CN115819613A - 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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CN115819613A
CN115819613A CN202210507054.1A CN202210507054A CN115819613A CN 115819613 A CN115819613 A CN 115819613A CN 202210507054 A CN202210507054 A CN 202210507054A CN 115819613 A CN115819613 A CN 115819613A
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赵旭东
郭婧
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West China Hospital of Sichuan University
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Abstract

The invention provides a preparation method and application of a chimeric antigen receptor immune cell constructed based on MSLN precursor protein. In particular, the invention provides a Chimeric Antigen Receptor (CAR) engineered based on the MSLN precursor protein, said CAR comprising an extracellular binding domain capable of specifically targeting MSLN binding proteins including MUC16. The CAR immune cell has high specificity and high killing capacity, and is high in 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 a chimeric antigen receptor immune cell constructed based on MSLN precursor protein.
Background
With the increase of medical level, the prevention and treatment of tumors have been greatly advanced, but there are still a great number of tumors with great difficulty in diagnosis, for example, because early ovarian cancer usually has no obvious symptoms, so the disease is often diagnosed at the late stage of cancer spread (for example, spread to the liver or lung), the five-year survival rate is low, and the prognosis is very poor. How to diagnose early stage tumor rapidly and accurately and provide new treatment method for patients lacking surgical treatment indication is always the exploration direction of medical workers. With the rapid development of imaging and molecular biology, new tumor cell specific targets are emerging continuously, and the specific targets are the basis of accurate diagnosis and treatment of tumors.
MUC16 (CA 125) is an important target for tumor treatment, the expression level of MUC16 is usually low in normal tissues, and the abnormal expression of MUC16 is often a cause 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 influenced by complex regulation, the expression of which is usually limited by epithelial polarity. However, during the course of canceration, following loss of cell polarity, MUC16 is expressed on the surface of almost all epithelial cells and interacts with a variety of growth factors, regulating its downstream signaling pathways, inducing the development of cancer.
Recently, the MUC16 mab drug oregovmab has been used for primary ovarian cancer patients. Secondly, an anti-MUC 16 antibody drug (JCAR-020), CAR-T, is in phase one clinical trial, also for targeted therapy of ovarian cancer. In addition, a number of agents targeting MUC16, including bispecific antibodies (BiTE) and antibody-conjugated drugs (ADC), are being further developed.
CAR-T cell therapy has achieved relatively successful clinical outcomes in the field of hematologic malignancies as one of the most popular targeted therapies today. Its function is to redirect T cells to recognize and eliminate cells expressing a particular target antigen. Binding of CARs to target antigens 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 signal domain (usually derived from CD8, CD28, OX-40, or 4-1 BB). Recent studies have found that chimeric antigen receptor T cells are constructed by means of natural selection in the body, which facilitates differentiation between malignant cancer cells (tumor cells) and healthy cells (non-tumor cells). By improving the persistence, survival and proliferation of chimeric antigen receptor T cells, a better anti-tumor effect is expected. For example, endogenous ligand-receptor recognition by each other has been through long natural selection, and thus endogenous receptor-ligand as target recognition region is also an important choice for CAR construction.
During the formation of the tumor, MSLN is combined with ovarian cancer antigen (MUC 16) to promote the adhesion of cancer cells, thereby promoting the pleuroperitoneal membrane planting and metastatic spread of the tumor. Whereas anti-MSLN antibodies recognize the CA125 binding domain and block mesothelin-MUC 16-dependent cell attachment on cancer cells. In addition, the study demonstrated that the region consisting of 64 amino acids at the N-terminus of mesothelin (residues 296-359) on the cell surface was the smallest fragment with full binding activity to MUC16 (CA 125).
Therefore, there is an urgent need in the art to develop CAR-T therapies targeting MUC16 (CA 125). Whether the chimeric antigen receptor constructed based on the MUC16 ligand MSLN precursor protein has the activity of recognizing MUC16 and activating immune effector cells or not is worthy of study.
Disclosure of Invention
The invention aims to provide a chimeric antigen receptor immune cell which is constructed based on MSLN precursor protein and takes MSLN binding protein including MUC16 as a target spot, 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 comprising the structure of the MSLN precursor protein or a fragment thereof based on the amino acid sequence shown in SEQ ID NO:1,
and, said extracellular binding domain is capable of specifically binding to 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, said 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 the MSLN precursor protein.
In another preferred embodiment, the extracellular binding domain comprises the MSLN precursor protein or a fragment thereof.
In another preferred embodiment, the extracellular binding domain comprises a fragment of 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 as shown in 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 membrane-bound mature MSLN of the MSLN precursor protein.
In another preferred embodiment, the amino acid sequence of the N-terminal extension fragment is as shown in positions 287 to 295 (preferably 295 positions 290) of the sequence of SEQ ID NO: 1.
In another preferred embodiment, the amino acid sequence of the extracellular binding domain is as shown in SEQ ID NO 1 at positions 290 to 362.
