CN110669138A - Double-chimeric antigen receptor, T cell, construction method and application thereof - Google Patents

Abstract

The invention discloses a dual chimeric antigen receptor, a T cell, a construction method and application thereof, and belongs to the field of tumor cell immunotherapy. The present invention specifically relates to the specific structure and construction method of the dual chimeric antigen receptor T cell (dCAR-T cell), and preliminarily discusses the in vivo and in vitro activities of the dCAR-T cell. The selected tumor-associated antigens were mesothelin and carcinoembryonic antigen, which studies have shown are simultaneously expressed on the surface of solid tumors, such as pancreatic cancer. The antigen receptor constructed by the present invention, through in vitro and in vivo tests, confirms that the constructed dCAR-T cells can be permanently and effectively activated only when they recognize two antigens at the same time, and have efficient anti-tumor activity, and can further It exerts specific tumor killing function and improves the application of CAR-T cell immunotherapy.

Description

A kind of double chimeric antigen receptor, T cell and its construction method and application

technical field

The present invention belongs to the field of cellular immunotherapy of tumors, and more particularly, to the field of cellular immunotherapy of tumors. Construction of a dual-chimeric antigen receptor (dual-receptor CAR, dCAR), T cells and construction methods and applications thereof.

Background technique

With the continuous development of cellular immunology, the role of immune T cells in relieving tumor development and clearing tumors has been paid more and more attention by scientists. Studies have found that when using endogenous T cells for tumor immunotherapy, the target antigens of tumor cells need to be processed and presented by the main histocompatibility complex (MHC) on their surface before they can be displayed on the surface of T cells in vivo. T cells are recognized by the receptors, so that the tumor cells are eliminated, that is, the function of T cells has the characteristics of "MHC restriction". However, the process of tumor immunoediting can significantly reduce the expression of MHC molecules on the surface of tumor cells, destroy the processing and presentation of antigens, reduce the immunogenicity of peptide fragments, hinder the recognition and activation of T cells, and make tumor cells successfully Avoiding the attack of T cells eventually leads to the rapid proliferation of tumors, which is the immune escape mechanism of tumors. In order to improve the ability of T cells to kill tumors, gene recombination technology is used to fuse a specific chimeric antigen receptor (CAR) targeting tumor antigens into the genome of T cells (hereinafter referred to as CAR-T cells). The pathway has shown good targeting, killing activity and persistence in in vitro and clinical trials, providing a new and effective solution for adoptive cellular immunotherapy, showing great application potential and development prospects.

Compared with traditional T cell surface receptors (TCRs), the structure and mechanism of action of CARs have been greatly simplified. CAR is mainly composed of three parts: extracellular antigen recognition region, transmembrane region and intracellular signal transduction region. The extracellular antigen recognition region is a single chain antibody variable region (single chain fragment variable, scFv) composed of the light chain (VL) and heavy chain (VH) and flexible hinge region of monoclonal antibodies. The source or heterologous CD3, CD8 or CD28 dimer membrane protein composition, the intracellular signal transduction domain is the immunoreceptor tyrosine-based activation motifs (immunoreceptor tyrosine-based activation motifs, ITAMs), usually CD3ζ. Once the extracellular scFv recognizes tumor-associated antigen (TAA), it activates T cells through the signal transduction pathway to release granzyme and perforin, and finally achieve the effect of killing tumor cells. At present, clinical CAR-T cell therapy has shown good therapeutic effect in the treatment of acute lymphocytic leukemia, chronic lymphocytic leukemia, lymphoma, melanoma, liver cancer, lung cancer and other malignant hematological tumors and solid tumors, especially for blood tumor.

