CN107475275B - Chimeric antigen receptor and expression gene thereof, double-antigen-regulated chimeric antigen receptor modified T cell and application thereof - Google Patents

Chimeric antigen receptor and expression gene thereof, double-antigen-regulated chimeric antigen receptor modified T cell and application thereof Download PDF

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CN107475275B
CN107475275B CN201710792918.8A CN201710792918A CN107475275B CN 107475275 B CN107475275 B CN 107475275B CN 201710792918 A CN201710792918 A CN 201710792918A CN 107475275 B CN107475275 B CN 107475275B
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chimeric antigen
expression gene
antigen receptor
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CN107475275A (en
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黄彪
陈雪梅
刘韬
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Shenzhen Huayun Biotechnology Co., Ltd.
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells

Abstract

The invention relates to a chimeric antigen receptor and an expression gene thereof, a chimeric antigen receptor modified T cell regulated by double antigens and application thereof. The chimeric antigen receptor expression gene includes a first fusion protein expression gene and a second fusion protein expression gene. The first fusion protein expression gene comprises a mucin-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcriptional activator protein expression gene which are sequentially connected. The second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and a mesothelin antibody expression gene which are connected in sequence. The chimeric antigen receptor expression gene with the innovative design can be successfully introduced into T cells to form chimeric antigen receptor modified T cells, and immunoreaction can be generated under the condition that mucin-1 and mesothelin double-antigen signals exist simultaneously, so that the chimeric antigen receptor immunoreaction is controllable, few treatment side effects are caused, and the specificity is high.

Description

Chimeric antigen receptor and expression gene thereof, double-antigen-regulated chimeric antigen receptor modified T cell and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a chimeric antigen receptor, an expression gene thereof, a chimeric antigen receptor modified T cell regulated by double antigens and application thereof.
Background
Chimeric Antigen Receptors (CARs) are transmembrane molecules encoded by artificially constructed fusion genes. Generally comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain. CAR-T, i.e., Chimeric Antigen Receptor (CAR) modified T cells. CAR-T cells have shown excellent efficacy and great potential for use in cancer therapy. Phase 2 clinical data of its CAR-T targeting CD19 molecule (CTL019) in treatment refractory, relapsed Acute Lymphatic Leukemia (ALL) were published on the ASH conference by novain on 12/5/2015, with phase 2 data similar to phase 1 data published in 2014, with a complete remission rate (CR) profile up to 93% (55/59) and 92% (36/39).
Conventional CARs can be largely classified into three generations, as shown in fig. 1, and the first generation CARs generally use a signal chain, called "signal 1" (signal 1). First generation CAR-T cells showed limited efficacy in clinical trials, possibly due to activation-induced cell death (AICD) of transplanted T cells, or lack of long-term T cell expansion. Second generation CARs utilize the first generation CARs as a pillar, adding an additional co-stimulatory signal domain, referred to as "signal 2"; thus, the same receptor needs to transmit "signal 1" and "signal 2" to optimize T cell activation. Second generation CAR-T has enhanced persistence and amplifiability for reinfusion back into non-hodgkin lymphoma patients compared to first generation CAR-T. But which one is the best second signal has yet to be determined. Another problem to be solved is whether a threshold of persistence and/or expansion is necessary to produce an effective clinical result when comparing different CAR-T cell designs. The signaling domain of third generation CARs comprises two co-stimulatory domains, and preclinical studies have shown that third generation CAR-T cells have greater anti-tumor efficacy than second generation CAR-T cells.
However, CAR-T cells, when applied in cancer therapy, while exhibiting therapeutic efficacy, are associated with toxicity and risk, even leading to patient death. Intermediate analysis of phase II clinical data of KTE-C19 was published by Kite at 26.9.2016. The complete remission rate of the treatment reaches 47 percent. However, of the 62 patients enrolled in this trial, 1/3 developed severe neurotoxicity, 18% were affected by cytokine release syndrome, and 2 died from the adverse effects associated with KTE-C19.
Among them, Cytokine Release Syndrome (CRS) is the most significant toxicity, which is the first safety risk. Cytokine release syndrome is based on the activation of T cells, a response to T cell activation activity, so side effects are clinical responses that are positively correlated with CAR-T therapeutic mechanisms. The highly proliferating T cells can cause CRS, manifested as hyperpyrexia and myalgia, unstable hypotension and respiratory failure. This is an unexpected result because similar symptoms did not appear in preclinical animal models. A key point was found from the CRS observation that IL-6, in addition to the expected effector cytokine INF-gamma, also rapidly elevated during the exponential proliferation of CART treated cells. CRS may be directly associated with another toxicity, macrophage activation syndrome.