In another preferred embodiment, the MSLN binding protein is of human or non-human mammalian origin.
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, in addition to the first extracellular domain directed against the MSLN binding protein, a second extracellular domain directed against an additional target.
In another preferred embodiment, the additional target is a tumor specific target.
In another preferred embodiment, said extracellular binding domain comprises a MSLN precursor protein or a fragment thereof, said MSLN precursor protein or fragment thereof having the amino acid sequence as shown in SEQ ID NO.1, or having the amino acid sequence from position 290 to 362 of the sequence as shown in SEQ ID NO. 1.
In another preferred embodiment, the amino acid sequence of said extracellular binding domain is selected from the group consisting of:
(i) The sequence as shown in the 290 th to 362 th positions of the sequence shown in SEQ ID NO. 1; and
(ii) 1, or 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are added at the N-terminus or C-terminus thereof, thereby obtaining an amino acid sequence; and the obtained amino acid sequence has a sequence identity of more than or equal to 85% (preferably more than or equal to 90%, more preferably more than or equal to 95%, such as more than or equal to 96%, more than or equal to 97%, more than or equal to 98% or more than or equal to 99%) with the sequence as shown in SEQ ID NO.1 at positions 290 to 362; 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 formula (I), the compound is shown in the specification,
each "-" is independently a linker peptide or a peptide bond;
l is a null or signal peptide sequence;
EB is an extracellular binding domain that specifically binds to MSLN binding protein;
h is a none or hinge region;
TM is a transmembrane domain;
c is a no or co-stimulatory signal molecule;
CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ;
RP is a null or reporter protein.
In another preferred embodiment, the reporter protein RP further comprises a self-splicing recognition site at its N-terminus, preferably a T2A sequence.
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 example, 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, said L is a signal peptide derived from CD 8.
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, said H is a CD 8-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 domain.
In another preferred embodiment, the amino acid sequence of TM is shown in SEQ ID NO. 5.
In another preferred embodiment, C is a costimulatory signal molecule for 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 a combination thereof.
In another preferred embodiment, C is a co-stimulatory signaling molecule from 4-1 BB.
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 ζ is as set forth in SEQ ID NO 7.
In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor CAR is shown as 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 as 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, pLJM1, pHCMV, pLBS.CAG, pHR, pLV and the like.
In another preferred embodiment, the vector is a pTomo lentiviral vector.
In another preferred embodiment, the vector further comprises one or more selected from the group consisting of: promoter, transcription enhancing element WPRE, long terminal repetitive sequence LTR, etc.
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 having integrated therein 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 having integrated therein 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: transducing 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 the engineered immune cell.
In another preferred embodiment, the method further comprises the step of performing function and effectiveness detection 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 said engineered immune cells in said formulation is 1 × 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 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 use in 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, said MSLN binding protein comprises MUC16.
In another preferred embodiment, the diseases associated with high expression of MSLN binding protein include, but are not limited to, tumors, aging, obesity, cardiovascular diseases, diabetes, neurodegenerative diseases, infectious diseases, and the like.
In another preferred embodiment, said diseases associated with high expression of MSLN binding protein comprise: tumor, aging, cardiovascular diseases, obesity, etc.
In another preferred embodiment, the disease is a malignancy with high expression of MSLN binding protein.
In another preferred embodiment, the MSLN binding protein is highly expressed, i.e., the ratio of the amount of expression of the MSLN binding protein (F1) to the amount of expression under normal physiological conditions (F0) (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 includes a solid tumor and a hematologic 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 cancer and esophageal cancer, glioma, lung cancer, prostate cancer, nasopharyngeal carcinoma or a combination thereof.
In another preferred embodiment, the hematological tumor is selected from the group consisting of: acute Myeloid Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoid Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof.
In a ninth aspect of the invention, there is provided a use of the engineered immune cell according to the fifth aspect of the invention, or the pharmaceutical composition according to the seventh aspect of the invention, for the prevention and/or treatment of cancer or tumor.
In a tenth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof 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 with high expression of MSLN binding protein.
In another preferred embodiment, the MSLN binding protein is expressed more than or equal to 1.5 times, preferably more than or equal to 2 times, and more preferably more than or equal to 2.5 times the amount of MSLN binding protein expressed under normal physiological conditions.
In another preferred embodiment, the disease is cancer or a tumor.
In another preferred embodiment, the engineered immune cell or the CAR immune cell comprised in the pharmaceutical composition is a cell derived from the subject (autologous cell).
In another preferred embodiment, the engineered immune cell or the CAR immune cell comprised in the pharmaceutical composition is a cell derived from a healthy individual (allogeneic cell).
In another preferred embodiment, the method may 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 to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows a schematic representation of the MSLN-CAR vector construction.
