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CN109097396B - Method for preparing mesothelin-targeted CAR-T cells - Google Patents

Method for preparing mesothelin-targeted CAR-T cells Download PDF

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CN109097396B
CN109097396B CN201811050979.8A CN201811050979A CN109097396B CN 109097396 B CN109097396 B CN 109097396B CN 201811050979 A CN201811050979 A CN 201811050979A CN 109097396 B CN109097396 B CN 109097396B
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mesothelin
antigen
antibody
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金华君
何江川
江芏青
刘祥箴
黄晨
钱其军
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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Abstract

The invention provides a method for preparing a mesothelin-targeted CAR-T cell, comprising the steps of: 1) Introducing a nucleic acid comprising a CAR coding sequence targeting a mesothelin antigen into PBMCs to obtain naive cells; 2) Contacting the primary cells in 1) with a mesothelin antigen and an anti-CD 28 antibody, wherein the state of the mesothelin antigen and the anti-CD 28 antibody is non-immobilized. The method of the invention does not need to coat fixed or suspended substrates, greatly simplifies the operation process and reduces the cost; the obtained mesothelin-targeted CAR-T cells have obviously increased positive rate, proliferation level and ratio of effector memory T cells, and have the same level of tumor cell lethality as the CAR-T cells prepared by a mesothelin antigen and CD28 antibody coating method, but the cytokine level is obviously reduced, so that the potential risk of Cytokine Release Syndrome (CRS) is reduced.

Description

Method for preparing mesothelin-targeted CAR-T cells
Technical Field
The invention relates to the field of medical immunology, in particular to a method for preparing a mesothelin-targeted CAR-T cell.
Background
Adoptive immune cell therapy technologies represented by Chimeric Antigen Receptor (CAR) T cell therapy technologies have become a hot spot for the development of new current tumor therapy technologies, and adoptive immune therapy based on T lymphocytes achieves certain effects in some tumors. Kymeriah by nova and yescatta by kat were approved in the united states for marketing in 2017, but their pricing was as high as $ 47.5 million and $ 37.3 million, respectively. The main reason for the high price is that the current two CAR-T cell drugs adopt an autologous treatment scheme, PBMC cells are required to be isolated from blood of a patient and then prepared again for a new patient, and the complicated operation process causes extremely high cost and is difficult to control. The current mature product CAR-T cells are generally prepared by a process comprising the steps of collecting blood from a patient, isolating T cells, activating T cells, genetically modifying T cells with CAR to obtain CAR-T cells, amplifying the CAR-T cells, and then formulating the amplified CAR-T cells into a preparation for reinfusion into the patient (Clinical management of CAR T cells: foundation of a breeding therapy. Wang X, rivi of re I Mol therapeutics. Jun 15). This may also include processes such as cryopreservation, resuscitation and cold chain transport of CAR-T cells. Any one of the whole preparation and use processes needs to be accompanied by strict quality control release inspection.
Ex vivo expansion of T cells requires continuous, proper activation. Activation of T cells requires a specific first signal (e.g., CD 3) and a costimulatory second signal, such as CD28, 4-1BB, or OX40, to proceed through the TCR. The most commonly used T cell stimulation methods today are stimulation after coating multiwell plates with CD3 and anti-CD 28 antibodies, stimulation with Artificial Antigen Presenting Cells (AAPCs), or stimulation with beads that mimic APC cells, such as clinical grade magnetic or nanobeads coated with CD3 and anti-CD 28 antibodies. Wherein Dynabeads CD3/CD28 are identical superparamagnetic beads covalently coupled to a CD3 antigen and an anti-CD 28 antibody. Dynabeads CD3/CD28, the first generation of readily available clinical grade CD3+ T cell selection and activation reagents, has been widely used in clinical trials in different laboratories. Several biotech companies have developed readily available clinical grade bead-based T cell activation reagents, including CTS Dynabeads CD3/28 by Invitrogen, miltenyi MACS GMP ExpAct Treg beads and Miltenyi MACS GMP TransAct CD3/28 beads by american day and whirlpool, and Juno Stage express owner technology by cinno. Miltenyi MACS GMP ExpAct Treg beads are paramagnetic beads covalently coupled to CD 3-biotin, CD28 and an anti-biotin antibody. By adjusting the magnetic bead-T cell ratio, miltenyi MACS GMP expoct Treg beads can be used to expand regulatory T cells and conventional T cell lineages. At the end of the CAR-T cell production run it is usually necessary to remove the magnetic beads, e.g. by using a Dynal clinexivivo MPC magnet. Miltenyi MACS GMP TransAct CD3/28 beads are polymeric nanomatrix conjugated with CD3 or anti-CD 28 antibodies, which are biodegradable and therefore do not need to be removed prior to the formation of the CAR-T cell formulation.
Mesothelin is a glycoprotein anchored to the plasma membrane of a cell via the phosphatidylinositol domain (GPI), and the mesothelin gene, highly expressed in a variety of tumor tissues, encodes a 69kDa precursor protein that is processed to form a 40kDa membrane-bound protein and a 31kDa split-off fragment called Megakaryocyte Promoting Factor (MPF) that is released extracellularly, and is commonly referred to as the membrane-anchored fragment and is classified into regions Region I, II, and III based on its protein structure. On one hand, the GPI structural domain can activate NF domain kinase, MAPK and PI3K intracellular signal pathways to promote cell proliferation and resist cell apoptosis; on the other hand, the interaction with the receptor CA125/MUC16 leads to abnormal cell adhesion and promotes cancer cell metastasis. Mesothelin is an extremely potential tumor-specific therapeutic target due to its overexpression in a variety of malignancies (mesothelioma, ovarian, pancreatic, gastric, cholangiocarcinoma, etc.).
The current conventional CAR-T cell preparation procedure usually comprises sorting T cells, performing stimulation amplification in vitro, and then introducing the CAR gene. And the first and second signals used for in vitro stimulation are also CD3 and CD28 as described above. Existing clinical grade magnetic bead or nanobead based T cell activation reagents are also covalently coupled to CD3 and CD28 designed for use. A problem with this approach is that indiscriminate stimulation of CD3 and anti-CD 28 antibodies tends to result in depletion of T cells. Furthermore, if the T cell activating method is stimulated by coating the substrate with the CD3 antigen and the anti-CD 28 antibody, the operation of the coating step will increase the complexity of the preparation process, and may bring more uncertain factors. If the commercially available clinical-grade magnetic beads or nano-beads coupled with CD3 and/or anti-CD 28 antibodies are adopted for stimulation, the preparation cost of the CAR-T cells is difficult to reduce, and the popularization of the CAR-T cells in clinical application is influenced. Thus, there is still a lack of methods for producing CAR-T cells, such as for producing mesothelin-targeted CAR-T cells, that include steps that are simple to handle, cost-controllable, and capable of efficiently activating T cells.
Disclosure of Invention
The invention aims to solve the technical problems that the T cell exhaustion can be caused by the operation of activating the T cells by using CD3 and CD28 as a first signal and a second signal respectively in the current CAR-T cell preparation process, the complexity of the preparation process can be increased by using a matrix coated with a stimulating agent to stimulate the T cells, and the preparation cost is high and is difficult to control due to the use of a commercialized clinical-grade magnetic bead or nano bead reagent covalently coupled with a CD3 antigen and/or an anti-CD 28 antibody, so that a novel method for preparing the mesothelin-targeted CAR-T cells is provided. The method adopts the opposite operation of firstly activating T cells and then introducing CAR genes, after sorting the obtained T cells, firstly introducing the CAR genes aiming at the mesothelin into the T cells, then co-stimulating the T cells by using the mesothelin antigen and an anti-CD 28 antibody, and directly adding the mesothelin antigen and the anti-CD 28 antibody into a system in a non-immobilized form to stimulate the CAR-T cells aiming at the mesothelin without coating matrixes such as pore plates or using magnetic beads or nano-bead materials coupled with the mesothelin antigen and/or the anti-CD 28 antibody, thereby greatly simplifying the process of preparing the mesothelin CAR-T cells and reducing the cost, the mesothelin CAR-T cells prepared by the preparation method containing the activation step have higher positive rate and the killing power of the CAR-T cells which is equivalent to the level of the conventional method, the expression level of partial cytokines has lower positive rate and lower killing power of the CAR-T cells compared with the CAR-T cells prepared by the CD28 antibody coating stimulation, and the potential CRS generation of the pore plates is reduced.
Specifically, the present invention solves the technical problem to be solved by the present invention through the following technical solutions.
In one aspect, the invention provides a method of making a CAR-T cell, comprising the steps of: 1) Introducing a nucleic acid comprising a Chimeric Antigen Receptor (Chimeric Antigen Receptor) coding sequence targeting a mesothelin Antigen into Peripheral Blood Mononuclear Cells (PBMCs) to obtain primary cells; 2) Contacting the primary cells in 1) with the mesothelin antigen and an anti-CD 28 antibody, in culture, wherein the state of the mesothelin antigen and the anti-CD 28 antibody is non-immobilized.
In a preferred embodiment, the mesothelin antigen is full length mesothelin.
In a preferred embodiment, the mesothelin antigen is mesothelin domain III, the amino acid sequence of which is shown at positions 21-131 of SEQ ID NO. 2.
In a preferred embodiment, the mesothelin antigen preferably comprises an IgG Fc, preferably the Ig Fc is an IgG4Fc.
In a preferred embodiment, the mesothelin antigen is fused to the IgG Fc via a hinge, preferably a CD8 hinge.
In a preferred embodiment, the mesothelin antigen further comprises a signal peptide at the N-terminus, preferably a light chain signal peptide.
In a preferred embodiment, the mesothelin antigen is a mesothelin domain III antigen fused to a light chain signal peptide at the N-terminus and IgG4Fc through a CD8 hinge at the C-terminus, and the amino acid sequence is shown in SEQ ID NO. 2.
In a preferred embodiment, the nucleic acid is a nucleic acid construct or an mRNA, preferably a nucleic acid construct.