In addition to CRS, there is also "targeted" toxicity caused by the antigen specificity of the engineered T cells. Such as oncolytic syndrome, which is directly caused by lysis of tumor cells. When CARs are targeted to a target expressed on the surface of B cells, such as CD19, B cell dysplasia results, a "targeted" toxicity, but the wrong challenge is to normal tissue cells. B-cell dysplasia is not improved as long as CD19CAR-T cells are present for a long time. B cell dysplasia, like treatment with monoclonal antibodies specific for CD20, causes severe hypogammaglobulinemia and requires intravenous immunoglobulin injection. Recently 2 cases of lethal toxicity induced by infusion of engineered T cells were reported, with one patient receiving HER2-CAR therapy and two patients receiving TCR-T cell therapy targeting MAGE-A3. In these 2 cases, both are due to the expression of these targets by normal tissues, resulting in acute irreversible cardiopulmonary toxicity. All targeted toxicities are due to the inability of engineered T cells to distinguish between normal and tumor cells expressing the targeted antigen.
CAR-T treatment of leukemia causes neurological symptoms with neurotoxicity. Several groups have reported that these symptoms are diverse but resolve themselves, such as delirium, dyslexia, dyskinesia, mutism, and seizures. Although somewhat temporally correlated with the occurrence of systemic CRS, it was of course also correlated with the presence of CAR-T in cerebrospinal fluid. The mechanisms and target tissues for these symptoms remain to be identified.
In addition, CAR-T cells have potential risks for cancer therapy, such as the risk of autoimmune disease caused by infusion of activated T cells and the potential risk of host transplantation disease against allogeneic T cells. This may raise concerns for patients who have received allogeneic hematopoietic stem cell transplantation.
In conclusion, the traditional chimeric antigen receptor has the problems of more side effects, poor specificity and the like in treatment.
Disclosure of Invention
Accordingly, there is a need for a chimeric antigen receptor with less therapeutic side effects and high specificity, and an expression gene and use thereof.
In addition, a need exists for a dual antigen-regulated chimeric antigen receptor modified T cell and uses thereof.
A chimeric antigen receptor expressing gene comprising a first fusion protein expressing gene and a second fusion protein expressing gene;
the first fusion protein expression gene comprises a mucin-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcriptional activator protein expression gene which are sequentially connected;
the second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and a mesothelin antibody expression gene which are connected in sequence.
In one embodiment, the mucin-1 antibody expression genes include: (a) a nucleotide sequence shown as SEQ ID No.1, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.1, or (c) a nucleotide sequence shown as SEQ ID No.1 in which one or more bases are deleted, substituted or added; and/or the presence of a catalyst in the reaction mixture,
the notch receptor expression gene includes: (a) a nucleotide sequence shown as SEQ ID No.2, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.2, or (c) a nucleotide sequence shown as SEQ ID No.2 in which one or more bases are deleted, substituted or added; and/or the presence of a catalyst in the reaction mixture,
the expression gene of the Gal4-VP64 transcriptional activator protein comprises: (a) a nucleotide sequence shown as SEQ ID No.3, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.3, or (c) a nucleotide sequence shown as SEQ ID No.3 in which one or more bases are deleted, substituted or added.
In one embodiment, the Gal4-UAS promoter expresses a gene comprising: (a) a nucleotide sequence shown as SEQ ID No.4, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.4, or (c) a nucleotide sequence shown as SEQ ID No.4 in which one or more bases are deleted, substituted or added; and/or the presence of a catalyst in the reaction mixture,
the mesothelin antibody expression gene expression genes include: (a) a nucleotide sequence shown as SEQ ID No.5, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.5, or (c) a nucleotide sequence shown as SEQ ID No.5 in which one or more bases are deleted, substituted or added.
In one embodiment, the second fusion protein expression gene further comprises a terminator expression gene linked to the mesothelin antibody expression gene, the terminator expression gene comprising: (a) a nucleotide sequence shown as SEQ ID No.6, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.6, or (c) a nucleotide sequence shown as SEQ ID No.6 in which one or more bases are deleted, substituted or added.
An expression vector comprising the chimeric antigen receptor-expressing gene according to any one of the above.
A chimeric antigen receptor comprising a first fusion protein and a second fusion protein;
the first fusion protein comprises a mucin-1 antibody, a notch receptor and Gal4-VP64 transcriptional activator protein which are connected in sequence;
the second fusion protein comprises a Gal4-UAS promoter and a mesothelin antibody which are connected in sequence.
In one embodiment, the mucin-1 antibody comprises: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.1, (b) a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.1, or (c) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.1 in which one or more bases are deleted, substituted or added; and/or the presence of a catalyst in the reaction mixture,
the notch receptors include: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 2; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.2, wherein one or more bases are deleted, substituted or added, and encodes a polypeptide; and/or the presence of a catalyst in the reaction mixture,
the Gal4-VP64 transcriptional activator protein comprises: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 3; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 3; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.3, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide.
In one embodiment, the second fusion protein further comprises a terminator, the terminator being linked to the mesothelin antibody, wherein in the second fusion protein,
the Gal4-UAS promoter includes: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 4; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 4; a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.4, wherein one or more bases are deleted, substituted or added, and encodes a polypeptide; and/or the presence of a catalyst in the reaction mixture,
the mesothelin antibody comprises: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 5; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 5; a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.5, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide; and/or the presence of a catalyst in the reaction mixture,
the terminator includes: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 6; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 6; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.6, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide.