Wherein, A is a schematic sequence diagram of the MSLN precursor protein, wherein 1-34AA is a signal peptide, 34-596AA is an extracellular domain, 34-286AA is a megakaryocyte enhancement factor domain, and 296-598AA is a membrane-bound mature MSLN; b is a structural schematic diagram of control group plasmids MOCK-CAR and MSLN-CAR (wherein the antigen binding domain of the MSLN-CAR is A picture 290-362 AA), 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, and mKate2 is a fluorescent marker for detecting CAR expression; c is the HindIII enzyme digestion identification of the pTomo-MSLN-CAR vector.
Figure 2 shows CAR transfection efficiency assay results.
Wherein A is a cell fluorescence expression result of T cells infected by MOCK-CAR and MSLN-CAR for 72 hours, BF is a bright field, and mKate2 is CAR fluorescence expression; b is the flow detection fluorescence expression result.
FIG. 3 shows the killing effect of MSLN-CAR-T on different tumor cell lines.
Among them, A, ovarian cancer. And B, cervical cancer. C, breast cancer.
FIG. 4 shows the results of MUC16 expression assays for different ovarian cancer cell lines.
Wherein, A is WB for detecting the expression of MUC16 protein in different ovarian cancer cell lines. B is qPCR for detecting MUC16 mRNA level. And C is the expression level of MUC16 on the cell membrane detected by immunofluorescence.
FIG. 5 shows the results of gradient killing of different ovarian cancer cell lines by MSLN-CAR-T.
FIG. 6 shows the results of IFN- γ release following killing of different ovarian cancer cell lines by MSLN-CAR-T.
Figure 7 shows that overexpression of MUC16 in the ovarian cancer cell line SKOV3 enhances killing by MSLN-CAR.
Wherein, A is WB for detecting MUC16 protein expression. B is qPCR assay for MUC16 mRNA levels. And C is the expression level of MUC16 on the cell membrane detected by immunofluorescence. D is the killing detection of the MSLN-CAR on SKOV3-MUC 16. E is the result of detecting the release of the cytokine IFN-gamma.
FIG. 8 shows the results of killing of the normal cell line HEK-293T by MSLN-CAR-T.
Detailed Description
The inventor develops a chimeric antigen receptor immune cell constructed based on MSLN precursor protein for the first time through extensive and intensive research and a large amount of screening. Single chain antibodies scFv or endogenous receptors/ligands can serve as target recognition regions for CARs, but CAR recognition targets and activation of intracellular signals are influenced by a number of factors, and the work of the obtained CARs requires a lot of research. The inventor finds that the constructed CAR-T cell can specifically bind to MUC16 positive target cells (such as tumor cells) by using a specific fragment (namely amino acid sequences from 290 to 362) of the mesothelin precursor protein as an extracellular binding domain of the CAR, and has strong killing capacity and high safety. The present invention has been completed on the basis of this finding.
Term(s) for
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined 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 the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies 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, since 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, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (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 terms "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 terms as used herein refer to the process of delivering an exogenous polynucleotide into a host cell, transcription and translation to produce a polypeptide product, including the introduction of the exogenous polynucleotide into the host cell (e.g., E.coli) using a plasmid molecule.
"Gene expression" or "expression" refers to the process of transcription, translation and post-translational modification of a gene to produce the RNA or protein product of the gene.
"Polynucleotide" refers to a polymeric form of nucleotides of any length, including Deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, 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 interchangeable single-and double-stranded molecules. Unless otherwise indicated, a polynucleotide in any of the embodiments described herein includes a double-stranded form and two complementary single strands that are known or predicted to make up 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. In addition, the invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.
Those skilled in the art will readily understand the meaning of all parameters, dimensions, materials and configurations described herein. The actual parameters, dimensions, materials, and/or configurations will depend upon the specific application for which the invention is being used. It will be understood by those skilled in the art that the embodiments or claims are given by way of example only and that within the scope of equivalents or claims, the embodiments of the invention may be covered without limitation to the specifically described and claimed scope.
All definitions, as defined and used herein, should be understood to exceed dictionary definitions or definitions in documents incorporated by reference.
All references, patents, and patent applications cited herein are hereby incorporated by reference with respect to the subject matter to 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 disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal cord or other parenteral routes of administration, e.g., by injection or infusion.
MSLN binding protein MUC16 (carbohydrate associated 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 known as 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 carcinoma cells. The first cDNA clone, reported in 2001, had an average molecular weight between 250 and 500 million daltons and was also 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-cosylations), and a C-terminal region with a short cytoplasmic tail. The SEA domain with high levels of O-glycosylation in the TR repeat can bind to MSLNs.
MUC16 is a tumor specific antigen overexpressed in ovarian cancer, is the most widely clinically used at present and is an important serum biomarker for diagnosing ovarian cancer. 90% of ovarian cancer patients serum MUC16 is associated with disease progression and is therefore also commonly used as a marker to monitor disease progression and recurrence. CA125 (MUC 16) inhibits the cytolytic response of ovarian cancer natural killer cells and suppresses the immune response against ovarian cancer cells.