In a preferred embodiment, the nucleic acid construct is an expression vector, optionally a viral vector, preferably a lentiviral vector derived from type I HIV, such as pWPT, pwxl, and the like.
In a preferred embodiment, the nucleic acid construct is an expression vector, optionally a transposon system vector, preferably a modified or unmodified Sleeping Beauty transposon system vector or PiggyBac transposon system vector, more preferably a modified or unmodified PiggyBac transposon system vector, most preferably a modified PiggyBac transposon system vector, such as the pNB328 transposon system vector disclosed in CN 105154473A.
In a preferred embodiment, the nucleic acid is an mRNA comprising a 5' end cap structure, a 5' UTR, an Open Reading Frame (ORF) encoding the chimeric antigen receptor, a 3' UTR and a polyA tail.
In a preferred embodiment, the method of introduction is any one selected from the group consisting of viral particle infection, electroporation, lipofection, calcium phosphate transfection and gene gun, preferably any one selected from the group consisting of viral particle infection, electroporation and lipofection, more preferably viral particle infection or electroporation, most preferably electroporation.
In a preferred embodiment, the anti-CD 28 antibody is an anti-CD 28 monoclonal or polyclonal antibody, preferably an anti-CD 28 monoclonal antibody, more preferably a humanized monoclonal antibody, even more preferably a humanized murine or rabbit anti-CD 28 monoclonal antibody, most preferably a humanized murine anti-CD 28 monoclonal antibody.
In a preferred embodiment, the temperature of the culture is 37 ℃; and/or, said cultured CO 2 The concentration is 5%; and/or the culture medium used for culturing is AIM-V containing 2% fetal bovine serum; and/or, the culturing time is 5-10 days.
In a preferred embodiment, the culture medium used for the culture further comprises cytokines; preferably, the cytokine is IL-2 with a final concentration of 500IU/mL; and/or, the cytokine is added to the culture medium when the culturing is carried out for 0-6 hours; preferably, the cytokine is added to the medium at the time the culture is performed for 6 hours.
In a preferred embodiment, said contacting is such that said mesothelin antigen and said anti-CD 28 antibody are present directly in a system containing said starting cells, and interact directly with the surface of said starting cells. Preferably, the mesothelin antigen is mesothelin domain III and the anti-CD 28 antibody is a monoclonal antibody.
In a preferred embodiment, both the mesothelin antigen and the anti-CD 28 antibody are in solution; preferably, the final concentration of the mesothelin antigen is 0.1-3 μ g/mL and the final concentration of the anti-CD 28 antibody is 0.1-3 μ g/mL.
In a preferred embodiment, the mesothelin antigen and the anti-CD 28 antibody are both in solution; the final concentration of the mesothelin antigen is 0.1-1 mug/mL, and the final concentration of the anti-CD 28 antibody is 0.1-1 mug/mL.
In a preferred embodiment, the mesothelin antigen and the anti-CD 28 antibody are both in solution; the final concentration of the mesothelin antigen is 0.5-1 mug/mL, and the final concentration of the anti-CD 28 antibody is 0.5-1 mug/mL.
In a preferred embodiment, the mesothelin antigen and the anti-CD 28 antibody are both in solution; the final concentration of the mesothelin antigen is 0.1 μ g/mL, and the final concentration of the anti-CD 28 antibody is 0.1 μ g/mL.
In a preferred embodiment, the mesothelin antigen and the anti-CD 28 antibody are both in solution; the final concentration of the mesothelin antigen is 0.5 μ g/mL, and the final concentration of the anti-CD 28 antibody is 0.5 μ g/mL.
In a preferred embodiment, the mesothelin antigen and the anti-CD 28 antibody are both in solution; the final concentration of the mesothelin antigen is 1 μ g/mL, and the final concentration of the anti-CD 28 antibody is 1 μ g/mL.
In a preferred embodiment, the mesothelin antigen and the anti-CD 28 antibody are both in solution; the final concentration of the mesothelin antigen is 3 μ g/mL, and the final concentration of the anti-CD 28 antibody is 3 μ g/mL.
Drawings
FIG. 1: schematic structural diagram of Meso3CAR and Muc1CAR genes;
FIG. 2A: positive rate results for fMeso3CAR-T-C329 cells made under conditions of cone-C329, cMeso3CAR-T-C329 and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 2B: positive results for fMeso3CAR-T-D70 cells prepared under conditions of cone-D70, cMeso3CAR-T-D70 and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 2C: positive rate results for fMeso3CAR-T-D71 cells prepared under conditions of cone-D71, cMeso3CAR-T-D71 and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 3A: proliferation profiles of fMeso3CAR-T-D70 cells prepared under conditions of ConT-D70, cMarso 3CAR-T-D70, and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 3B: proliferation curves of fMeso3CAR-T-D71 cells prepared under conditions of ConT-D71, cMyso 3CAR-T-D71 and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 4A: levels of cytokine IL-2 secretion by fMeso3CAR-T-D70 and fMeso3CAR-T-D71 cells prepared under conditions stimulated by cMeso3CAR-T-D70, cMeso3CAR-T-D71 and varying concentrations of mesothelin III antigen + anti-CD 28 antibody following contact with PANC-1 cells and SKOV-3 cells;
FIG. 4B: levels of cytokine IL-4 secretion by fMeso3CAR-T-D70 and fMeso3CAR-T-D71 cells prepared under conditions stimulated by various concentrations of mesothelin III antigen + anti-CD 28 antibody following contact with PANC-1 cells and SKOV-3 cells, cMeso3CAR-T-D70, cMeso3CAR-T-D71 and;
FIG. 4C: levels of cytokine IL-10 secreted by fMeso3CAR-T-D70 and fMeso3CAR-T-D71 cells prepared under conditions stimulated by cMeso3CAR-T-D70, cMeso3CAR-T-D71 and varying concentrations of mesothelin III antigen + anti-CD 28 antibody after contact with PANC-1 cells and SKOV-3 cells;
FIG. 4D: levels of cytokine IFN- γ secretion by fMeso3CAR-T-D70 and fMeso3CAR-T-D71 cells prepared under conditions of stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody following contact with PANC-1 cells and SKOV-3 cells, cMeso3CAR-T-D70, cMeso3 CAR-T-D71;
FIG. 5: results of detection of expression levels of fMeO 3CAR-T-C329 cell T cell depleting factor PD-1 produced under the conditions of ConT-C329, cMetho 3CAR-T-C329, and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 6A: results of flow-based detection of effector T cells from fMeso3CAR-T-D70 cells prepared under conditions of ConT-D70, cMarso 3CAR-T-D70, and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 6B: flow assay results for effector T cells in fMeso3CAR-T-D71 cells prepared under conditions of ConT-D71, cMarso 3CAR-T-D71, and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody;
FIG. 6C: histogram of the statistics of the percentage of effector T cells in FIGS. 6A and 6B;
FIG. 7A: killing curves of fMeso3CAR-T-D70 cells prepared under the conditions of ConT-D70, cMetho 3CAR-T-D70 and stimulation of different concentrations of mesothelin III antigen + anti-CD 28 antibody on pancreatic cancer cell line PANC-1 respectively;
FIG. 7B: killing curves of fMeso3CAR-T-D70 cells prepared under the conditions of ConT-D70, cMetho 3CAR-T-D70 and stimulation of mesothelin III antigen + anti-CD 28 antibody with different concentrations on ovarian cancer cells SKOV-3 respectively;
FIG. 7C: killing curves of fMeso3CAR-T-D71 cells prepared under the conditions of ConT-D71, cMetho 3CAR-T-D71 and stimulation of different concentrations of mesothelin III antigen + anti-CD 28 antibody on pancreatic cancer cell line PANC-1 and ovarian cancer cell SK-OV-3 respectively;
FIG. 7D: killing curves of fMeso3CAR-T-D71 cells prepared under the conditions of ConT-D71, cMetho 3CAR-T-D71 and stimulation of different concentrations of mesothelin III antigen + anti-CD 28 antibody on pancreatic cancer cell line PANC-1 and ovarian cancer cell SK-OV-3 respectively;
FIG. 8A: positive rate results for cMuc1CAR-T-D42 and fMuc1CAR-T-D42 cells;
FIG. 8B: positive rate results for cMUc1CAR-T-D43 and fMuc1CAR-T-D43 cells.
Detailed Description
The steps of current state-of-the-art methods of making CAR-T cells include isolation of PBMCs, activation of expanded T cells with CD3 and anti-CD 28 antibodies, introduction of transgenic CARs into T cells with viral particles or electroporation. In the process of obtaining CAR-T cells through the above-mentioned operation, the operation of activating T cells using CD3 and CD28 as the first signal and the second signal, respectively, may cause T cell depletion, and the stimulation of T cell expansion after coating the matrix with CD3 and anti-CD 28 antibodies as the stimulating agents increases the complexity of the preparation process, while the use of commercially available clinical grade magnetic beads or nanobead reagents covalently coupled with CD3 antigen and/or anti-CD 28 antibodies causes high preparation cost. Through intensive research and repeated experiments, the inventor finds that the method is opposite to the operation of firstly activating T cells and then introducing CAR genes adopted in the prior art, namely, after T cells are obtained through sorting, the mesothelin CAR genes are firstly introduced into the T cells, then the mesothelin antigen and the anti-CD 28 antibody are used for jointly stimulating the T cells, and the mesothelin antigen and the anti-CD 28 antibody are directly added into a system in a non-immobilized form to stimulate the mesothelin-targeting CAR-T cells without coating a fixed or suspended matrix, so that the operation flow for preparing the mesothelin-targeting CAR-T cells is greatly simplified, and the cost is reduced. The mesothelin-targeted CAR-T cells prepared by the method are obviously improved in the aspects of positive rate, proliferation level and proportion of effector memory T cells, and have the same level of tumor cell killing power as the CAR-T cells prepared by a mesothelin antigen and anti-CD 28 antibody coating method, the expression level of cytokines is obviously reduced compared with the CAR-T cells prepared by coating a pore plate with the mesothelin antigen and anti-CD 28 antibody, and the potential risk of Cytokine Release Syndrome (CRS) is reduced on the premise of not reducing the tumor killing capacity, so that the invention is completed.