A chimeric antigen receptor-modified T cell with dual antigen modulation, wherein the chimeric antigen receptor expression gene of any one of the above is introduced into the T cell, or the expression vector of the above is transfected into the T cell, or the T cell can express the chimeric antigen receptor of any one of the above.
Use of the chimeric antigen receptor expressing gene of any one of the above, the expression vector of any one of the above, the chimeric antigen receptor of any one of the above, or the dual antigen regulated chimeric antigen receptor modified T cell of claim 9, in the preparation of a medicament for treating a tumor.
The chimeric antigen receptor expression gene includes a first fusion protein expression gene and a second fusion protein expression gene. The first fusion protein expression gene comprises a mucin-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcriptional activator protein expression gene which are sequentially connected. The second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and a mesothelin antibody expression gene which are connected in sequence. The chimeric antigen receptor expression gene which is innovatively designed can be successfully introduced into T cells to form chimeric antigen receptor modified T cells, and the chimeric antigen receptor expression gene can be used for treating tumors and the like. The first fusion protein is capable of recognizing mucin-1 (muc-1), a marker on the surface of tumor cells, after expression, so as to specifically target the chimeric antigen receptor to a corresponding focal site. And after the first fusion protein is contacted with the mucin-1, the notch receptor is subjected to self-cleavage to release Gal4-VP64 transcriptional activator protein. The Gal4-VP64 transcriptional activator protein activates the Gal4-UAS promoter of the second fusion protein, which in turn initiates the expression of mesothelin antibody. When two tumor markers, namely mucin-1 (muc-1) and mesothelin (mesothelin), are expressed on the surface of tumor cells, dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin) are provided, and the chimeric antigen receptor initiates immune attack to kill the tumor cells. When the dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin) are absent or only one of them, the chimeric antigen receptor does not initiate an immune attack and is in the off state. Therefore, the dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin) are the 'switch' of the behavior of the chimeric antigen receptor in the T cell, and the immune response is generated under the condition of the specific dual antigen signals, so that the controllable immune response of the chimeric antigen receptor is realized, the treatment side effect is less, and the specificity is high.
Drawings
FIG. 1 is a schematic diagram of the CARs structure according to an embodiment;
FIG. 2 is a schematic representation of intracellular signaling of one embodiment of a dual antigen modulated chimeric antigen receptor modified T cell in the presence of dual antigen signals;
FIG. 3 is a flow chart of one embodiment of a method for producing a dual antigen-modulated chimeric antigen receptor-modified T cell;
FIG. 4 is an electrophoretogram of a cleavage product of the first expression vector constructed in example 1;
FIG. 5 is an electrophoretogram of a cleavage product of the second expression vector constructed in example 1;
fig. 6 is a picture of observing the chimeric antigen receptor-modified T cell (JurkatCAR) obtained in example 1 under a microscope;
FIG. 7 is a graph comparing the proliferation results of the chimeric antigen receptor-modified T cells obtained in example 1 under different conditions;
FIG. 8 is a graph comparing the results of the secretion of IFN γ by the chimeric antigen receptor-modified T cells obtained in example 1 under different conditions;
FIG. 9 is a graph comparing the flow scatter plots of the chimeric antigen receptor-modified T cells obtained in example 1 for killing target cells under different conditions;
FIG. 10 is a comparison graph of the results of statistics of the killing rate of the chimeric antigen receptor-modified T cells obtained in example 1 against target cells under different conditions.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
A chimeric antigen receptor expressing gene comprising a first fusion protein expressing gene and a second fusion protein expressing gene. The first fusion protein expression gene comprises a mucin-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcriptional activator protein expression gene which are sequentially connected. The second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and a mesothelin antibody expression gene which are connected in sequence.
The chimeric antigen receptor expressing gene is capable of expressing a Chimeric Antigen Receptor (CAR) and is therefore useful in the treatment of tumors.
Specifically, the mucin-1 antibody-expressing gene is used to express mucin-1 (muc-1). Mucin-1 is expressed in high levels in tumor cells, particularly solid tumor cells, but is expressed in low or almost no levels in normal tissues, and thus mucin-1 (muc-1) can serve as an "ideal antigen" for tumor therapy. By designing a mucin-1 antibody (anti muc-1) in a Chimeric Antigen Receptor (CAR), T cells transfected with the Chimeric Antigen Receptor (CAR) can be specifically targeted to tumor tissue parts, so that the specificity is improved, the damage to other tissues is reduced, and the side effect of cancer treatment is effectively reduced.
In one embodiment, the mucin-1 antibody expression genes include: (a) a nucleotide sequence shown as SEQ ID No.1, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.1, or (c) a nucleotide sequence shown as SEQ ID No.1 in which one or more bases are deleted, substituted or added. The above nucleotide sequence can be expressed smoothly to obtain the mucin-1 antibody (anti muc-1).
Specifically, the notch receptor expression gene is used for expressing the notch receptor. The notch receptor is linked to the mucin-1 antibody at one end and to the Gal4-VP64 transcriptional activator protein at the other end. After binding of the mucin-1 antibody to mucin-1, the notch receptor undergoes self-cleavage to release Gal4-VP64 transcriptional activator protein.