MUC16 is also overexpressed in tumors other than ovarian cancer, including cervical, fallopian tube, pancreatic, colon, peritoneal, nasopharyngeal, lung, breast, and gastric cancers. Therefore, the compound can be used as a target for treating tumors, especially various solid tumors.
Mesothelin (MSLN) and MSLN precursor proteins
The MSLN gene is located on chromosome 1p13.3 and is 8kD in length. The gene comprises 1884bp open reading frame, 17 coded exons and 628 amino acids. The precursor protein of MSLN is a glycoprotein of about 69kD in length anchored to the cell membrane with a glycosyl phospholipid peptide inositol, which can be hydrolyzed by proteolytic enzymes into 2 parts, a soluble protein of 31kD at the N-terminus, has megakaryocyte stimulating activity, and is called megakaryocyte-stimulating factor (MPF); while the C-terminus is a membrane-bound protein of approximately 40kD in length, with cellular adhesion, called MSLN, whose N-terminus (residues 296-359) can bind CA 125. The MUC16-MSLN interaction plays a role in cancer cell adhesion, and anti-MSLN antibodies can eliminate the binding of MSLN to MUC 16-expressing positive cells, and block MUC 16/mesothelin-dependent cell attachment.
Based on the method, the MSLN precursor protein fragment is integrated into the CAR vector in a genetic engineering mode for the first time, and related immune cells are modified, so that specific killing of MUC16 positive cells is realized, and the method can be used for treating related diseases. The present invention uses the N-terminal fragment of MSLN (SEQ ID NO. 1AA296-362) and its N-terminal linked 6 amino acids to construct CAR from MSLN precursor protein. The CARs of the invention are constructed based on MSLN precursor protein fragments, capable of binding to MSLN receptors including MUC16.
Chimeric Antigen Receptors (CAR) of the invention
A Chimeric Antigen Receptor (CAR) consists of an extracellular antigen recognition region, a transmembrane region, and an intracellular costimulatory signal region.
The extracellular domain of the CAR recognizes a specific antigen and subsequently transduces this signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity and secretion of cytokines, which in turn clear the target cell. Autologous cells from the patient (or a heterologous donor) are first isolated, activated and genetically engineered to produce immune cells for CAR production, and then injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by immune cells in a non-MHC restricted manner.
CAR-immune cell therapy has achieved very high clinical response rates in the treatment of hematological malignancies, which rates were previously unattainable by any therapeutic approach, and have triggered a hot surge of clinical research in the world.
Specifically, the Chimeric Antigen Receptors (CARs) of the invention comprise 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 based on specific binding of an antigen-antibody or 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 invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence from position 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. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.
A linker may be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain or a 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 its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the CD28 signaling domain, and CD3 zeta signaling domain in combination.
In the present invention, the extracellular binding domain of the CAR of the invention also includes sequence-based conservative variants, which refer to polypeptides formed by substituting up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids having similar or similar properties, as compared to the amino acid sequence from position 290 to 362 of SEQ ID No. 1.
In the present invention, the number of amino acids to be 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 number of amino acids in the original amino acid sequence.
In the present invention, the number of the amino acids to be added, deleted, modified and/or substituted is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.
For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.
The intracellular domain in the CAR of the invention includes the 4-1BB co-stimulatory domain and the signaling domain of CD3 ζ.
In one embodiment of the invention, the CAR is a CAR that can specifically target MUC16.
Chimeric antigen receptor immune cells (CAR-immune cells)
In the present invention, a chimeric antigen receptor immune cell is provided, which comprises the chimeric antigen receptor of the present invention having specific targeting to MUC16.
The chimeric antigen receptor immune cells of the invention may be CAR-T cells, and may also be CAR-NK cells, CAR-macrophages. Preferably, the chimeric antigen receptor immune cell of the invention is a CAR-T cell.
As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to a CAR-T cell according to the fifth aspect of the invention.
CAR-T cells have the following advantages over other T cell-based therapies: (1) the action process of CAR-T cells is not restricted by MHC; (2) Given that many tumor cells express the same tumor marker, the CAR gene construction for a certain tumor marker can be widely utilized once it is completed; (3) The CAR can utilize a tumor protein marker and a glycolipid non-protein marker, so that the target range of the tumor marker is expanded; (4) The use of patient autologous cells reduces the risk of rejection; (5) The CAR-T cell has an immunological memory function and can survive in vivo for a long time.
As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to a CAR-NK cell according to the fifth aspect of the invention. The CAR-NK cells of the invention 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 through non-antigen specific pathways. By engineering (genetically modifying) NK cells it is possible to obtain new functions, including the ability to specifically recognize tumor antigens and having an enhanced anti-tumor cytotoxic effect.
CAR-NK cells also have the following advantages compared to CAR-T cells, for example: (1) Directly kills tumor cells by releasing perforin and granzyme, but has no killing effect on normal cells of an organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storm; (3) Is easy to be amplified in vitro and can be developed into ready-made products. Otherwise, similar to CAR-T cell therapy.