Some of the terms to which the invention relates are defined below.
Unless otherwise noted, this application uses technical terms in accordance with conventional usage. Common terms in molecular biology may be defined in Benjamin lewis, genes X, published by Jones & Bartlett Publishers,2009; and Meyers et al (eds.), the Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 169olumes, 2008 and other similar references.
In the present invention, the term "antigen" is defined herein to mean any substance which, when introduced into the body, is recognized by the immune system. For example, a protein, a fragment or domain of a protein, such as the extracellular domain of a cell transmembrane protein.
The term "Chimeric Antigen Receptor" (CAR) is defined herein as an artificially engineered Receptor that is capable of anchoring a specific molecule (e.g., an antibody) that recognizes a cell surface Antigen to an immune cell (e.g., a T cell) such that the immune cell recognizes the Antigen and kills the cell expressing the Antigen it recognizes. The CAR typically comprises, in order, an optional signal peptide, a polypeptide that binds to a cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signal region. In general, the polypeptide that binds to a cell membrane antigen can be a natural polypeptide or an artificially synthesized polypeptide; preferably, the artificially synthesized polypeptide is a single chain antibody or a Fab fragment.
The term "coding sequence" is defined herein as the portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (e.g., CAR). The boundaries of the coding sequence are generally determined by a ribosome binding site immediately upstream of the 5 'open reading frame of the mRNA (for prokaryotic cells) and a transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA. A coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
The term "Peripheral Blood Mononuclear Cell" (PBMC) is defined herein as any Cell in Peripheral Blood having a round single nucleus, including but not limited to T cells, B cells, NK cells, macrophages and monocytes; red blood cells, platelets, neutrophils, eosinophils or basophils are excluded.
The term "nucleic acid construct" is defined herein as an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue. Typically comprises a DNA construct, meaning a DNA insertion of a nucleotide sequence encoding a protein of interest that has been subcloned into a vector.
The term "expression vector" is defined herein as a nucleic acid construct or vector (e.g., an exogenous nucleic acid or transgene) that is produced recombinantly or synthetically for expression of a nucleic acid of interest in a target cell. Typically, the nucleic acid of interest expresses a protein of interest.
The term "antibody" is defined herein as an immunoglobulin molecule that specifically binds to an antigen, and may be a whole immunoglobulin derived from a natural source or from a recombinant sourceProteins, and may be the immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of the invention may exist in a variety of forms including, but not limited to, polyclonal, monoclonal, fv, fab, F (ab)' 2 scFv, humanized antibodies, heavy chain antibodies and single domain antibodies.
The term "polyclonal antibody" is defined herein as a mixture of antibodies containing a plurality of antibodies secreted by different B cell lineages in vivo. Each of these B cell lineages secrete antibodies directed against the same specific antigen, but each antibody is directed against a different epitope on that specific antigen.
The term "monoclonal antibody" is defined herein as an antibody that exhibits a single binding specificity, binding only a single epitope of an antigen. It includes antibodies comprising a heavy chain-light chain tetramer, in the traditional sense, produced by the same immune cell derived from a clone of only one parent cell. It also includes Fv, fab ', F (ab') 2 scFv, humanized antibodies, heavy chain antibodies and single domain antibodies.
The term "humanized antibody" is defined herein as a non-human antibody whose sequence has been modified to increase its similarity to its native human antibody sequence. Wherein at least one antibody binding site (complementarity determining region, CDR), such as CDR3, preferably all six CDRs, is replaced by a CDR from a human antibody having the desired specificity, and/or at least one FR region thereof is replaced by a FR region of a human antibody, optionally a non-human constant region thereof is replaced by a constant region of a human antibody.
The term "non-immobilized" is defined herein as protein antigens or antibodies that are not covalently or non-covalently linked to any substrate. The substrate may be a fixed substrate, such as a solid well plate, a petri dish, a glass slide, etc., or a suspended substrate, such as magnetic beads, nanoparticles, etc.
The term "immobilized" is defined herein as an antibody, such as an anti-CD 28 antibody, covalently or non-covalently attached to a substrate, which may be a fixed substrate, such as a solid well plate, a petri dish, a glass slide, etc., or a suspended substrate, such as a magnetic bead, a nanoparticle, etc.
The invention thus provides a method of making a mesothelin-targeted CAR-T cell, comprising 1) introducing a nucleic acid comprising a chimeric antigen receptor coding sequence targeted to a mesothelin antigen into Peripheral Blood Mononuclear Cells (PBMCs) to obtain naive cells; 2) Contacting the primary cells in 1) with the mesothelin antigen and an anti-CD 28 antibody, in culture, wherein the states of the mesothelin antigen and the anti-CD 28 antibody are non-immobilized.
In the present invention, the mesothelin antigen is a full-length mesothelin antigen or a mesothelin domain III antigen, preferably, a mesothelin domain III antigen; preferably, the mesothelin domain III antigen comprises an IgG Fc. Preferably, the IgG Fc is IgG4Fc, and the amino acid sequence of the IgG Fc is shown in positions 144-250 of SEQ ID NO. 2. The mesothelin domain III antigen may be fused to the IgG Fc via a hinge, preferably the hinge is a CD8 hinge, the amino acid sequence of which is shown in SEQ ID No. 3 at positions 132-143.
In the present invention, the mesothelin antigen may further comprise a signal peptide at the N-terminus, and preferably, the signal peptide is a light chain signal peptide having an amino acid sequence shown in positions 2-20 of SEQ ID No. 2.
In the invention, the mesothelin domain III antigen can be fused with a light chain signal peptide at the N end and fused with IgG4Fc at the C end through a CD8 hinge, and the amino acid sequence of the mesothelin domain III antigen is shown in SEQ ID NO. 2.
The chimeric antigen receptor in the above-described method of preparing a mesothelin-targeted CAR-T cell provided by the present invention may be a chimeric antigen receptor conventional in the art, comprising an extracellular antigen-binding region, a hinge region, a transmembrane region, an intracellular costimulatory signal region, and an intracellular signal region.
In the present invention, the extracellular antigen-binding region may be an antigen-binding molecule conventional in the art, preferably an antibody, such as a single chain antibody (scFv) or a single domain antibody.
In certain embodiments, the extracellular antigen-binding region is an scFv against mesothelin, preferably an scFv against mesothelin domain III. The DNA coding sequence for the scFv for the mesothelin domain III is preferably represented by SEQ ID NO 7 at positions 64-813.
In the present invention, the hinge region refers to the region between the CH1 and CH2 functional regions of the immunoglobulin heavy chain. The hinge region suitable for use in the present invention may be selected from any one or more of an extracellular hinge region of CD8a, an IgG1Fc CH2CH3 hinge region, an IgD hinge region, an extracellular hinge region of CD28, an IgG4Fc CH2CH3 hinge region, and an extracellular hinge region of CD 4. In certain embodiments, a CD8 hinge region or an IgG4Fc CH2CH3 hinge region is used herein.
In the present invention, the transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3 zeta transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region and a DAP10 transmembrane region; preferably the CD8 transmembrane domain.
In the present invention, the intracellular costimulatory signal region can be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cell costimulatory factor (ICOS), and the intracellular domain of DNAX activator protein 10 (DAP 10). In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, and the sequence thereof can be that of the CD28 intracellular domain as is conventional in the art. In certain embodiments, the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD137/4-1BB, which can be of a sequence conventional in the art for the intracellular domain of CD137/4-1 BB.
In certain embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus: a scFv targeting the mesothelin antigen, a CD8 hinge region, a CD8 transmembrane region, the intracellular domain of CD137/4-1BB, and a CD3 zeta intracellular signal domain. In certain embodiments, the chimeric antigen receptor further comprises a signal peptide, preferably the signal peptide is a CD8 signal peptide whose coding sequence is shown in SEQ ID NO 7, positions 4-63. In certain embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus: optionally a CD8 signal peptide, scFv, CD8 hinge region, CD8 transmembrane region, intracellular domain of CD137/4-1BB, and CD3 zeta intracellular signal domain.
In the present invention, the source of the Peripheral Blood Mononuclear Cells (PBMC) may be a source conventional in the art, for example, a source isolated from the blood of a donor subject by Ficoll density gradient centrifugation, or a commercially available PBMC may be purchased.
In the present invention, the nucleic acid containing the coding sequence of the chimeric antigen receptor targeting the mesothelin antigen may be a nucleic acid conventional in the art, including DNA and mRNA. When the nucleic acid is DNA, it can be a nucleic acid construct containing the coding sequence of the chimeric antigen receptor targeting the mesothelin antigen, which comprises the coding sequence of the mesothelin antigen-targeting CAR and a promoter operably linked thereto, preferably further comprising an enhancer sequence operably linked to the promoter. The nucleic acid construct may be a nucleic acid construct as conventional in the art, preferably an expression vector, more preferably a viral system expression vector, such as a type I HIV-based lentiviral system expression vector, an adenoviral expression vector or an adeno-associated virus (AAV) expression vector, or a transposon system expression vector, such as a Sleeping Beauty transposon system expression vector or a PiggyBac transposon system expression vector. When the nucleic acid is mRNA it comprises a 5' end cap structure, a 5' UTR, an Open Reading Frame (ORF) encoding the chimeric antigen receptor, a 3' UTR and a polyA tail.
In certain embodiments, the nucleic acid containing a mesothelin antigen-targeting chimeric antigen receptor coding sequence is the pNB328 transposon system vector disclosed in CN105154473A, comprising a mesothelin domain III targeting chimeric antigen receptor coding sequence as shown in SEQ ID No. 7, positions 64-813.
In the present invention, the introduction method may be any method conventional in the art, including any one of viral particle infection, electroporation, lipofection, calcium phosphate transfection and gene gun transfection. Preferably, the introduction method is electroporation, and more preferably, the electroporation is performed under the condition that the number U014 is selected using Lonza nucleobacterium 2 b.