In one embodiment, the notch receptor expression gene comprises (a) a nucleotide sequence shown as SEQ ID No.2, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.2, or (c) a nucleotide sequence shown as SEQ ID No.2 in which one or more bases are deleted, substituted or added. The inventors design the synthetic sequence shown in SEQ ID No.2 to be able to express smoothly to obtain notch receptor.
Specifically, the Gal4-VP64 transcriptional activator expression gene was used to express Gal4-VP64 transcriptional activator. The Gal4-VP64 transcriptional activator protein was more able to activate the Gal4-UAS promoter in the second fusion protein, thereby inducing mesothelin antibody expression in the second fusion protein.
In one embodiment, the Gal4-VP64 transcriptional activator expression genes include: (a) a nucleotide sequence shown as SEQ ID No.3, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.3, or (c) a nucleotide sequence shown as SEQ ID No.3 in which one or more bases are deleted, substituted or added.
In one embodiment, the first fusion protein expression gene comprises a nucleotide sequence shown as SEQ ID No.1, a nucleotide sequence shown as SEQ ID No.2 and a nucleotide sequence shown as SEQ ID No.3 which are connected in sequence. The expression of the polypeptide coded by the polynucleotide consisting of the nucleotide sequence is anti muc-1-synNotch-Gal4VP 64.
Specifically, the Gal4-UAS promoter expression gene is used for expressing the Gal4-UAS promoter. The Gal4-UAS promoter promotes the expression of mesothelin antibody.
In one embodiment, the Gal4-UAS promoter expresses a gene comprising: (a) a nucleotide sequence shown as SEQ ID No.4, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.4, or (c) a nucleotide sequence shown as SEQ ID No.4 in which one or more bases are deleted, substituted or added.
Specifically, the mesothelin antibody expression gene is used to express mesothelin antibody. Mesothelin (mesothelin) is expressed in higher amounts in tumor cells and in lower or hardly expressed in normal tissues, and thus mesothelin can be used as an "ideal antigen" for treating tumors. Mucin-1 (muc-1) and mesothelin (mesothelin) constitute a dual antigen signal, and in the presence of the dual antigen signal, the chimeric antigen receptor initiates immune attack, killing the tumor cells.
In one embodiment, the mesothelin antibody expressing gene expression gene comprises: (a) a nucleotide sequence shown as SEQ ID No.5, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.5, or (c) a nucleotide sequence shown as SEQ ID No.5 in which one or more bases are deleted, substituted or added. The nucleotide sequence can be expressed smoothly to obtain the mesothelin antibody.
In one embodiment, the second fusion protein expression gene further comprises a terminator expression gene linked to the mesothelin antibody expression gene, the terminator expression gene comprising: (a) a nucleotide sequence shown as SEQ ID No.6, (b) a nucleotide sequence having at least 95% homology with the nucleotide sequence shown as SEQ ID No.6, or (c) a nucleotide sequence shown as SEQ ID No.6 in which one or more bases are deleted, substituted or added. A terminator is provided to further regulate the expression of the second fusion protein.
In one embodiment, the second fusion protein expression gene comprises a nucleotide sequence shown as SEQ ID No.4, a nucleotide sequence shown as SEQ ID No5 and a nucleotide sequence shown as SEQ ID No.6 which are connected in sequence. The expression of the polypeptide coded by the polynucleotide consisting of the nucleotide sequence is Gal4-UAS promoter-anti mesothelin CAR-SV40_ PA _ terminator.
The chimeric antigen receptor expression gene which is innovatively designed can be successfully introduced into T cells to form chimeric antigen receptor modified T cells, and the chimeric antigen receptor expression gene can be used for treating tumors.
An expression vector containing the above chimeric antigen receptor expression gene.
Specifically, the expression vector may be, for example, a lentiviral vector pLV-IRES-Puro into which the chimeric antigen receptor expression gene is inserted, and the first fusion protein expression genes of the first fusion protein expression genes are inserted into the lentiviral vector and transfected into T cells, thereby obtaining chimeric antigen receptor-modified T cells.
The Chimeric Antigen Receptor (CAR) of one embodiment includes a first fusion protein and a second fusion protein. The first fusion protein comprises a mucin-1 antibody, a notch receptor and Gal4-VP64 transcriptional activator protein which are connected in sequence. The second fusion protein comprises the Gal4-UAS promoter and the mesothelin antibody which are connected in sequence.
Specifically, the specific functions and properties of the mucin-1 antibody, notch receptor, Gal4-VP64 transcription activator protein, Gal4-UAS promoter and mesothelin antibody can be described above and will not be described herein.
In one embodiment, the mucin-1 antibody comprises: (a) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.1, (b) a polypeptide encoded by a polynucleotide having at least 98% homology with a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.1, or (c) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.1 in which one or more bases are deleted, substituted or added.
In one embodiment, the notch receptor comprises: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 2; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 2; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.2, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide.
In one embodiment, the Gal4-VP64 transcriptional activator proteins comprise: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 3; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 3; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.3, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide.
In one embodiment, the Gal4-UAS promoter comprises: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 4; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 4; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.4, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide.
In one embodiment, the mesothelin antibody comprises: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 5; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 5; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.5, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide.