Carrier
Nucleic acid sequences encoding the 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, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.
In brief summary, an 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 into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.
The expression constructs of the invention may also be used for 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 can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, molecular cloning. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise 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 gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, 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 that promoter function is maintained 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 apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.
An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). 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 cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.
To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells 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 a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.
Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of 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 an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al, 2000FEBS letters 479. In one embodiment of the invention, the reporter gene is a gene encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.
Methods for introducing 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 by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a 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, e.g., sambrook et al (2001, molecular cloning. A preferred method for 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 for 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. Pat. Nos. 5,350,674 and 5,585,362.
Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).
In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linker 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 as a suspension in the lipid, contained in or complexed with a 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 bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets, which occur naturally in the cytoplasm as well as such compounds that contain 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.
Preparation
The 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 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 CAR-T cells are present in the formulation at a concentration of 1X 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, 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 formulations of the present invention are preferably formulated for intravenous administration.
Therapeutic applications
The invention includes therapeutic applications of cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding 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 cell immune response, so that the killing efficiency of the 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 to the mammal the CAR-cells of the invention.
In one embodiment, the invention includes a class of cell therapy in which autologous T cells (or allogeneic donors) from a patient are isolated, activated, genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. In this way, the probability of graft versus host disease is very low and antigens are recognized by T cells in an MHC-unrestricted manner. Furthermore, one CAR-T can treat all cancers expressing this antigen. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained 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. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, CAR-T cells of MUC16 elicit a cell-specific immune response against MUC16.
Although the data disclosed herein specifically disclose lentiviral vectors comprising the MSLN precursor protein or fragment thereof, the hinge and transmembrane regions, and the 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 substantially vascularized, as well as vascularized tumors. Cancers include non-solid tumors (such as hematological tumors, e.g., leukemias and lymphomas) and solid tumors. The types of cancer treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.
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 cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.
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 a vector expressing a CAR 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 using 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, a pharmaceutical composition 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 compositions 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 an "effective amount", "immunologically effective amount", "anti-tumor 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, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10 4 To 10 9 Dosage of individual cells/kg body weight, preferably 10 5 To 10 6 Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion 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 those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.
Administration of the subject composition may be carried out 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, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. 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 conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatment for specific tumor patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with 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, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.
The dosage 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 proportion of doses administered to a human can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment 6 1 to 10 10 The CAR-T cells of the invention are administered to a patient, for example, by intravenous infusion.
The main advantages of the invention include:
(a) And (3) specific target spots: MUC16 is not substantially expressed on the cell membrane of normal cells, but is highly expressed on the cell membrane of a part of tumors. The CAR immune cells of the invention are directed only to malignant cells whose cell membranes highly express MUC16, and have little effect on other cells that do not express or that express MUC16.
(b) The present invention utilizes the mode of action of the ligand in combination with the receptor rather than scfv in the traditional sense. The selectivity and affinity of receptor-ligand interactions has been long-term and naturally selected, and the conservation of receptor-ligand interactions determines that safety tests in animals, particularly primates, are more responsive to safety in humans.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Reagents, plasmids, and cells in the examples of the present application are commercially available unless otherwise specified. Table 1 summarizes the sequences of the present invention.
TABLE 1 summary of sequences to which the invention relates
Figure BDA0003636463550000201
Figure BDA0003636463550000211
Table 2 shows the cell lines used in the examples.
TABLE 2 cell lines
Cell lines Type (B)
OVCAR3 Ovarian cancer cells
SKOV3 Ovarian cancer cells
Hela Cervical cancer cells
MDA-MB468 Breast cancer cell
MDA-MB231 Breast cancer cell
Example 1: preparation of MSLN-CAR vector
Based on the nucleotide sequence of MSLN (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 domain were synthesized and the nucleotide sequence of the CAR molecule was cleaved by AgeI (Thermo) and NheI (Thermo) enzymes in a double-restriction mode and inserted into the lentiviral vector pTomo into which the CD8 transmembrane region, the 4-1BB co-stimulatory domain, the CD3 zeta signaling region had been inserted, by T4 DNA ligase (NEB) ligation. Competent E.coli (Stbl 3) was transformed.
Sequencing the recombinant plasmid, and comparing sequencing results to confirm whether the plasmid is correct, wherein the sequencing primer is a universal sequencing primer. Both sequencing and restriction analysis showed that the CAR coding sequence was correctly inserted into the predetermined position of the plasmid (figure 1C).
All plasmids were extracted using a QIAGEN endotoxin-free macroextraction kit, and purified plasmids were packaged with lentiviruses transfected into HEK-293T cells using Bilongtian lipo6000.