In certain embodiments, a pNB328 transposon system vector comprising a coding sequence for a chimeric antigen receptor targeting mesothelin domain III as shown in SEQ ID No. 7, positions 64-813 is introduced by electroporation into PBMCs isolated from the blood of a donor subject to obtain naive cells.
In the present invention, the contacting is such that the mesothelin antigen and the anti-CD 28 antibody are present directly in the system containing the naive cells, producing a direct interaction with the surface of the naive cells. Preferably, the mesothelin antigen is mesothelin domain III and the anti-CD 28 antibody is a murine anti-CD 28 monoclonal antibody.
In the present invention, the non-immobilization refers to a state in which the mesothelin antigen or the anti-CD 28 antibody is not linked to any matrix in a covalent or non-covalent manner. The substrate is conventional in the art and includes stationary substrates such as well plates, petri dishes, glass slides, etc., as well as suspended substrates such as magnetic beads, nanoparticles, etc.
In certain embodiments, the contacting is by adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the chimeric antigen receptor targeting mesothelin III is introduced, directly present in solution in the initial cell system, and binding to the mesothelin domain III targeting CAR and the CD28 antigen, respectively, on the surface of the initial cells, the final concentration of the mesothelin domain III antigen being 0.1-3 μ g/mL, preferably 0.1-1 μ g/mL, more preferably 0.5-1 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 0.1-3. Mu.g/mL, preferably 0.1-1. Mu.g/mL, more preferably 0.5-1. Mu.g/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In certain embodiments, the contacting is adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the mesothelin III-targeted chimeric antigen receptor is introduced, directly present in solution in the naive cell system, and binding to the mesothelin domain III-targeted CAR and the CD28 antigen, respectively, on the naive cell surface, the final concentration of the mesothelin domain III antigen being 0.1 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 0.1 mug/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In certain embodiments, the contacting is adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the mesothelin III-targeted chimeric antigen receptor is introduced, directly present in solution in the naive cell system, and binding to the mesothelin domain III-targeted CAR and the CD28 antigen, respectively, on the naive cell surface, the final concentration of the mesothelin domain III antigen being 0.5 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 0.5 mug/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In certain embodiments, the contacting is adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the mesothelin III-targeted chimeric antigen receptor is introduced, directly present in solution in the naive cell system, and binding to the mesothelin domain III-targeted CAR and the CD28 antigen, respectively, on the naive cell surface, the final concentration of mesothelin domain III antigen being 1 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 1 mug/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In certain embodiments, the contacting is adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the mesothelin III-targeted chimeric antigen receptor is introduced, directly present in solution in the naive cell system, and binding to the mesothelin domain III-targeted CAR and the CD28 antigen, respectively, on the naive cell surface, the final concentration of mesothelin domain III antigen being 3 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 3 mug/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In certain embodiments, the contacting is by adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the chimeric antigen receptor targeting mesothelin III is introduced, directly present in solution in the initial cell system, and binding to the mesothelin domain III targeting CAR and the CD28 antigen, respectively, on the surface of the initial cell, the final concentration of the mesothelin domain III antigen being 0.1 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 0.5 mug/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In certain embodiments, the contacting is adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the mesothelin III-targeted chimeric antigen receptor is introduced, directly present in solution in the naive cell system, and binding to the mesothelin domain III-targeted CAR and the CD28 antigen, respectively, on the naive cell surface, the final concentration of the mesothelin domain III antigen being 0.5 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 1 mug/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In certain embodiments, the contacting is adding the mesothelin domain III antigen and the murine anti-CD 28 monoclonal antibody directly to the PBMC system into which the mesothelin III-targeted chimeric antigen receptor is introduced, directly present in solution in the naive cell system, and binding to the mesothelin domain III-targeted CAR and the CD28 antigen, respectively, on the naive cell surface, the final concentration of mesothelin domain III antigen being 1 μ g/mL; the final concentration of the murine anti-CD 28 antibody is 0.5 mug/mL; wherein the mesothelin III-targeted chimeric antigen receptor is introduced into PBMCs by electroporation of the PB transposon vector pNB328-Meso3CAR containing its coding sequence at the final concentration formed upon contact of the initial cells in 1) with the mesothelin antigen and the anti-CD 28 antibody.
In the present invention, the culture may be a T cell culture condition which is conventional in the art. Preferably, the medium used for the culture further contains cytokines; more preferably, the cytokine is IL-2; even more preferably, said cytokine is added to said medium at a time when said culturing is carried out for 0-6 hours; most preferably, the cytokine is added to the medium at the time the culture is performed for 6 hours.
In certain embodiments, the temperature of the culture is 37 ℃, and the CO of the culture 2 The concentration is 5%, the culture medium used for culturing is AIM-V containing 2% fetal bovine serum, and the culture time is 5-10 days.
In certain embodiments, the temperature of the culturing is 37 ℃, and the CO of the culturing is 2 The concentration is 5%, the culture medium used for culturing is AIM-V containing 2% fetal bovine serum and IL-2 with the final concentration of 500IU/mL, and the culturing time is 5-7 days; the IL-2 was added at 6 hours of incubation.
The positive progress effects of the invention are as follows: after T cells are obtained through sorting, the CAR gene targeting mesothelin is firstly introduced into the T cells, then the mesothelin antigen and the anti-CD 28 antibody are used for jointly stimulating the T cells, and the mesothelin antigen and the anti-CD 28 antibody are directly added into a system in a non-immobilized form to stimulate the CAR-T cells targeting mesothelin without coating a fixed or suspended matrix, so that the operation flow of preparing the CAR-T cells targeting mesothelin is greatly simplified, and the cost is reduced; the mesothelin-targeted CAR-T cells prepared by the method have obviously improved positive rate, proliferation level and proportion of effector memory T cells, and have tumor cell killing power with the same level as mesothelin-targeted CAR-T cells prepared by a mesothelin antigen and anti-CD 28 antibody coating method, while the expression level of cytokines is obviously reduced compared with the mesothelin-targeted CAR-T cells prepared after the mesothelin antigen and anti-CD 28 antibody are coated on a pore plate for stimulation, so that the potential risk of Cytokine Release Syndrome (CRS) is reduced on the premise of not reducing the tumor killing capacity.
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental procedures, for which specific conditions are not indicated in the following examples, were selected according to conventional methods and conditions (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, compiled by J. SammBruk et al, huang Peitang et al), or according to commercial instructions. The reagents or instruments used in the present invention are not indicated by manufacturers, and are all conventional products available on the market.
Some of the instruments and reagents used in the present invention are derived from the following sources:
the "room temperature" mentioned in the examples refers to the temperature between the operations of carrying out the test, and is generally 25 ℃.
Synthesis of gene sequences is entrusted to Shanghai Czeri;
commercial PBMC were purchased from the navy military medical university affiliated long-sea hospital blood station, no. 329;
FBS (fetal bovine serum) was purchased from Gibco;
AIM-V was purchased from Gibco:
the electrotransformation instrument is Lonza nucleoactor 2b, available from Lonza;
CCK8 detection kits were purchased from: sigmaaldrich, lot number: 96992;
the mesothelin domain III-Fc (Meso 3-Fc) antigen was prepared as follows: synthesizing the DNA fragment with the sequence of SEQ ID NO. 1, expressing the DNA fragment with the amino acid sequence of SEQ ID NO. 2 and containing a light chain signal peptide at the N terminalThe Meso3-Fc fusion protein aims to ensure that the Meso3-Fc fusion protein can be effectively secreted out of eukaryotic cells, thereby facilitating the subsequent purification. The 5 'end and the 3' end of SEQ ID NO are respectively connected with EcoRI and XbaI enzyme cutting site linkers and then connected to a pCDNA3.4 vector to construct an expression vector for over-expressing the Meso3-Fc fusion protein. According to ExpicHO TM Instructions for expression System, using ExpicHO TM After the expression system over-expresses the fusion protein, purifying an expression product by using MabSelect affinity chromatography resin of GE Healthcare according to the operation steps of the instruction, so as to prepare a purified Meso3-Fc fusion protein;
the MUC1-Fc antigen was prepared by the same method as the Meso3-Fc antigen, synthesizing a DNA fragment having the sequence shown in SEQ ID NO. 3, encoding a Muc1-Fc fusion protein having the amino acid sequence of SEQ ID NO. 4 and a light chain signal peptide at the N-terminus, ligating the DNA fragment to a pCDNA3.4 vector according to the above procedures, and then ligating the pCDNA3.4 vector with ExpicCHO TM The expression system expresses and purifies to obtain purified MUC1-Fc fusion protein;
the preparation method of recombinant mesothelin (rMSLN) antigen is the same as that of Meso3-Fc antigen, the DNA fragment with the sequence shown in SEQ ID NO. 5 is synthesized, the mesothelin antigen with the coded amino acid sequence shown in SEQ ID NO. 6 and containing light chain signal peptide at the N end is connected to pCDNA3.4 carrier according to the operation, and then ExpicCHO is used for connecting the DNA fragment to the pCDNA3.4 carrier TM The expression system expresses and purifies to obtain a purified recombinant mesothelin antigen;
the mesothelin domain III-Fc antigen-biotin (Meso 3-Fc-biotin) and the Muc1-Fc antigen-biotin (Muc 1-Fc-biotin) were prepared by labeling the 3 prepared fusion protein antigens by Kinsley with a biotin labeling method that is conventional in the art;
streptavidin labeled with PE fluorescein (PE-streptavidin): purchased from underwriters biology, inc;
SKOV-3 cell line, PANC-1 cell line: (all purchased from American type culture Collection ATCC);
anti-CD 28 antibodies were purchased from: merck Millipore, cat #: CBL517;
donor subjects No. 42, no. 43, no. 70, and No. 71 were all healthy adults.