In one embodiment, the second fusion protein further comprises a terminator, the terminator being linked to the mesothelin antibody. The terminator includes: (a) polypeptide coded by polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 6; (b) the polypeptide coded by the polynucleotide with at least 98 percent of homology with the polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 6; or (c) a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No.6, wherein the polynucleotide in which one or more bases are deleted, substituted or added encodes the resulting polypeptide.
Further, the first fusion protein comprises a mucin-1 antibody, a notch receptor, and Gal4-VP64 transcriptional activator protein, which are linked in this order. The expression of the first fusion protein is anti muc-1-synNotch-Gal4VP 64.
Further, the second fusion protein comprises Gal4-UAS promoter, mesothelin antibody and terminator connected in sequence. The expression of the second fusion protein is Gal4-UAS promoter-anti mesothelin CAR-SV40_ PA _ terminator.
It is understood that due to polymorphisms and variations in the expressed genes of the chimeric antigen receptor, there may exist various forms of expressed genes encoding the same protein. If the base is deleted, substituted or added in the expressed gene, or the amino acid is deleted, inserted, substituted or otherwise varied, the amino acid sequence of the protein is caused to have one or more amino acids deleted, substituted or added. A polypeptide or protein that is expressed as a protein that is different from the corresponding protein but has no significant functional difference from the protein is referred to as a functionally equivalent variant. Therefore, an expressed gene capable of expressing a polypeptide or protein that has no significant functional difference from the chimeric antigen receptor should be considered as an expressed gene equivalent to the chimeric antigen receptor expressed gene in the present application. Polypeptides or proteins that do not have a significant functional difference from the chimeric antigen receptor should be considered proteins that are equivalent to the chimeric antigen receptor in the present application.
Through continuous research and exploration, the Chimeric Antigen Receptor (CAR) with the functional structure is successfully designed, and the Chimeric Antigen Receptor (CAR) can be specifically expressed in T cells so as to be used for treating tumors and the like. When two tumor markers, namely mucin-1 (muc-1) and mesothelin (mesothelin), are expressed on the surface of tumor cells, dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin) are provided, and the chimeric antigen receptor initiates immune attack to kill the tumor cells. When the dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin) are absent or only one of them, the chimeric antigen receptor does not initiate an immune attack and is in the off state. Therefore, the dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin) are the 'switch' of the behavior of the chimeric antigen receptor in the T cell, and the immune response is generated under the condition of the specific dual antigen signals, so that the controllable immune response of the chimeric antigen receptor is realized, the treatment side effect is less, and the specificity is high.
The Chimeric Antigen Receptor (CAR) can be applied to anti-tumor drugs.
In one embodiment, the Chimeric Antigen Receptor (CAR) is used in a medicament against breast cancer.
One embodiment of the double antigen-regulated chimeric antigen receptor-modified T cell (CAR-T) is a T cell into which the above-described chimeric antigen receptor expression gene is introduced. Or the T cell is transfected with the above expression vector. Or the T cell is capable of expressing a chimeric antigen receptor as described above.
The expression genes and structures of the Chimeric Antigen Receptor (CAR) are described above and not described herein.
In one embodiment, referring to FIG. 2, the mucin-1 antibody in the chimeric antigen receptor-modified T cell binds to mucin-1 on the tumor cell, and the notch receptor undergoes self-cleavage, releasing Gal4-VP64 transcriptional activator protein. The Gal4-VP64 transcriptional activator protein activates the Gal4-UAS promoter of the second fusion protein, which in turn initiates the expression of mesothelin antibody. When two tumor markers, namely mucin-1 (muc-1) and mesothelin (mesothelin), are expressed on the surface of the tumor cell, the tumor cell has dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin), and the dual antigen signals are respectively combined with a mucin-1 antibody and a mesothelin antibody, so that T cells modified by chimeric antigen receptors are activated, and further immune attack is initiated to kill the tumor cell.
When the dual antigen signals of mucin-1 (muc-1) and mesothelin (mesothelin) are absent or only one of them, the chimeric antigen receptor does not initiate an immune attack and is in the off state.
In one embodiment, the T cell is a Jurkat cell. Jurkat cells belong to one of T lymphocytes, have a strong proliferative capacity, and are suitable as host cells for Chimeric Antigen Receptors (CAR).
Experimental results show that the double-antigen-regulated chimeric antigen receptor modified T cell (CAR-T) has specific and efficient killing effect on tumor cells expressing double-antigen signals of specific mucin-1 (muc-1) and mesothelin (mesothelin). The chimeric antigen receptor does not initiate immune attack on cells which do not express mucin-1 (muc-1) and mesothelin (mesothelin) or cells which express only one of the antigens, is in a closed state, and has little killing effect on normal cells. Therefore, the chimeric antigen receptor modified T cell (CAR-T) is expected to be applied to anti-tumor drugs and provides a thought for tumor treatment. The T cell modified by the chimeric antigen receptor realizes controllable immune response of the chimeric antigen receptor and has less treatment side effect.
The double antigen-regulated chimeric antigen receptor modified T cell (CAR-T) can be applied to anti-tumor drugs or drugs for resisting virus infection.
