and collecting supernatant solution for concentration measurement; protein samples were loaded at 50. Mu.g and subjected to protein electrophoresis to detect the expression of MUC16 in target cells.
(3) qPCR: collecting 6-well plate cells, removing culture medium, adding 1ml Trizol to lyse cells, standing at room temperature for 5min, adding 200 μl/1ml chloroform of Trizol, mixing for 6-8 times, and standing at room temperature for 5min;12000g, centrifuging at 4 ℃ for 15 minutes, and sucking the supernatant into another centrifuge tube; adding isopropanol with equal volume, mixing upside down, and standing at room temperature for 10min;12000g, centrifuging at 4 ℃ for 10 minutes, and discarding the supernatant; 1ml of 70% ethanol (with RNase free H 2 O) washing, 7500g, centrifuging at room temperature for 5min; removing the supernatant, standing at room temperature for 10min, drying RNA, and adding 30 μl RNase free water to dissolve RNA; nandrop 2000 measured the concentration of RNA and 1% agarose gel electrophoresis was used to detect the integrity of RNA and the accuracy of quantification. cDNA was synthesized and mRNA levels were measured according to the instructions of RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific).
Results: the results of MUC16 expression assays for each cell line are shown in FIG. 4. The expression of target cell MUC16 was detected by WB (FIG. 4A) and qPCR (FIG. 4B), and the results consistently indicate that OVCAR3 highly expressed MUC16 and SKOV3 lowly expressed MUC16. Further, high expression of MUC16 on OVCAR3 cell membrane was confirmed by immunofluorescence localization, and MUC16 on SKOV3 cell membrane was not substantially expressed (FIG. 4C).
The results of gradient killing of MSLN-CAR-T against different ovarian cancer cell lines are shown in figure 5. (a) gradient killing effect of MSLN-CAR-T on SKOV 3; (B) gradient killing effect of MSLN-CAR-T on OVCAR 3. The results show that the killing effect of MSLN-CAR-T cells on MUC16 high expression tumor cells gradually increases with the increase of the effective target ratio (E: T).
Example 8: CAR-T targeting ovarian cancer cells is accompanied by cytokine IFN-gamma 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.
The method comprises the following steps: IFN-y 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 OVCAR3, SKOV3 target cells (E: T ratio 4:1).
The standard was dissolved with Standard Dilution Buffer and diluted in a gradient to 1000pg/ml, 500 pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0 pg/ml.
Mu. l Incubation buffer, 50. Mu.l of the test sample and 50. Mu.l of IFN-. Gamma. biotin conjugated solution were added to each well, and the mixture was allowed to stand at room temperature for 90 minutes after mixing.
Then sequentially operating according to the following steps:
(1) Wells were washed 4 times with 1 XWash Buffer, each for 1 min.
(2) Mu.l of 1 x strepitavidin-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) 100 mu l Stabilized chromogen was added thereto, and the mixture was allowed to stand at room temperature for 30 minutes.
(5) Mu.l of Stop solution was added to each well and mixed well.
(6) Absorbance was measured at 450 nm.
The results are shown in FIG. 6. The cytokines are obviously increased after MSLN-CAR-T kills OVCAR3, and SKOV3 is not obviously changed. The results show that the killing effect of MSLN-CAR-T cells on tumor cells is accompanied by IFN-gamma release, suggesting that the killing effect is related to IFN-gamma release.
Example 9: effect on killing of MSLN-CAR-T tumors after MUC16 overexpression
Based on the CDS region sequence of MUC16, MUC16 overexpressing plasmids (EX-Y1397-Lv 183, ORF lentiviral expression clone) were purchased from brocade organisms to construct SKOV3 stable overexpressing MUC16 cell lines.
The Lenti-MUC16-EGFP-NeoR lentiviral packaging procedure was the same as in example 2. SKOV3-MUC16 cells were successfully obtained by screening with Neomycin (3. Mu.g/ml) for 2 weeks after virus infection of SKOV3 cells. The over-expression efficiency is detected at the protein and gene level, and MSLN-CAR-T killing detection is performed.
Will be different from ovaryCell density was adjusted to 2.5X10 after the cell digest count of the cancer cell line SKOV3-Vector, SKOV3-MUC16-luciferase 4 /ml. Mu.l of luciferase cells were seeded in 96-well plates and the CAR-T/NT cells were adjusted to a cell density of 1X 10 5 The wells were plated in black 96-well plates at 4:1 for E:T, with 100 μl each. The target cells and the T cells were mixed uniformly and incubated in an incubator for 24 hours.
Results: the results of killing of the ovarian cancer cell line SKOV3 after overexpression of MUC16 by MSLN-CAR-T are shown in figure 7. FIG. 7A shows the WB assay for MUC16 protein expression. FIG. 7B shows qPCR detection of MUC16 mRNA levels. FIG. 7C shows the immunofluorescence of MUC16 on cell membrane. The results all showed successful construction of SKOV3 cells over-expressing MUC 16.
FIG. 7D shows the killing effect of MSLN-CAR-T on MUC16 over-expression of SKOV 3. FIG. 7E shows IFN-y release from MSLN-CAR-T killing after overexpression of MUC16 by SKOV 3. The results show that both the killing rate and IFN-gamma release of SKOV3-MUC16 cells over-expressing MUC16 by MSLN-CAR-T cells were significantly increased compared to the control SKOV 3-Vector. The result shows that the killing effect of the MSLN-CAR-T cells on MUC16 over-expression tumor cells is obviously enhanced.
Example 10: killing of non-tumor cells by MSLN-CAR-T
HEK-293T cells are human embryonic kidney cell lines, the human embryonic kidney cell lines are inoculated into a black 96-well plate according to an effective target ratio of 4:1, MSLN-CAR-T cells are incubated with HEK-293T-luciferase cells, and the killing of the HEK-293T cells by the MSLN-CAR-T cells is detected through fluorescence value change.
The results are shown in FIG. 8. FIG. 8A shows the WB assay for MUC16 protein expression. FIG. 8B shows qPCR detection of MUC16 mRNA levels. FIG. 8C shows the immunofluorescence of MUC16 on cell membrane. FIG. 8D is the killing effect of MSLN-CAR-T on HEK-293T. FIG. 8E is IFN- γ release from MSLN-CAR-T killing by HEK-293T.
The results show that the expression level of MUC16 in non-tumor cells HEK-293T is low, and MSLN-CAR-T has no remarkable killing effect on HEK-293T.
Discussion of the invention
The single chain antibody scFv or endogenous receptor/ligand can serve as the target recognition region of the CAR, but CAR recognition targets and activation intracellular signaling are affected by a number of factors, and much research is required to determine whether the resulting CAR works. Previous studies suggest that there is an interaction between MSLN and MUC16 that can bind to each other, and the present invention uses a specific fragment (i.e., amino acid sequence 290-362) of the MSLN fragment and its N-terminal portion extension as the extracellular binding domain of CAR in the MSLN precursor protein. The present inventors have found that CAR-T cells constructed using this fragment as the extracellular binding domain of CAR are able to specifically bind MUC16 positive target cells (e.g. tumor cells), have strong killing ability and high safety.
All documents mentioned in this disclosure are incorporated by reference in this disclosure 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 application as defined in the appended claims.
Sequence listing
<110> Huaxi Hospital at university of Sichuan
<120> preparation of chimeric antigen receptor immune cells constructed based on MSLN precursor protein and application thereof
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atggccctgc ccgtcaccgc tctgctgctg ccccttgctc tgcttcttca tgcagcaagg 60
ccgcggccgc ggttccggcg ggaagtggag aagacagcct gtccttcagg caagaaggcc 120
cgcgagatag acgagagcct catcttctac aagaagtggg agctggaagc ctgcgtggat 180
gcggccctgc tggccaccca gatggaccgc gtgaacgcca tccccttcac ctacgagcag 240
ctggacgtcc taaagcataa actggatgag ctctacccac aaaccacgac gccagcgccg 300
cgaccaccaa caccggcgcc caccatcgct agccagcccc tgtccctgcg cccagaggcg 360
tgccggccag cggcgggggg cgcagtgcac acgagggggc tggacttcgc ctgtgatatc 420
tacatctggg cgcccttggc cgggacttgt ggggtccttc tcctgtcact ggttatcacc 480
ctttactgca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 540
ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 600
ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtacaagcag 660
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 720
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 780
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 840
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 900
gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg c 951