Abbreviations:
meso3CAR: a chimeric antigen receptor targeting mesothelin, wherein the extracellular antigen-binding region is a single chain antibody directed against domain III of mesothelin protein;
muc1CAR: a chimeric antigen receptor targeting MUC1, wherein the extracellular antigen-binding region is a single chain antibody directed against MUC 1;
PBMC: peripheral blood mononuclear cells;
rMSLN: recombinant mesothelin antigen;
meso3: mesothelin III antigen;
muc1: a Muc1 antigen;
aCD28: an anti-CD 28 antibody;
d70: donor subject No. 70;
d71: donor subject No. 71.
Example 1 construction of expression vectors containing genes encoding Meso3CAR and Muc1CAR
The coding sequence of meso3CAR (SEQ ID NO: 7) and the coding sequence of Muc1CAR (SEQ ID NO: 8) were synthesized and loaded between EcoRI and SalI cleavage sites of the PB transposon system based vector pNB328 (the structure and sequence of pNB328 is described in CN201510638974.7, which is incorporated herein by reference in its entirety), respectively, and the constructed recombinant expression vectors were named pNB328-meso3CAR and pNB328-Muc1CAR, the structural schematic of both of which is shown in FIG. 1. The promoter sequence and polyA-tailed signal sequence, located between the 5'LTR and signal peptide sequences and before the 3' LTR, respectively, are not shown in FIG. 1.
Example 2 preparation of Meso3CAR-T cells and Muc1CAR-T cells by immobilization of activating Agents
Meso3 antigen and anti-CD 28 antibody coated 6-well plates: 2mL of DPBS (Hyclone) was added to one well of a 6-well plate, and the volume of 10. Mu.g of each was calculated based on the concentration on the package of Meso3 and anti-CD 28 antibody, and then each was aspirated by a gun and added to the well, followed by mixing by the "Cross-mix method" so that the coating concentration of Meso3 antigen and anti-CD 28 antibody were 5. Mu.g/mL. The 6-well plate was placed in a 4 ℃ freezer for coating overnight (or in a 37 ℃ cell incubator for 4 h).
Number taking device329 commercial PBMC cells, adjusted to 5X 10 concentration with physiological saline 6 one/mL. Taking 2 1.5mLEP tubes for each numbered cell, numbered a and b, adding 5 × 10 to each tube 6 Each cell was centrifuged at 1200rmp for 3min, the supernatant was discarded, and an electrotransfer reagent (from Lonza) was added to each of tubes a and b in a proportion of 100. Mu.L. Adding 4 mu g of pNB328 no-load plasmid into the tube a, adding 4 mu g of pNB328-meso3CAR plasmid constructed in the embodiment 1 into the tube b, transferring the mixed solution into an electric rotating cup, putting into a Lonza nucleofector 2b electric rotating instrument, and selecting a program with the number of U014 for electric shock; transferring the electroporated cell suspension to a six-well plate coated with meso3 antigen and anti-CD 28 antibody (the culture medium is 2% FBS-containing AIM-V culture medium) to which the culture medium has been added, mixing, and placing at 37 deg.C, 5% CO 2 After 5-7 days of culture, the cells were centrifuged again, the supernatant was discarded, the cell pellet was resuspended in fresh 2% FBS-containing AIM-V medium, culture was continued for 3-5 days, the total number of days of culture was made to 10 days, and the growth of the cells was observed to obtain control T cells and Meso3CAR-T cells derived from commercial PBMC, which were designated ConT-C329 and cMarso 3CAR-T-C329, respectively.
Blood from donor subjects numbered 70 and 71 were taken and PBMCs were obtained from each separated by Ficoll separation. Culturing PBMC for 2-4h in adherent manner, wherein nonadherent suspension cells are initial T cells, collecting the suspension cells in a 15ml centrifuge tube, centrifuging for 3min at 1200rmp, discarding the supernatant, adding physiological saline, centrifuging for 3min at 1200rmp, discarding the physiological saline, and repeating the steps; adjusting the concentration to 5 × 10 with physiological saline 6 one/mL. Taking 2 1.5mLEP tubes, numbered a and b, for each numbered cell, and adding 5 × 10 to each tube 6 The cells were centrifuged at 1200rmp for 3min, the supernatant was discarded, and the cells were taken out of the electrotransfer kit (purchased from Lonza) and the tubes a and b were dosed with the electrotransfer reagent in a total of 100. Mu.L. Adding 4 mu g of pNB328 no-load plasmid into the tube a, adding 4 mu g of pNB328-meso3CAR plasmid constructed in the embodiment 1 into the tube b, transferring the mixed solution into an electric rotating cup, putting into a Lonza nucleofector 2b electric rotating instrument, and selecting a program with the number of U014 for electric shock; transferring the cell suspension to the culture medium with meso3 antigen and anti-CD 28 antibody by using a micropipette in the kitMixing the coated six-well plates (the culture medium is an AIM-V culture medium containing 2% FBS), placing at 37 deg.C, and 5% CO 2 After 5-7 days of culture, the cells were centrifuged again, the supernatant was discarded, the cell pellet was resuspended in fresh 2-vol fbs-containing AIM-V medium, culture was continued for 3-5 days to reach 10 days in total days of culture, and the growth of the cells was observed to obtain control T cells and Meso3CAR-T cells derived from donor subjects No. 70 and No. 71, which were designated as cone-D70, cone-D71, cMeso3CAR-T-D70 and cMeso3CAR-T-D71, respectively.
Similarly, the donor subject blood numbers 42 and 43 were taken, and Muc1CAR-T cells derived from donor subject numbers 42 and 43 were prepared and named ConT-D42, conT-D43, cMUC1CAR-T-D42, and cMUC1CAR-T-D43, respectively, according to the same procedure as described above.
Example 3 activation reagent suspension method for preparation of Meso3CAR-T cells and Muc1CAR-T cells
The commercial PBMC cells designated 329 were treated separately and adjusted to 5X 10 concentration with physiological saline 6 One per mL. Taking 4 1.5mLEP tubes with numbers of c, d, e and f, adding 5 × 10 6 The cells were centrifuged at 1200rmp for 3min, the supernatant was discarded, the electrotransfer kit (from Lonza) was prepared, 100. Mu.L of electrotransfer reagent was added to each c-f tube in a proportion, 4. Mu.g of pNB328-meso3CAR plasmid constructed in example 1 was added to each tube, the mixture was transferred to an electrotransfer cup, a Lonza nucleofector 2b electrotransfer was placed in the cuvette, an electric shock was applied to the program No. U014, and the electrically transferred cell suspension was transferred to the wells of 4 six-well plates to which the culture medium (AIM-V culture medium containing 2 FBS) was added using a micropipette in the kit, mixed, and placed at 37 ℃ and 5 CO 2 Culturing in an incubator, adding stimulation factor IL-2 to each well of 4 wells after 6 hours till the final concentration is 500IU/mL, and adding meso3 antigen and anti-CD 28 antibody according to the following final concentration combinations respectively: 0.1. Mu.g/mL + 0.1. Mu.g/mL, 0.5. Mu.g/mL + 0.5. Mu.g/mL, 1. Mu.g/mL + 1. Mu.g/mL, 3. Mu.g/mL + 3. Mu.g/mL, 37 ℃,5% CO% 2 After culturing for 5-7 days, the cells were centrifuged again, the supernatant was discarded, the cell pellet was resuspended in fresh 2-vol FBS-containing AIM-V medium, culturing was continued for 3-5 days until the total culture time reached 10 days, and the growth of the cells was observed, and the obtained Meso3CAR-T cells were named fMeso3CAR-T-C329。
Blood from donor subjects numbered 70 and 71 were collected, and Meso3CAR-T cells derived from donor subjects numbered 70 and 71 were prepared according to the same procedure as described above and named fMeso3CAR-T-D70 and fMeso3CAR-T-D71, respectively.
Similarly, the donor subject blood numbers 42 and 43 were taken and, following the same procedure as above, muc1CAR-T cells derived from donor subject number 42 and 43 were prepared, designated fmec 1CAR-T-D42 and fmec 1CAR-T-D43, respectively, wherein fmec 1CAR-T-D42 and fmec 1CAR-T-D43 were suspension stimulated with 0.5 μ g/mL Muc1 antigen +0.5 μ g/mL anti-CD 28 antibody.
Example 4 comparison of the Positive Rate of Meso3CAR-T cells prepared by the activation reagent suspension method and the activation reagent immobilization method
1. Dissolving Meso3-Fc-biotin and PE-stretavidin in PBS to prepare 100 multiplied stock solution with the concentration of 10.0mg/mL, and diluting 100 times with PBS before use to obtain Meso3-Fc diluent labeled by PE fluorescein
2. Each of the Meso3CAR-T cells prepared in examples 2, 3 and 4 and each of the control T cells were taken at 1X 10 6 Centrifuging at 1000rpm for 3min, discarding the upper culture medium, resuspending with 1mL of fresh culture medium, adding 1, 2 and 5 μ L of PE fluorescein-labeled Meso3-Fc diluent of step 1 into all cells, and incubating at 37 ℃ for 1h;
3. each of the incubated Meso3CAR-T cells was washed three times with cold PBS, resuspended in 1mL of cold PBS per wash, and centrifuged at 1000rpm for 3min. After three times the fluorescence intensity of the cells was measured by flow cytometry and the positive rates of ConT-C329, cMetho 3CAR-T-C329 and fMeso3CAR-T-C329 and ConT-D70, conT-D71, cMetho 3CAR-T-D70, cMetho 3CAR-T-D71 cells and fMeso3CAR-T-D70 and fMeso3CAR-T-D71 cells were analyzed and compared.