Example 2: virus package
HEK-293T cells were cultured in 15cm dishes for virus packaging. 2ml OPTIMEM solubilized plasmid mixture (20. Mu.g core plasmid, 8.9. Mu.g pCMV. DELTA.R, 4. Mu.g PMD2. G) was prepared for transfection with HEK-293T cells at about 80% -90% confluency; in another centrifuge tube, 2ml OPTI MEM and 68. Mu.l lipo6000 were added. Standing at room temperature for 5min, adding the plasmid complex into 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. Add again the pre-warmed complete medium. After collecting the virus supernatants for 48 hours and 72 hours, they were centrifuged at 3000rpm at 4 ℃ for 20 minutes, filtered through a 0.45 μm filter and then centrifuged at 25000rpm at 4 ℃ for 2.5 hours to concentrate the viruses. After the concentrated virus was dissolved overnight in 30. Mu.l of a virus dissolving solution, the virus titer was measured 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 Ficool separation and purified CD3 was obtained from Rosettesep Human T Cell Enrichment Cocktail (Stemcell technologies) + T cells. T cells were activated with CD3/CD28 magnetic beads (Life technology), and then a final concentration of 200U/ml IL2 (PeproTech) was added to stimulate culture for 48 hours before virus infection. CAR-T cells were prepared by infecting T cells with lentiviruses in the presence of lentiboost at MOI = 20. The medium was changed one day after infection.
Example 4: positive rate of detecting CAR-T cell infection by flow cytometry
CAR-T cells and NT cells (control group) after 72 hours of virus infection were collected by centrifugation, washed once with PBS, and the supernatant was discarded, and the cells were resuspended in PBS containing 2% FBS, and the positive rate was determined by flow-assay.
As a result: the results of transfection efficiency are shown in FIG. 2.
As shown in FIG. 2A, after the CAR-T2A-mKate2 fusion protein expressed by the CAR-T cell is cut, the formed mKate2 protein shows red fluorescence in cells.
As shown in FIG. 2B, flow cytometry was used to demonstrate a positive expression rate of CAR or mKate2 CAR-T of about 60%.
Example 5: construction of target cells carrying luciferase
The pTomo-CMV-Luciferase-IRES-Puro lentivirus packaging procedure was the same as in example 2.
After OVCAR3, SKOV3, hela, MDA-MB-468, MDA-MB-231 and HEK-293T cells are infected by viruses, puromycin (1 mug/ml) is used for screening for 2 weeks, and OVCAR3, SKOV3, hela, MDA-MB-468, MDA-MB-231 and HEK-293T-lucifease cells are successfully obtained.
Example 6: CAR-T cell killing
In this example, the killing ability of the CAR-T cells of the invention against different target cells was examined. Target cells employed included: target cells highly expressing MUC 16: OVCAR3; target cells that do not express or under-express MUC 16: SKOV3, hela, MDA-MB-468, MDA-MB-231.
Digesting and counting OVCAR3, SKOV3, hela, MDA-MB-468, MDA-MB-231, HEK-293T-luciferase cells, and adjusting cell density to 2.5 × 10 4 And/ml. Mu.l of luciferase cells were seeded in 96-well plates and CAR-T and NT cells were adjusted to a cell density of 1X 10 5 Perml, inoculated into a black 96-well plate at an effective target ratio E: T of 4:1, at 100. Mu.l per well. The target cells and T cells were mixed well and incubated in an incubator for 24 hours.
After OVCAR3 and SKOV3-luciferase cell digestion and counting, the cell density is adjusted to be 2.5 multiplied by 10 4 And/ml. Mu.l of OVCAR3 and SKOV3-luciferase cells were seeded in 96-well plates and CAR-T and NT cells were adjusted to a cell density of 2.5X 10 4 、5×10 4 、1×10 5 、2×10 5 Perml, according to E: T1:1, 2:1, 4:1, 8:1 were inoculated into black 96-well plates, 100. Mu.l per well. The target cells and T cells were mixed and incubated in an incubator for 24 hours.
Cell supernatants were collected and stored at-80 ℃ for IFN-. Gamma.release (see example 8). Cell killing was detected using the promega fluorescence detection kit, cells were first treated with 30 μ l of 1 × plb lysate for 20 minutes, and 30 μ l of substrate was added to each well and immediately detected using a BioTek plate reader.
Cytotoxic killer cell% = (1-fluorescence of target cell with effector cell/fluorescence of target cell without effector cell) × 100%
As a result: the results of the killing effect of MSLN-CAR-T on different tumor cell lines are shown in figure 3. Results in ovarian cancer cells (A), cervical cancer cells (B) and breast cancer cells (C) show that the MSLN-CAR-T has good killing effect on various tumor cell lines.
Example 7: expression of MUC16 in ovarian cancer cells and CAR-T cytotoxicity test
(1) Cellular immunofluorescence: target cells were plated on discs in 24-well plates, 24 hours later cells were fixed with 4% Paraformaldehyde (PFA) for 20 minutes, and PBST was washed three times for 5 minutes each; blocking with 10% goat serum for 1 hour at room temperature, and incubating overnight at four degrees with an antibody that specifically recognizes NUC 16. The following day, three washes with PBST for five minutes each. Secondary antibodies specifically recognizing the primary antibodies were labeled with CY3 and incubated for 1 hour at room temperature. After three PBST washes, DAPI stained nuclei. And (4) imaging by using a confocal microscope.