Claims (9)

1. A Chimeric Antigen Receptor (CAR), characterized in that the CAR has the structure according to formula I:
L-EB-H-TM-C-CD3ζ-RP (I)
in the method, in the process of the application,
each "-" is independently a connecting peptide or peptide bond;
l is an absent or signal peptide sequence;
EB is an extracellular binding domain with the amino acid sequence shown in positions 290 to 362 of SEQ ID NO. 1;
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.
2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 8.
3. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.
4. A vector comprising the nucleic acid molecule of claim 3.
5. A host cell comprising the vector or chromosome of claim 4 integrated with an exogenous nucleic acid molecule of claim 3 or expressing the CAR of claim 1.
6. An engineered immune cell comprising the vector or chromosome of claim 4 integrated with an exogenous nucleic acid molecule of claim 3 or expressing the CAR of claim 1.
7. A method of preparing the engineered immune cell of claim 6, comprising the steps of: transduction of the nucleic acid molecule according to claim 3 or the vector according to claim 4 into an immune cell, thereby obtaining said engineered immune cell.
8. A pharmaceutical composition comprising the CAR of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, the host cell of claim 5, and/or the engineered immune cell of claim 6, and a pharmaceutically acceptable carrier, diluent or excipient.
9. Use of the CAR of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, the host cell of claim 5, and/or the engineered immune cell of claim 6 for the preparation of a medicament or formulation for preventing and/or treating a disease of high expression of MUC16 selected from the group consisting of: ovarian cancer, cervical cancer, or a combination thereof.
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Publication number Priority date Publication date Assignee Title
CN110527667A (en) * 2018-05-25 2019-12-03 深圳宾德生物技术有限公司 A kind of Chimeric antigen receptor and Chimeric antigen receptor T cell and its preparation method and application targeting MUC16

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Publication number Priority date Publication date Assignee Title
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CA3101641A1 (en) * 2018-06-04 2019-12-12 Intrexon Corporation Muc16 specific chimeric antigen receptors and uses thereof
US20210040174A1 (en) * 2019-08-08 2021-02-11 Navrogen, Inc. Composition and Use of Humoral Immune Suppressor Antagonists for the Treatment of Humoral Immune Suppressed Diseases
WO2021155034A1 (en) * 2020-01-29 2021-08-05 TCR2 Therapeutics Inc. Compositions and methods for tcr reprogramming using muc16 specific fusion proteins
CN111925990B (en) * 2020-09-02 2022-08-26 北京立康生命科技有限公司 Anti-MUC16 CAR-T cell aiming at ovarian cancer and preparation method thereof
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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110527667A (en) * 2018-05-25 2019-12-03 深圳宾德生物技术有限公司 A kind of Chimeric antigen receptor and Chimeric antigen receptor T cell and its preparation method and application targeting MUC16

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Ligand-based adoptive T cell targeting CA125 in ovarian cancer;Haihong Zhao et al;Research Square;第1-20页 *

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