The results are shown in FIGS. 2A-2C. FIG. 2A shows the positive rate of fMeso3CAR-T-C329 cells produced under conditions of ConT-C329, cMyso 3CAR-T-C329 and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody; FIG. 2B shows the positive rate of fMeso3CAR-T-D70 cells produced under conditions of ConT-D70, cMyso 3CAR-T-D70 and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody; FIG. 2C shows the positive rates of fMeso3CAR-T-D71 cells produced under conditions of ConT-D71, cMyso 3CAR-T-D71 and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody. 0.1+0.1, 0.5+0.5, and 3+3 in FIGS. 2A-2C respectively represent stimulation of cells in solution with final concentrations of 0.1. Mu.g/mL Meso3+ 0.1. Mu.g/mL anti-CD 28 antibody, 0.5. Mu.g/mL Meso3+ 0.5. Mu.g/mL anti-CD 28 antibody, and 3. Mu.g/mL Meso3+ 3. Mu.g/mL anti-CD 28 antibody.
The results shown in figures 2A-2C demonstrate that compared to Meso3CAR-T cells prepared by stimulation of Meso3 antigen and anti-CD 28 antibody coated well plates, the positive rate of various Meso3CAR-T cells prepared by suspension stimulation of Meso3 antigen and anti-CD 28 antibody added directly to each concentration combination was significantly higher than the former, and the same effect was observed in commercial PBMC-derived Meso3CAR-T cells (329) and in different donor subjects (donor 70 and donor 71) blood-derived Meso3CAR-T cells.
Example 5 comparison of proliferation levels of Meso3CAR-T cells prepared by the activation reagent suspension method and the activation reagent immobilization method
The proliferation level of the Meso3CAR-T cells prepared by an activating reagent suspension method and an activating reagent immobilization method is detected by a CCK8 proliferation test, and the steps are as follows:
(1) After the T cells are activated and in a proliferation vigorous stage, the volume of the T cells under a microscope is larger, and the T cells under the state are selected for a proliferation experiment;
(2) Taking 10 mu L T cell suspension, staining with trypan blue, adding into a disposable counting plate, and counting;
(3) Take 6.4X 10 5 Each T cell was fixed in 800. Mu.L of AIM-V complete medium to give a cell count of 8X 10 4 100 μ L, then diluted in gradient to 1250/100 μ L for a total of 7 concentration gradients (8X 10) 4 、4×10 4 、2×10 4 、1×10 4 、0.5×10 3 、0.25×10 3 And 0.125X 10 3 /100μL)；
(4) Cells were plated in 96-well plates at 3 wells per concentration, 100. Mu.L per well. Then, adding 10 mu L of CCK8 into each hole, putting the hole into a cell culture box, incubating for 4h, reading the OD value under the wavelength of 450nm, and calculating a standard curve;
(5) Taking 3 pieces of 96-well plate, each plateMiddle berth with 6 multiple holes, each hole 1X 10 4 Placing the T cells in a cell culture box for culture;
(6) One plate was removed at 24h,48h, and 72h, respectively, 10. Mu.L of CCK8 was added, incubated in a cell incubator for 4h, and OD was read at 450 nm. The number of T cells in each time period was calculated from the standard curve to prepare a proliferation curve.
The results are shown in FIGS. 3A and 3B. FIG. 3A shows proliferation curves of fMeso3CAR-T-D70 cells prepared under conditions stimulated by ConT-D70, cMyso 3CAR-T-D70 and varying concentrations of mesothelin III antigen + anti-CD 28 antibody; FIG. 3B shows proliferation curves of fMeso3CAR-T-D71 cells prepared under conditions of ConT-D71, cMyso 3CAR-T-D71 and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody. 0.1+0.1, 0.5+0.5, 1+1 and 3+3 respectively represent the stimulation of cells in solution with final concentrations of 0.1. Mu.g/mL Meso3+ 0.1. Mu.g/mL anti-CD 28 antibody, 0.5. Mu.g/mL Meso3+ 0.5. Mu.g/mL anti-CD 28 antibody, 1. Mu.g/mL Meso3+ 1. Mu.g/mL anti-CD 28 antibody and 3. Mu.g/mL Meso3+ 3. Mu.g/mL anti-CD 28 antibody.
The results shown in figures 3A-3B demonstrate that the proliferation levels of Meso3CAR-T cells produced by stimulation with Meso3 antigen + anti-CD 28 antibody added in suspension directly at each concentration combination were significantly higher than those produced by immobilization of Meso3CAR-T cells with their respective control T cells and activating agents, compared to Meso3CAR-T cells produced by stimulation with Meso3 antigen and anti-CD 28 antibody coated well plates. Among these, the increase in proliferation levels of Meso3CAR-T cells produced by the combined stimulation of two concentrations of 0.5 μ g/mL Meso3 antigen +0.5 μ g/mL anti-CD 28 antibody and 1 μ g/mL Meso3 antigen +1 μ g/mL anti-CD 28 antibody was particularly pronounced.
Example 6 comparison of cytokine secretion levels of Meso3CAR-T cells prepared by the activation reagent suspension method and the activation reagent immobilization method
The method comprises the following steps of taking the cells of fMeso3CAR-T-D70 and fMeso3CAR-T-D71 which are prepared under the stimulation condition of different concentrations of mesothelin III antigen + anti-CD 28 antibody, and detecting the Cytokine secretion of the Meso3CAR-T cells prepared by different methods by using the CBA Human Th1/Th2Cytokine Kit II of BD, wherein the specific steps are as follows:
(1) The recombinant MSLN antigen (rMSLN) obtained as described above was diluted to 5. Mu.g/mL with PBS, coated onto 6-well plates, and 3mL was added to each well overnight at 4 ℃. Removing the antigen coating solution the next day, and washing for 2-3 times by PBS;
(2) The various Meso3CAR-T cells were counted and 1 × 10 was taken 6 Centrifuging the Meso3CAR-T cells at 1500rpm for 5min, and discarding the supernatant;
(3) Resuspending T cells in AIM-V complete medium without IL-2, 1X 10 6 Meso3 CAR-cells plated in rMSLN-coated wells;
(4) Collecting cell suspension after 24h, centrifuging at 3000rpm for 5min, and collecting supernatant;
(5) 10 μ L of each sample was taken. Human IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma capture magnetic beads in the Th1/Th2Cytokine Kit II are mixed uniformly, 40 mu L of each capture magnetic bead is added, and the mixture is incubated for 3 hours in a dark place at 4 ℃;
(6) Adding 3mL of PBS into each reaction system, washing for 1-2 times at 4000rpm, centrifuging for 5min, and removing supernatant;
(7) Adding 300. Mu.L PBS for re-suspending the reacted magnetic beads, and detecting on a machine. Finally, the concentration of the four cytokines was calculated by FCAP Array v3 software.
The results are shown in FIGS. 4A-4D. FIGS. 4A-4D show the levels of four cytokines IL-2, IL-4, IL-10 and IFN- γ secreted by fMeO 3CAR-T-D70 and fMeO 3CAR-T-D71 cells, respectively, produced under stimulation with cMyso 3CAR-T-D70, cMyso 3CAR-T-D71 and varying concentrations of mesothelin III antigen + anti-CD 28 antibody following contact with PANC-1 cells and SKOV-3 cells. 0.1+0.1, 0.5+0.5 and 3+3 respectively represent the stimulation of cells in solution with final concentrations of 0.1. Mu.g/mL Meso3+ 0.1. Mu.g/mL anti-CD 28 antibody, 0.5. Mu.g/mL Meso3+ 0.5. Mu.g/mL anti-CD 28 antibody and 3. Mu.g/mL Meso3+ 3. Mu.g/mL anti-CD 28 antibody.
According to the results shown in FIGS. 4A-4D, the secretion levels of some of the above cytokines were significantly reduced in various Meso3CAR-T cells prepared by the activated reagent suspension method, compared to Meso3CAR-T cells prepared by the coated well plate method, with the reduction levels of IL-10 and IFN- γ being particularly significant. Cytokine Release Syndrome (CRS) is a major side effect of CAR-T cell therapy, and significantly reduced levels of cytokine secretion can greatly reduce the risk of side effects.
Example 7 comparison of the level of depleting factor in Meso3CAR-T cells prepared by the activated reagent suspension method and the activated reagent immobilization method
fMeso3CAR-T-C329 cells prepared under the conditions of ConT-C329, cMyso 3CAR-T-C329 and stimulation with different concentrations of mesothelin III antigen + anti-CD 28 antibody were counted at 1X 10 6 Adding each cell/tube into 1.5ml EP tube, washing twice with PBS, centrifuging at 1200rpm for 5min, and discarding supernatant; adding a flow antibody anti-PD-1-PE for detecting T cell exhaustion phenotype, and flicking to precipitate to uniformly mix; after incubation for 30min in the dark at room temperature, PBS was washed once, centrifuged at 1200rpm for 5min, the supernatant was discarded and 400. Mu.L of physiological saline was added, the cells were transferred to a flow tube and tested on a computer.
The results are shown in figure 5, and compared with control T cells and Meso3CAR-T cells prepared by the coated well plate method, the expression level of PD-1 in fMeso3CAR-T-C329 cells prepared by the activated reagent suspension method is significantly higher, indicating that dual suspension stimulation with two reagents, meso3 antigen and anti-CD 28 antibody, can activate CAR-T cells more effectively.
Example 8 detection of memory T cell phenotype of Meso3CAR-T cells prepared by activation reagent suspension and activation reagent immobilization
The samples of fMeso3CAR-T-D70 and fMeso3CAR-T-D71 were taken from ConT-D70, conT-D71, cMyso 3CAR-T-D70, cMyso 3CAR-T-D71 and different concentrations of mesothelin III antigen + anti-CD 28 antibody, and counted at 1X 10 6 Adding each cell/tube into 1.5ml EP tube, washing twice with PBS, centrifuging at 1200rpm for 5min, and discarding supernatant; respectively adding flow antibodies anti-CD197-FITC + anti-CD62L-PE for detecting the phenotype of the memory T cells, and flicking to precipitate so as to uniformly mix the materials; after incubation for 30min in the dark at room temperature, PBS was washed once, centrifuged at 1200rpm for 5min, the supernatant was discarded and 400. Mu.l of physiological saline was added, the cells were transferred to a flow tube and tested on a computer.