(2) Immunoblotting: collecting 6cm culture dish cells, 5500r/min, and centrifuging for 5min at 4 ℃; removing supernatant, adding RIPA cell lysate containing protease inhibitor PMSF according to cell number, performing ice lysis for 20min, 14,000r/min, centrifuging at 4 deg.C for 30min, collecting supernatant, and measuring concentration; the protein sample was loaded in an amount of 50. Mu.g, subjected to protein electrophoresis, and the expression of MUC16 in the target cell was detected.
(3) qPCR: collecting 6-well plate cells, removing culture medium, adding 1ml Trizol to crack cells, standing at room temperature for 5min, adding 200 μ l/1ml Trizol chloroform, reversing and mixing uniformly for 6-8 times, and standing at room temperature for 5min;12000g, centrifuging for 15 minutes at 4 ℃, and sucking supernatant liquid into another centrifuge tube; adding isopropanol with equal volume, mixing by inversion, and standing at room temperature for 10min;12000g, centrifuging for 10 minutes at 4 ℃, and discarding the supernatant; adding 1ml 70% ethanol (with RNase free H) 2 O preparation), washing, 7500g, centrifuging for 5min at room temperature; removing supernatant, standing at room temperature for 10min, drying RNA, and adding 30 μ l RNase free water to dissolve RNA; nandrop 2000 measures the concentration of RNA and detects the integrity of the RNA and the accuracy of the quantification by electrophoresis on a 1% agarose gel. cDNA was synthesized according to RevertAIdTM First Strand cDNA Synthesis Kit (Thermo Scientific) and tested for mRNA levels.
As a result: the results of MUC16 expression assays for each cell line are shown in FIG. 4. The expression of MUC16 in target cells is detected by WB (figure 4A) and qPCR (figure 4B), and the results show that the expression of MUC16 is high in OVCAR3 and low in SKOV 3. It was further verified by immunofluorescence localization that MUC16 on OVCAR3 cell membranes was highly expressed and MUC16 on SKOV3 cell membranes was not substantially expressed (fig. 4C).
The results of the 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 is gradually enhanced along with the increase of the effective target ratio (E: T).
Example 8: CAR-T targeting ovarian cancer cells with cytokine IFN-gamma release
In this example, cytokine release was examined in the case of CAR-T cells of the invention incubated with target cells. The cell supernatants were co-incubated in a cell killing experiment for detection.
The method comprises the following steps: cell supernatants from CAR-T cells of the invention in example 7 co-incubated with OVCAR3, SKOV3 target cells (E: T ratio 4:1) were assayed for IFN- γ according to the IFN gamma Human ELISA Kit (life technology).
The Standard was dissolved in Standard Dilution Buffer and gradient diluted to 1000pg/ml, 500 pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 0 pg/ml.
Add 50. Mu.l of incorporation buffer, 50. Mu.l of detection sample, and 50. Mu.l of IFN-. Gamma.biotin conjugated solution to each well, mix well, and then stand at room temperature for 90 minutes.
Then the operation is carried out according to the following steps in sequence:
(1) Wash the wells 4 times with 1 × wash Buffer, each time for 1 minute.
(2) Mu.l of 1 streptavidin-HRP solution was added to each well and allowed to stand at room temperature for 45 minutes.
(3) Wash the wells 4 times with 1 × wash Buffer, each time for 1 minute.
(4) Mu.l of Stabilized chromogen was added and the mixture was allowed to stand at room temperature for 30 minutes.
(5) Add 100. Mu.l of Stop solution to each well and mix well.
(6) Absorbance at 450nm was measured.
The results are shown in FIG. 6. After the OVCAR3 is killed by MSLN-CAR-T, the cytokine is obviously increased, and the SKOV3 has no obvious change. 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 the IFN-gamma release.
Example 9: effect on tumor killing of MSLN-CAR-T after overexpression of MUC16
According to the CDS region sequence of MUC16, a MUC16 overexpression plasmid (EX-Y1397-Lv 183, ORF lentivirus expression clone) is purchased from a brocade organism, and a SKOV3 stable overexpression MUC16 cell line is constructed.
The Lenti-MUC16-EGFP-NeoR lentivirus packaging procedure was the same as in example 2. After the SKOV3 cells are infected by the virus, the SKOV3-MUC16 cells are successfully obtained by screening with Neomycin (3 mu g/ml) for 2 weeks. The overexpression efficiency was tested at the protein and gene level and the MSLN-CAR-T killing test was performed.
After digesting and counting different ovarian cancer cell lines SKOV3-Vector and SKOV3-MUC16-luciferase, the cell density is adjusted to be 2.5 multiplied by 10 4 And/ml. Mu.l of luciferase cells were seeded in a 96-well plate, and CAR-T/NT cells were adjusted to a cell density of 1X 10 5 The cells were plated at 4:1E: T in black 96-well plates, 100. Mu.l per well. The target cells and T cells were mixed well and incubated in an incubator for 24 hours.