In one embodiment, the chimeric antigen receptor-modified T cell is administered to a patient in need of treatment for breast cancer.
Referring to fig. 3, one embodiment of a method for preparing a chimeric antigen receptor-modified T cell with dual antigen modulation includes the following steps S110 to S130.
S110, providing a chimeric antigen receptor expression gene, wherein the chimeric antigen receptor expression gene comprises a first fusion protein expression gene and a second fusion protein expression gene, the first fusion protein expression gene comprises a mucin-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcriptional activator protein expression gene which are sequentially connected, and the second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and a mesothelin antibody expression gene which are sequentially connected.
Specifically, the structure of the Chimeric Antigen Receptor (CAR) is described above and will not be described herein.
In one embodiment, the second fusion protein further comprises a terminator, the terminator being linked to the mesothelin antibody.
In one embodiment, the encoded first fusion protein is expressed as anti muc-1-synNotch-Gal4VP 64. The expression of the encoded second fusion protein is Gal4-UAS promoter-anti mesothelin CAR-SV40_ PA _ terminator.
Specifically, the nucleic acid sequence of anti muc-1-synNotch-Gal4VP64 and the nucleic acid sequence of Gal4-UAS promoter-anti mesothelin CAR-SV40_ PA _ terminator were synthesized by means of gene synthesis. Obtaining the chimeric antigen receptor expression gene.
S120, connecting the first fusion protein expression gene obtained in S110 to a lentiviral vector to obtain a first expression vector, and connecting the second fusion protein expression gene obtained in S110 to the lentiviral vector to obtain a second expression vector.
Specifically, the obtained first fusion protein expression gene and the second fusion protein expression gene are subjected to double enzyme digestion by restriction enzymes, and then are connected to a lentiviral vector subjected to double enzyme digestion treatment by the same restriction enzymes.
In one embodiment, the restriction enzyme is, for example, Nde I, Xho I, EcoR I or BamH I, or the like.
In one embodiment, the lentiviral vector is pLV-IRES-Puro.
S130, packaging the first expression vector and the second expression vector obtained in the S120 into lentivirus and transfecting the lentivirus into T cells to obtain the chimeric antigen receptor modified T cells regulated by double antigens.
Specifically, the first expression vector and the second expression vector obtained in S120 are transfected into host cells such as 239T cells, and amplified to obtain a large amount of lentiviruses. A viral titer of lentivirus is then transfected into T cells to obtain dual antigen-regulated chimeric antigen receptor modified T cells (CAR-T).
In one embodiment, the expression vector is packaged as a lentivirus and transfected into a T cell, and the viral MOI value is about 10.
The method for preparing the chimeric antigen receptor modified T cell regulated by the double antigens has simple and convenient operation, and the prepared chimeric antigen receptor modified T cell generates immunoreaction under the condition of the existence of the specific double antigens, thereby realizing controllable immunoreaction of the chimeric antigen receptor and having less treatment side effect.
The following is a detailed description of the embodiments.
In the following examples, unless otherwise specified, the experimental procedures without specifying the specific conditions are usually carried out according to conventional conditions, for example, the conditions described in the molecular cloning's Experimental guidelines [ M ] (Beijing: scientific Press, 1992) by Sammbruke, EF Friech, T Mannich, et al (decoded by gold winter goose, Rimeng maple, et al) or the procedures recommended by the manufacturers of the kits. The reagents used in the examples are all commercially available.
Example 1
Preparation of Dual antigen-modulated chimeric antigen receptor-modified T cells
A first fusion protein expression gene (comprising a nucleotide sequence shown as SEQ ID No.1, a nucleotide sequence shown as SEQ ID No.2 and a nucleotide sequence shown as SEQ ID No.3 which are connected in sequence) for coding a first fusion protein is synthesized by a gene company respectively, wherein the expression of the first fusion protein is anti muc-1-synNotch-Gal4VP 64.
The first fusion protein expression gene is subjected to double enzyme digestion by EcoR I and BamH I, and is cloned into a lentiviral vector pLV-IRES-Puro to obtain a first expression vector. The constructed first expression vector EcoR I and BamH I are identified by double enzyme digestion, and the electrophoresis picture of the digestion product is shown in FIG. 4 (wherein, the 2.4kb fragment in FIG. 4 is the target fragment, and the 8.1kb fragment is the vector fragment). The size of the target fragment is consistent with the expectation, which indicates that the vector construction is successful.
Respectively synthesizing a second fusion protein expression gene (comprising a nucleotide sequence shown as SEQ ID No.4, a nucleotide sequence shown as SEQ ID No.5 and a nucleotide sequence shown as SEQ ID No.6 which are connected in sequence) for encoding a second fusion protein by a gene company. The expression of the second fusion protein is Gal4-UAS promoter-anti mesothelin CAR-SV40_ PA _ terminator.
The second fusion protein expression gene is subjected to double enzyme digestion by Nde I and Xho I, and is cloned into a lentiviral vector pLV-IRES-Puro to obtain a second expression vector. The constructed second expression vector Nde I and Xho I were subjected to double digestion and the electrophoresis pattern of the digestion products is shown in FIG. 5 (in FIG. 5, the 2kb fragment is the target fragment, and the 9.6kb fragment is the vector fragment). The size of the target fragment is consistent with the expectation, which indicates that the vector construction is successful.