At present, the development of CAR-T cell technology has mainly gone through four generations. As early as 1989, the first generation of CAR was formed by fusion of immunoglobulin scFv and CD3 complex (ζ chain) intracellular domain to form a chimeric receptor, and the engineered T cells bound tumor antigens in an antigen-dependent, MHC-independent manner. , initiate and activate specific tumor-killing responses. Although there are many studies on first-generation CAR-T cells, most of the tests are still insufficient in terms of cell expansion, in vivo survival time, cytokine secretion, etc., and have not achieved the expected clinical effect. Studies have shown that the complete activation of T cells depends on its dual signaling pathways, namely antigen recognition signaling pathway and co-stimulatory signaling pathway, mainly its surface receptor TCR recognizes antigen peptide-MHC complexes and CD28 costimulatory molecules on target cells. B7 molecules interact to promote the synthesis of IL-2, so that T cells are fully activated. CAR-T cells containing only the CD3ζ signaling domain cannot be fully activated, thus greatly weakening the function of CAR-T cells. Therefore, according to the dual-signal theory of T cell activation, the second-generation CAR structurally adds a costimulatory molecule, such as CD28 and CD137 (4/1BB), to enhance the cytotoxicity of T cells and prolong the duration in vivo. The third-generation CAR adds two co-stimulatory molecules, which clinically show that this CAR-T cell has a strong tumor cell clearance ability, but faces serious safety problems, mainly cytokine release syndrome. CD28 molecule plays an important role in regulating lymphocyte proliferation and survival, especially for the initial activity of CAR-T cells, which has a strong explosive force; 4/1BB provides signals for maintaining T cell responses, which can greatly improve the CAR-T cells in CAR-T cells. duration in the body.

At present, CAR-T cell technology has a good therapeutic effect in the treatment of chronic lymphocytic leukemia, acute leukemia and lymphoma, but the effect on solid tumors is not good. The reason is that this is closely related to the occurrence and development of solid tumors, and a microenvironment with strong immunosuppressive function will be formed around tumor cells. In this microenvironment, there are many regulatory T cells with inhibitory functions, M2 tumor-associated macrophages and myeloid-derived suppressor cells, some inhibitory molecules (eg PD-L1) that are abnormally highly expressed by the tumor itself, and secreted Inhibitory cytokines, etc. (eg, TGF-beta and IL-10). In this case, the ability of CAR-T cells to home to the tumor tissue site is greatly impaired. In response to this research bottleneck, the fourth-generation CAR-T co-expressed some cytokines and, on the basis of the second CAR-T, such as: IL-12 or HPSE, these factors can well overcome the immunosuppressive microenvironment, It is expected to become an important means in the treatment of solid tumors.

At present, a serious side effect of CART cell therapy in clinic is targeted non-tumor toxicity. The reason is that tumor-specific antigens targeting CAR structures are very rare and difficult to isolate. The tumor associated antigen (tumor associated antigen, TAA) not only exists on the surface of tumor cells, but also exists in some normal tissues. Therefore, after the CAR-T cells designed with TAA are reinfused into the body, they can not only kill tumor cells, but also cause damage to normal tissues. This toxic and side effect has attracted great attention from doctors and patients in clinical practice, and will also have a negative impact on the application of CAR-T cell technology. How to use the existing TAA to improve the safety of CAR-T cells is the primary problem facing the clinic.

Mesothelin (MSLN), a tumor-associated antigen with a molecular weight of 40 kDa, is often expressed in normal pleura, pericardium, and peritoneal mesothelial cells, but is also highly expressed in a variety of malignant tumor tissues. Studies have shown that mesothelin is widely expressed in solid tumors such as mesothelioma, pancreatic cancer, lung cancer, ovarian cancer, bile duct cancer, gastric cancer, and breast cancer. Therefore, mesothelin is often used as a broad-spectrum TAA in tumor immunotherapy. Carcion-embryonicantigen (CEA) is a polysaccharide protein complex with a molecular weight of 22kDa. CEA is a non-organ-specific tumor-associated antigen that is originally synthesized by fetal gastrointestinal epithelial tissue, pancreas and liver cells. The serum content was significantly reduced. There is a small amount of expression in the small intestine, liver, spleen and other tissues of normal adults, but the expression in colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, breast cancer, etc. will greatly increase with the degree of malignancy. Therefore, it can also be used as a A broad spectrum TAA. At present, it has been found by bioinformatics that multiple TAAs are simultaneously overexpressed in some tumor cells. For example, tumor types such as pancreatic cancer, lung cancer, and breast cancer all overexpress both MSLN and CEA antigens at the same time. Relatively speaking, the probability of expressing two TAAs at the same time is relatively low for normal tissues and even tumor-adjacent tissues, and they often only overexpress one TAA. s reason. To develop T cells with dual-targeting functions, T cells can be fully activated only on the premise of recognizing two antigens at the same time, thereby exerting an anti-tumor effect, while causing only slight or even no damage to normal tissues.