As a result: the over-expression efficiency and killing results of MSLN-CAR-T after MUC16 was over-expressed in ovarian cancer cell line SKOV3 are shown in FIG. 7. FIG. 7A shows the WB detection of MUC16 protein expression. Figure 7B is qPCR detection of MUC16 mRNA levels. FIG. 7C shows immunofluorescence detection of MUC16 expression levels on cell membranes. The results all show that SKOV3 cells over-expressing MUC16 were successfully constructed.
FIG. 7D is the killing effect of MSLN-CAR-T on SKOV3 after overexpression of MUC16. FIG. 7E is the IFN- γ release of MSLN-CAR-T on killing after overexpression of MUC16 by SKOV 3. The results show that compared with the SKOV3-Vector of the control group, the killing rate and IFN-gamma release amount of MSLN-CAR-T cells on SKOV3-MUC16 cells over-expressing MUC16 are obviously increased. The results indicate that the killing effect of MSLN-CAR-T cells on MUC16 over-expression tumor cells is obviously enhanced.
Example 10: killing effect of MSLN-CAR-T on non-tumor cells
HEK-293T cells are human embryonic kidney cell lines, are inoculated into a black 96-well plate according to an effective target ratio of 4:1, and are subjected to co-incubation with the HEK-293T-luciferase cells, and the killing of the HEK-293T cells by the MSLN-CAR-T is detected through the change of fluorescence values.
The results are shown in FIG. 8. FIG. 8A shows the WB detection of MUC16 protein expression. Figure 8B is qPCR detection of MUC16 mRNA levels. FIG. 8C shows immunofluorescence detection of MUC16 expression levels on cell membranes. FIG. 8D is the killing effect of MSLN-CAR-T on HEK-293T. FIG. 8E is the IFN- γ release of MSLN-CAR-T on HEK-293T killing.
The results show that the expression level of MUC16 in the HEK-293T non-tumor cells is low, and the MSLN-CAR-T has no obvious killing effect on the HEK-293T.
Discussion of the related Art
Single chain antibodies scFv or endogenous receptors/ligands can serve as target recognition regions for CARs, but CAR recognition targets and activation of intracellular signals are influenced by a number of factors, and the work of the obtained CARs requires a lot of research. Previous studies suggest that interaction between MSLN and MUC16 allows binding to each other, and the present invention uses a specific fragment of the MSLN precursor protein, which contains the MSLN fragment and its N-terminal partial extension (i.e., amino acid sequence 290-362), as the extracellular binding domain of the CAR. The inventor finds that the CAR-T cell constructed by using the fragment as the extracellular binding domain of the CAR can specifically bind to MUC16 positive target cells (such as tumor cells), and has strong killing capacity and high safety.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Sichuan university Hospital in western China
<120> preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor protein
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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 (10)

1. A Chimeric Antigen Receptor (CAR), wherein the CAR comprises an extracellular binding domain comprising the structure of the MSLN precursor protein or a fragment thereof based on the amino acid sequence shown in SEQ ID NO:1,
and, said extracellular binding domain is capable of specifically binding to MSLN binding proteins.
2. The chimeric antigen receptor of claim 1, wherein said extracellular binding domain comprises MSLN precursor protein or a fragment thereof, said MSLN precursor protein or fragment thereof having the amino acid sequence set forth in SEQ ID No.1, or having the amino acid sequence from position 290 to 362 of the sequence set forth in SEQ ID No. 1.
3. The chimeric antigen receptor according to claim 1, wherein said CAR has the structure of formula I:
L-EB-H-TM-C-CD3ζ-RP (I)
in the formula (I), the compound is shown in the specification,
each "-" is independently a linker peptide or a peptide bond;
l is a null or signal peptide sequence;
EB is the extracellular binding domain;
h is a none or hinge region;
TM is a transmembrane domain;
c is a no or co-stimulatory signal molecule;
CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ;
RP is a null or reporter protein.
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 having integrated therein an exogenous nucleic acid molecule of claim 4 or expressing the CAR of claim 1.
7. An engineered immune cell comprising the vector or chromosome of claim 5 having integrated therein the exogenous nucleic acid molecule of claim 4 or expressing the CAR of claim 1.
8. A method of making the engineered immune cell of claim 7, comprising the steps of: transferring the nucleic acid molecule of claim 4 or the vector of claim 5 into an immune cell, thereby obtaining the engineered immune cell.
9. A pharmaceutical composition comprising the CAR 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 the CAR 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, for the preparation of a medicament or formulation for the prevention and/or treatment of a disease with high expression of MSLN binding protein.
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WO2023217192A1 (en) * 2022-05-10 2023-11-16 四川大学华西医院 Preparation of chimeric antigen receptor immune cell constructed based on msln precursor protein and use thereof

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