The results are shown in FIGS. 6A-6C. FIG. 6A shows the results of flow-based detection of effector T cells in fMeso3CAR-T-D70 cells prepared under conditions of ConT-D70, cMyso 3CAR-T-D70, and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody; FIG. 6B shows ConT-D71, cMyso 3CAR-T-D71 and different concentrations of mesothelin III antigen + anti-CD 28 antibody(ii) effector T cell flow assay results in fMeso3CAR-T-D71 cells made under stimulated conditions; FIG. 6C is a statistical result of the percentage of T cells responding in FIGS. 6A and 6B. In Meso3CAR-T cells prepared by different methods from donor subject No. 70 and No. 71, potent memory T cells (T cells) were found in Meso3CAR-T cells prepared by the activating agent suspension method compared to control T cells and T cells prepared by the activating agent immobilization method EM ) The ratio of (a) to (b) is significantly increased.
Example 9 comparison of killing of tumor cells by Meso3CAR-T cells prepared by activation reagent suspension and immobilization
The killing effect of fMeso3CAR-T-D70 and fMeso3CAR-T-D71 cells on tumor cells in vitro, which are prepared under the condition of being stimulated by ConT-D70, conT-D71, cMyso 3CAR-T-D70, cMyso 3CAR-T-D71 and different concentrations of mesothelin III antigen + anti-CD 28 antibody, is detected by a real-time label-free cell function analyzer.
Specifically, pancreatic cancer cell line PANC-1 and ovarian cancer cell line SKOV3 are selected as target cells, and real-time unmarked cell function analyzer (RTCA) of the Essen company is used for detecting the in vitro killing activity of each Meso3CAR-T cell and the control T cell thereof, and the method specifically comprises the following steps:
(1) Zero setting: adding 50 mul DMEM or 1640 culture solution into each well, putting into an instrument, selecting step 1, and zeroing;
(2) Target cell plating: laying the ovarian cancer cell SKOV-3 and the pancreatic cancer cell PANC-1 in a plate containing a detection electrode according to the proportion of 104 cells/50 mu l per hole, standing for several minutes, placing the plate into an instrument after the cells are stabilized, starting step2, and culturing the cells;
(3) Adding effector cells: after 24h of target cell culture, step2 was suspended, effector cells (i.e., each Meso3CAR-T derived from the blood of donor subject 70 and donor subject 71 described above and its control T cells) were added at 50 μ L per well, the effector ratio was set to 1:1, step 3 was started with the control of cone T-D70 and cone T-D71 cells, respectively, and the cell proliferation curve was observed after 24h of further co-culture.
The results are shown in FIGS. 7A-7D. FIGS. 7A and 7B show the killing curves of fMeso3CAR-T-D70 cells prepared under conditions of ConT-D70, cMetho 3CAR-T-D70, and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody against pancreatic cancer cell line PANC-1 and ovarian cancer cell SKOV-3, respectively; FIGS. 4C and 4D show the killing curves of fMeso3CAR-T-D71 cells on pancreatic cancer cell line PANC-1 and ovarian cancer cell SK-OV-3, respectively, produced under conditions of ConT-D71, cMyso 3CAR-T-D71, and stimulation with varying concentrations of mesothelin III antigen + anti-CD 28 antibody. 0.1+0.1, 0.5+0.5, 1+1 and 3+3 respectively represent the stimulation of cells in solution with final concentrations of 0.1. Mu.g/mL Meso3+ 0.1. Mu.g/mL anti-CD 28 antibody, 0.5. Mu.g/mL Meso3+ 0.5. Mu.g/mL anti-CD 28 antibody, 1. Mu.g/mL Meso3+ 1. Mu.g/mL anti-CD 28 antibody and 3. Mu.g/mL Meso3+ 3. Mu.g/mL anti-CD 28 antibody.
The structures shown in FIGS. 7A-7D show that the lethality of each Meso3CAR-T cell prepared by the activator reagent suspension method to PANC-1 cells and SK-OV-3 cells was essentially at the same level, with no significant difference, compared to Meso3CAR-T cells prepared by the activator reagent immobilization method. In combination with the results of the significant reduction in cytokine secretion levels in example 6, meso3CAR-T cells prepared by the activation reagent suspension method of the present invention have higher safety while ensuring lethality to tumor cells.
Comparative example 1 comparison of Muc1CAR-T cell positivity rates obtained by activation reagent suspension method and activation reagent immobilization method
The positive rates of ConT-D42, conT-D43, cMuc1CAR-T-D42, cMuc1CAR-T-D43, and fMuc1CAR-T-D42 and fMuc1CAR-T-D43 cells prepared in examples 2 to 3 were analyzed by detecting the fluorescence intensity of the cells by flow cytometry, with reference to the procedure of example 4, and compared.
The results are shown in FIGS. 8A-8B. FIG. 8A shows the level of positive rate for cMuc1CAR-T-D42 and fMuc1CAR-T-D42 cells; FIG. 8B shows the level of positive rate for cMUc1CAR-T-D43 and fMuc1CAR-T-D43 cells. According to the results shown in FIGS. 8A-8B, the positive rate was not increased for fMuc1CAR-T-D42 and fMuc1CAR-T-D43 cells prepared by the activation reagent suspension method compared to cMuc1CAR-T-D42 and cMuc1CAR-T-D43 prepared by the activation reagent immobilization method, and was even lower for fMuc1CAR-T-D43 in Muc1CAR-T cells derived from donor subject number 43.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.
Sequence listing
<110> Shanghai cell therapy engineering research center group Co., ltd
SHANGHAI CELL THERAPY Research Institute
<120> a method for preparing mesothelin-targeted CAR-T cells
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atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60
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ttgaaggcgc tcagtcagca gaatgtgagc atggacttgg ccacgttcat gaagctgcgg 180
acggatgcgg tgctgccgtt gactgtggct gaggtgcaga aacttctggg accccacgtg 240
gagggcctga aggcggagga gcggcaccgc ccggtgcggg actggatcct acggcagcgg 300
caggacgacc tggacacgct ggggctgggg ctacagggcg gcatccccaa cggctacctg 360
gtcctagacc tcagcatgca agaggccctc tcggagtcca aatatggtcc cccatgccca 420
ccatgcccag ggcagccccg agagccacag gtgtacaccc tgcccccatc ccaggaggag 480
atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctaccc cagcgacatc 540
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 600
ctggactccg acggctcctt cttcctctac agcaggctaa ccgtggacaa gagcaggtgg 660
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Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
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Asp Thr Thr Gly Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe
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Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn
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Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val
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Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val
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Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile
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Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln
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Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu
115 120 125
Ala Leu Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly
130 135 140
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
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Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
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Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
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Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
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Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
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Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
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atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60
tctggtcatg caagctctac cccaggtgga gaaaaggaga cttcggctac ccagagaagt 120
tcagtgccca gctctactga gaagaatgct gtgagtatga ccagcagcgt actctccagc 180
cacagccccg gttcaggctc ctccaccact cagggacagg atgtcactct ggccccggcc 240
acggaaccag cttcaggttc agctgccctg tggggacagg atgtcacctc ggtcccagtc 300
accaggccag ccctgggctc caccaccccg ccagcccacg atgtcacctc agccccggac 360
aacaagccag ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 420
accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 480
accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 540
accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 600
accaggccgg ccccgggctc caccgccccc ccagcccacg gtgtcacctc ggccccggac 660
accaggccgg ccccgggctc caccgccccc ccagcccatg gtgtcacctc ggccccggac 720
aacaggcccg ccttgggctc caccgcccct ccagtccaca atgtcacctc ggcctcaggc 780
tctgcatcag gctcagcttc tactctggtg cacaacggca cctctgccag ggctaccaca 840
accccagcca gcaagagcac tccattctca attcccagcc accactctga tactcctacc 900
acccttgcca gccatagcac caagactgat gccagtagca ctcaccatag ctcggtacct 960
cctctcacct cctccaatca cagcacttct ccccagttgt ctactggggt ctctttcttt 1020
ttcctgtctt ttcacatttc aaacctccag tttaattcct ctctggaaga tcccagcacc 1080
gactactacc aagagctgca gagagacatt tctgaaatgt ttttgcagat ttataaacaa 1140
gggggttttc tgggcctctc caatattaag ttcaggccag gatctgtggt ggtacaattg 1200
actctggcct tccgagaagg taccatcaat gtccacgacg tggagacaca gttcaatcag 1260
tataaaacgg aagcagcctc tcgatataac ctgacgatct cagacgtcag cgtgagtgat 1320
gtgccatttc ctttctctgc ccagtctggg gagtccaaat atggtccccc atgcccacca 1380
tgcccagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1440
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1500
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Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys
20 25 30
Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys
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Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly
50 55 60
Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala
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Thr Glu Pro Ala Ser Gly Ser Ala Ala Leu Trp Gly Gln Asp Val Thr
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100 105 110
His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr
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Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
130 135 140
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
145 150 155 160
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr
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Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
180 185 190
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr
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Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
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Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
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Ser Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn
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Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro
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Phe Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser
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His Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro
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Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly
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Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn
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Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg
355 360 365
Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu
370 375 380
Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu
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Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr
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Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr
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Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln
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Ser Gly Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln
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Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
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Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
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Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
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Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
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Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
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Lys Ser Leu Ser Leu Ser Leu Gly Lys
565
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atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60
gaagtggaga agacagcctg tccttcaggc aagaaggccc gcgagataga cgagagcctc 120
atcttctaca agaagtggga gctggaagcc tgcgtggatg cggccctgct ggccacccag 180
atggaccgcg tgaacgccat ccccttcacc tacgagcagc tggacgtcct aaagcataaa 240
ctggatgagc tctacccaca aggttacccc gagtctgtga tccagcacct gggctacctc 300
ttcctcaaga tgagccctga ggacattcgc aagtggaatg tgacgtccct ggagaccctg 360
aaggctttgc ttgaagtcaa caaagggcac gaaatgagtc ctcaggtggc caccctgatc 420
gaccgctttg tgaagggaag gggccagcta gacaaagaca ccctagacac cctgaccgcc 480
ttctaccctg ggtacctgtg ctccctcagc cccgaggagc tgagctccgt gccccccagc 540
agcatctggg cggtcaggcc ccaggacctg gacacgtgtg acccaaggca gctggacgtc 600
ctctatccca aggcccgcct tgctttccag aacatgaacg ggtccgaata cttcgtgaag 660
atccagtcct tcctgggtgg ggcccccacg gaggatttga aggcgctcag tcagcagaat 720
gtgagcatgg acttggccac gttcatgaag ctgcggacgg atgcggtgct gccgttgact 780
gtggctgagg tgcagaaact tctgggaccc cacgtggagg gcctgaaggc ggaggagcgg 840
caccgcccgg tgcgggactg gatcctacgg cagcggcagg acgacctgga cacgctgggg 900
ctggggctac agggcggcat ccccaacggc tacctggtcc tagacctcag catgcaagag 960
gccctctcgt ga 972
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<213> Artificial Sequence (Artificial Sequence)
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Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys
20 25 30
Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu
35 40 45
Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val
50 55 60
Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys
65 70 75 80
Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His
85 90 95
Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp
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Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys
115 120 125
Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val
130 135 140
Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala
145 150 155 160
Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser
165 170 175
Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr
180 185 190
Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala
195 200 205
Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe
210 215 220
Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn
225 230 235 240
Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val
245 250 255
Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val
260 265 270
Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile
275 280 285
Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln
290 295 300
Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu
305 310 315 320
Ala Leu Ser
<210> 7
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<213> Artificial Sequence (Artificial Sequence)
<400> 7
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggaggtgc agctggtgga gtccggggga ggcctggtcc agcctggggg atccctgaga 120
ctctcctgcg cagcctctgg attcgacctc ggtttctact tttacgcctg ttgggtccgc 180
caggctccag ggaagggcct ggagtgggtc tcatgcattt atactgctgg tagtggtagc 240
acgtactacg cgagctgggc gaaaggccga ttcaccatct ccagagacaa ttcgaagaac 300
acgctgtatc tgcaaatgaa cagtctgaga gccgaggaca cggccgtgta ttactgtgcg 360
agatctactg ctaatactag aagtacttat tatcttaact tgtggggcca aggcaccctg 420
gtcaccgtct cctcaggcgg aggcggatca ggtggtggcg gatctggagg tggcggaagc 480
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtgggaga cagagtcacc 540
atcacttgcc aggccagtca gaggattagt agttacttat cctggtatca gcagaaacca 600
gggaaagttc ccaagctcct gatctatggt gcatccactc tggcatctgg ggtcccctcg 660
cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 720
gaagatgttg ccacttacta ctgtcagagt tatgcttatt ttgatagtaa taattggcat 780
gctttcggcg gagggaccaa ggtggagatc aaaaccacga cgccagcgcc gcgaccacca 840
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 960
gcgcccctgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac cctttactgc 1020
aaacggggca gaaagaagct cctgtatata ttcaaacaac catttatgag accagtacaa 1080
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1140
gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1200
cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1260
cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 1320
tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 1380
gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 1440
gacacctacg acgcccttca catgcaggcc ctgccccctc gctga 1485
<210> 8
<211> 1506
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgagcgagg tccagctgca gcagtcagga ggaggcttgg tgcaacctgg aggatccatg 120
aaactctcct gtgttgcctc tggattcact ttcagtaact actggatgaa ctgggtccgc 180
cagtctccag agaaggggct tgagtgggtt gctgaaatta gattgaaatc taataattat 240
gcaacacatt atgcggagtc tgtgaaaggg aggttcacca tctcaagaga tgattccaaa 300
agtagtgtct acctgcaaat gaacaactta agagctgaag acactggcat ttattactgt 360
acctttggta actcctttgc ttactggggc caagggacca cggtcaccgt ctcctcaggt 420
ggttctggtt ctggcggctc cggttccggt ggatccggct ctgatatcgt tgtgactcag 480
gaatctgcac tcaccacatc acctggtgaa acagtcacac tcacttgtcg ctcaagtact 540
ggggctgtta caactagtaa ctatgccaac tgggtccaag aaaaaccaga tcatttattc 600
actggtctaa taggtggtac caacaaccga gcaccaggtg ttcctgccag attctcaggc 660
tccctgattg gagacaaggc tgccctcacc atcacagggg cacagactga ggatgaggca 720
atatatttct gtgctctatg gtacagcaac cattgggtgt tcggtggagg aaccaaactg 780
actgtcctag gatccgagtt cgtgccggtc ttcctgccag cgaagcccac cacgacgcca 840
gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc cctgcgccca 900
gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 960
gatatctaca tctgggcgcc cctggccggg acttgtgggg tccttctcct gtcactggtt 1020
atcacccttt actgcaacca caaacggggc agaaagaagc tcctgtatat attcaaacaa 1080
ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1140
gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agacgccccc 1200
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1260
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1320
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1380
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1440
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1500
cgctga 1506

Claims (38)

1. A method of making a mesothelin-targeted CAR-T cell, comprising the steps of: 1) Introducing nucleic acid containing a chimeric antigen receptor coding sequence of a targeted mesothelin antigen into peripheral blood mononuclear cells to obtain primary cells; 2) Contacting the primary cells in 1) with mesothelin antigen and an anti-CD 28 antibody, culturing; wherein the mesothelin antigen and the anti-CD 28 antibody are non-immobilized in state; wherein said contacting is such that said mesothelin antigen and said anti-CD 28 antibody together are present directly in a system comprising said naive cell, producing a direct interaction with the surface of said naive cell.
2. The method of claim 1, wherein the mesothelin antigen is a full length mesothelin antigen or a mesothelin domain III antigen.
3. The method of claim 1, wherein the mesothelin antigen is a mesothelin domain III antigen.
4. The method of claim 3, wherein the mesothelin domain III antigen comprises an IgG Fc.
5. The method of claim 4, wherein the IgG Fc is IgG4Fc.
6. The method of claim 4, wherein the mesothelin domain III antigen is fused to the IgG Fc by a hinge.
7. The method of claim 6, wherein the hinge is a CD8 hinge.
8. The method of claim 2, wherein the mesothelin antigen further comprises a signal peptide at the N-terminus.
9. The method of claim 8, wherein the signal peptide is a light chain signal peptide.
10. The method of claim 1, wherein the mesothelin antigen is a mesothelin domain III antigen fused to a light chain signal peptide at the N-terminus and to IgG4Fc via a CD8 hinge at the C-terminus, and the amino acid sequence is set forth in SEQ ID No. 2.
11. The method of claim 1, wherein the nucleic acid is a nucleic acid construct or mRNA.
12. The method of claim 11, wherein the nucleic acid is a nucleic acid construct.
13. The method of claim 11, wherein the nucleic acid construct is an expression vector.
14. The method of claim 13, wherein the nucleic acid construct is a viral vector or a transposon system vector.
15. The method of claim 14, wherein the viral vector is a lentiviral vector derived from type I HIV.
16. The method of claim 15, wherein the viral vector is pWPT or pWPXL.
17. The method of claim 14, wherein the transposon system is a modified or unmodified Sleeping Beauty transposon system vector or PiggyBac transposon system vector.
18. The method of claim 17, wherein the transposon system is a modified or unmodified PiggyBac transposon system vector.
19. The method of claim 18, wherein the transposon system is a modified PiggyBac transposon system vector.
20. The method of claim 18, wherein the transposon system is a pNB328 transposon system vector.
21. The method of claim 11, wherein the nucleic acid is an mRNA comprising a 5' end cap structure, a 5' utr, an open reading frame encoding the antigen, a 3' utr and a polyA tail.
22. The method according to claim 1, wherein the introduction is performed by any one method selected from the group consisting of viral particle infection, electroporation, lipofection, calcium phosphate transfection, and particle gun transfection.
23. The method according to claim 22, wherein the introducing is performed by any one method selected from the group consisting of viral particle infection, electroporation, and lipofection.
24. The method of claim 22, wherein the introduction is by viral particle infection or electroporation.
25. The method of claim 22, wherein the introducing is by electrotransfer.
26. The method of claim 1, wherein the anti-CD 28 antibody is an anti-CD 28 monoclonal antibody or an anti-CD 28 polyclonal antibody.
27. The method of claim 26, wherein the anti-CD 28 antibody is an anti-CD 28 monoclonal antibody.
28. The method of claim 26, wherein the anti-CD 28 antibody is a humanized monoclonal antibody.
29. The method of claim 26, wherein the anti-CD 28 antibody is a humanized murine or rabbit anti-CD 28 monoclonal antibody.
30. The method of claim 6, wherein the anti-CD 28 antibody is a humanized murine anti-CD 28 monoclonal antibody.
31. The method of claim 1, wherein the temperature of the culturing is 37 ℃; and/or, said cultured CO 2 The concentration is 5%; and/or the culture medium used for culturing is AIM-V containing 2% fetal bovine serum; and/or, the culturing time is 5-10 days.
32. The method of claim 31, wherein the culture medium used for said culturing further comprises cytokines; and/or, the cytokine is added to the medium at a time when the culturing is performed for 0-6 hours.
33. The method of claim 32, wherein the cytokine is IL-2 at a final concentration of 500IU/mL.
34. The method of any one of claims 1-33, wherein the mesothelin antigen and the anti-CD 28 antibody are both in solution.
35. The method of claim 34, wherein the final concentration of mesothelin antigen is 0.1-3 μ g/mL and the final concentration of anti-CD 28 antibody is 0.1-3 μ g/mL.
36. The method of claim 35, wherein the final concentration of mesothelin antigen is 0.1-1 μ g/mL and the final concentration of anti-CD 28 antibody is 0.1-1 μ g/mL.
37. The method of claim 36, wherein the final concentration of mesothelin antigen is 0.5-1 μ g/mL and the final concentration of anti-CD 28 antibody is 0.5-1 μ g/mL.
38. The method of claim 37, wherein the final concentration of mesothelin antigen is 0.5 μ g/mL and the final concentration of anti-CD 28 antibody is 0.5 μ g/mL.
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