The first expression vector and the second expression vector obtained above were lentiviruses, and viruses (virus MOI values of 10, respectively) were transfected into Jurkat E6.1 cells (T lymphocyte cell line, purchased from american type collection, ATCC) to obtain double antigen-regulated chimeric antigen receptor-modified Jurkat cells (JurkatCAR).
After 2 days, the transfected Jurkat E6.1 cells were transferred to RPMI 1640 medium with puromycin and the cells were cloned by limiting dilution. Through 21-day screening, a puromycin resistant dual antigen regulated chimeric antigen receptor modified Jurkat E6.1 cell line (JurkatCAR) was established. As shown in FIG. 6, a photograph showing the growth of transfected Jurkat E6.1(Jurkat CAR) cells under a microscope shows that Jurkat E6.1 was well grown and transfection was successful.
Test one
Dual antigen regulated chimeric antigen receptor modified T cell proliferation assay
Will be 5X 105JurkatCAR cells prepared in example 1 were added to a culture medium containing U-2OS and U-2OS/muc-1+、U-2OS/muc-1+/mesothelin+、A431、A431/mesothelin+6-well plate (5X 10) of MCF7 cells5Per well). Wherein U-2OS is human osteosarcoma cell, A431 is human epidermal carcinoma cell, MCF7 is human breast cancer cell, and U-2OS, A431 and MCF7 are all purchased from ATCC. U-2OS/muc-1+It is shown that the muc-1 antigen is overexpressed by U-2OS by genetic modification of muc-1 on the basis of U-2OS cells. U-2OS/muc-1+/mesothelin+It is shown that the genetic modification of muc-1 and mesothelin on the basis of U-2OS cells results in the overexpression of muc-1 antigen and mesothelin antigen by U-2 OS. A431/mesothelin+It is shown that the gene modification of mesothelin on the basis of A431 cells allows A431 to overexpress the mesothelin antigen. In the above cells, U-2OS does not express muc-1 antigen and mesothelin antigen. U-2OS/muc-1+And A431 only expressed muc-1 antigen. U-2OS/muc-1+/mesothelin+、A431/mesothelin+And MCF7 express the muc-1 antigen and the mesothelin antigen.
Cell counts were performed on suspended JurkatCAR cells on days 3 (D3) and 7 (D7), respectively. As shown in FIG. 7, JurkatCAR proliferated in large amounts after contact with U-2OS/muc-1+/mesothelin +, A431/mesothelin +, MCF7 cells expressed by both muc-1 (mucin-1) and mesothelin antigens. While the other groups of jurkatcars did not substantially proliferate. The above results indicate that JurkatCAR (Dual antigen regulated chimeric antigen receptor modified T cell) can proliferate in large amounts when both antigenic signals, muc-1 (mucin-1) and mesothelin (mesothelin), are present. JurkatCAR (a dual antigen-regulated chimeric antigen receptor-modified T cell) does not substantially proliferate when dual signals are absent or only one signal is present.
Test two
Determination of IFN gamma secretion in Dual antigen-regulated chimeric antigen receptor-modified T cells
Will be 5X 105Each JurkatCAR cell prepared in example 1 was incubated with U-2OS, U-2OS/muc-1 in 24-well plates+、U-2OS/muc-1+/mesothelin+、A431、A431/mesothelin+MCF7 cell (5X 10)5/well) co-culture.
The supernatant was collected after 72 hours, and the secretion amount of IFN γ was measured by an ELISA detection kit for IFN γ (BD Biosciences).
The results are shown in FIG. 8, U-2OS/muc-1 expressed with both antigens muc-1 (mucin-1) and mesothelin (mesothelin)+/mesothelin+、A431/mesothelin+JurkatCAR can secrete IFN-gamma in a large amount after MCF7 cell coculture; the other groups secreted substantially no IFN-. gamma.. Above knotIt was shown that JurkatCAR (Dual antigen regulated chimeric antigen receptor modified T cell) is able to recognize tumor cell targets and, when activated, secrete more IFN-. gamma.when both muc-1 (mucin-1) and mesothelin (mesothelin) antigens are present. JurkatCAR (Dual antigen regulated chimeric antigen receptor modified T cell) secretes little or essentially no IFN- γ in the absence of dual antigen signals or in the presence of only one signal.
Test three
In vitro killing assay for dual antigen-regulated chimeric antigen receptor-modified T cells
JurkatCAR cells prepared in example 1 were incubated with target cells U-2OS, U-2OS/muc-1+、U-2OS/muc-1+/mesothelin+、A431、A431/mesothelin+MCF7 cells were co-cultured (1: 1 ratio of effective targets). And (3) detecting the in-vitro killing capacity of the genetically modified JurkatCAR cells on different types of tumor cells by using a CFSE/PI double-labeling method.
The method comprises the following steps: 1X 10 pairs of CFSE at a final concentration of 0.05. mu.M6Marking each target cell by living cell staining, and marking 1 × 106JurkatCAR cells and 1X 106Mixing the CFSE labeled target cells, centrifuging at 200g for 1min to contact the cells, and centrifuging at 5% CO2And incubating for 4 hours in an incubator at 37 ℃, adding 1 mu g/ml PI dye solution after the reaction is finished, mixing uniformly, incubating for 15min at room temperature in a dark place, and then carrying out flow detection.
The results are shown in FIGS. 9 and 10, where the upper right quadrant cell population in FIG. 9 is the proportion of killed cells. JurkatCAR can effectively kill U-2OS/muc-1 expressed by double antigens of muc-1 (mucin-1) and mesothelin (mesothelin)+/mesothelin+、A431/mesothelin+MCF7 cells, while the other groups of cells did not substantially kill. The above results indicate that JurkatCAR (chimeric antigen receptor modified T cell regulated by double antigens) can recognize and effectively kill target cells when two antigens, muc-1 (mucin-1) and mesothelin (mesothelin), are present simultaneously. JurkatCAR (a dual antigen-regulated chimeric antigen receptor-modified T cell) does not substantially kill target cells in the absence of dual antigen signals or in the presence of only one signal.
In conclusion, the double antigen-regulated chimeric antigen receptor-modified T cell (JurkatCAR) has specific and efficient killing effect on tumor cells expressing the double antigens of muc-1 (mucin-1) and mesothelin (mesothelin), and has little killing effect on cells when the signal of the double antigens of mucin-1 (muc-1) and mesothelin (mesothelin) does not exist or is only one. Therefore, the T cell modified by the chimeric antigen receptor can be used as an anti-tumor medicament and provides a thought for tumor treatment. The T cell modified by the chimeric antigen receptor generates immune reaction in the presence of specific muc-1 (mucin-1) and mesothelin (mesothelin) double antigens, so that the controllable immune reaction of the chimeric antigen receptor is realized, and the treatment side effect is less.
The above-mentioned embodiments only express one or several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shenzhen university
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Claims (9)

1. A chimeric antigen receptor expressing gene comprising a first fusion protein expressing gene and a second fusion protein expressing gene;
the first fusion protein expression gene comprises a mucin-1 antibody expression gene, a notch receptor expression gene and a Gal4-VP64 transcriptional activator protein expression gene which are sequentially connected, wherein the nucleotide sequence of the mucin-1 antibody expression gene is shown as SEQ ID No.1, the nucleotide sequence of the notch receptor expression gene is shown as SEQ ID No.2, and the nucleotide sequence of the Gal4-VP64 transcriptional activator protein expression gene is shown as SEQ ID No. 3;
the second fusion protein expression gene comprises a Gal4-UAS promoter expression gene and a mesothelin antibody expression gene which are sequentially connected, and the nucleotide sequence of the mesothelin antibody expression gene is shown as SEQ ID No. 5.
2. The chimeric antigen receptor expressing gene according to claim 1, wherein the nucleotide sequence of the expression gene of Gal4-UAS promoter is shown in SEQ ID No. 4.
3. The chimeric antigen receptor expression gene according to claim 1, wherein the second fusion protein expression gene further comprises a terminator expression gene, wherein the terminator expression gene is linked to the mesothelin antibody expression gene, and the nucleotide sequence of the terminator expression gene is shown as SEQ ID No. 6.
4. An expression vector comprising the chimeric antigen receptor-expressing gene according to any one of claims 1 to 3.
5. A chimeric antigen receptor comprising a first fusion protein and a second fusion protein;
the first fusion protein comprises a mucin-1 antibody, a notch receptor and Gal4-VP64 transcriptional activator protein which are sequentially connected, wherein the mucin-1 antibody is a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence shown in SEQ ID No.1, the notch receptor is a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence shown in SEQ ID No.2, and the Gal4-VP64 transcriptional activator protein is a polypeptide encoded by a polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 3;
the second fusion protein comprises a Gal4-UAS promoter and a mesothelin antibody which are connected in sequence, wherein the mesothelin antibody is a polypeptide obtained by encoding a polynucleotide consisting of a nucleotide sequence shown in SEQ ID No. 5.
6. The chimeric antigen receptor according to claim 5, wherein the Gal4-UAS promoter encodes a polypeptide consisting of a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 4.
7. The chimeric antigen receptor according to claim 5, wherein the second fusion protein further comprises a terminator, wherein the terminator is linked to the mesothelin antibody, and wherein the terminator in the second fusion protein is a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence shown in SEQ ID No. 6.
8. A chimeric antigen receptor-modified T cell with dual antigen modulation, wherein the chimeric antigen receptor expression gene of any one of claims 1 to 3 is introduced into the T cell, or the T cell is transfected with the expression vector of claim 4, or the T cell is capable of expressing the chimeric antigen receptor of any one of claims 5 to 7.
9. Use of the chimeric antigen receptor-expressing gene of any one of claims 1 to 3, the expression vector of claim 4, the chimeric antigen receptor of any one of claims 5 to 7, or the dual antigen-regulated chimeric antigen receptor-modified T cell of claim 8 for the preparation of an anti-tumor medicament.
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