SUMMARY OF THE INVENTION

1. The problem to be solved

Aiming at the toxicity of normal tissue damage in the current CAR-T cell therapy process, the present invention provides a dichimeric anti-human MSLN single-chain antibody (hMSLNscFv) and an anti-human CEA single-chain antibody (hCEAscFv) constructed. It is a method of transfecting T lymphocytes through a unique electroporation method by combining with antigen receptors, and separately coupling the CD3ζ and 4/1BB signaling domains of the T cell activation signaling pathway in the downstream. The efficacy and safety of the modified T cells were studied. Only when the two antigens, MSLN and CEA were recognized at the same time, could they be fully activated, which effectively reduced the damage to normal tissues and enhanced the safety of CAR-T cell therapy. To further explore its application in clinical treatment. This study reduces off-target effects in CAR-T therapy because it is a dual receptor, while avoiding the potential risks brought by the use of lentivirus, which greatly increases the safety of the treatment.

2. Technical solutions

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

A novel dual chimeric antigen receptor consisting of a first chimeric antigen receptor for MSLN and a second chimeric antigen receptor for CEA.

Further, the first chimeric antigen receptor includes the light chain and heavy chain variable regions hMSLNscFv and signaling domains of human MSLN monoclonal antibody; the MSLNscFv sequence is SEQID NO.1 in the sequence listing or a variant with 80% sequence similarity; the signaling domain is composed of a hinge region, a transmembrane region and an intracellular signaling domain in tandem.

Further, the light chain and heavy chain variable region MSLNscFv of the MSLN monoclonal antibody set a linker between the light chain and the heavy chain, and the linker can be a different amino acid sequence, preferably a GlyGlyGlyGlySerGlyGlyGlyGlySer SerGlyGlyGlySer connecting peptide .

Further, the hinge region of the first chimeric antigen receptor is a CD8α hinge; the transmembrane region is a human CD3 transmembrane region, a human CD8 transmembrane region or a human CD28 transmembrane region; the signaling cell The inner region consists of the costimulatory signaling domain CD137 (4/1BB).

Further, the upstream of the first chimeric antigen receptor contains a signal peptide, the amino acid sequence of the signal peptide is as shown in SEQ ID NO.2 in the sequence listing, and the corresponding nucleotide sequence is as follows: It is shown as SEQ ID NO. 3 in the sequence listing.

Further, the amino acid sequence of the signaling structure of the first chimeric antigen receptor is shown in SEQ ID NO.4 in the sequence listing, and its corresponding nucleotide sequence is shown in SEQ ID NO.4 in the sequence listing. 5 shown.

Further, the described second chimeric antigen receptor comprises the light chain and heavy chain variable region hCEAscFv and signaling domain of human CEA monoclonal antibody; the CEAscFv sequence is SEQ ID NO. 6 or a variant with 80% sequence similarity therewith; the signaling domain is composed of a hinge region, a transmembrane region and an intracellular signaling domain in series.

Further, the light chain and heavy chain variable region CEAscFv of the CEA monoclonal antibody set a linker between the light chain and the heavy chain, and the linker can be a different amino acid sequence, preferably a GlyGlyGlyGlySerGlyGlyGlyGlySer SerGlyGlyGlySer connecting peptide .

Further, the hinge region of the second chimeric antigen receptor is a CD8α hinge; the transmembrane region is a human CD3 transmembrane region, a human CD8 transmembrane region or a human CD28 transmembrane region; the signaling cell The inner region consists of the antigen recognition signal domain CD3ζ.

Further, the upstream of the second chimeric antigen receptor contains a signal peptide, the amino acid sequence of the signal peptide is shown in SEQ ID NO.7 in the sequence listing, and the corresponding nucleotide sequence is shown in the sequence SEQ ID NO. 8 in the list.

Further, the amino acid sequence of the signaling structure of the second chimeric antigen receptor is shown in SEQ ID NO.9 in the sequence listing, and its corresponding nucleotide sequence is shown in SEQ ID NO.9 in the sequence listing. 10 shown.

Further, the upstream of the DAC gene sequence contains a Kozak sequence, and its nucleotide sequence is shown in SEQ ID NO. 11 in the sequence listing. A linker sequence IRES is contained between the first chimeric antigen receptor and the second chimeric antigen receptor, and its nucleotide sequence is shown in SEQ ID NO. 12 in the sequence listing.

Preferably, the downstream of the first chimeric antigen receptor and the second chimeric antigen receptor contains a fluorescent protein tag GFP, whose amino acid sequence is shown in SEQ ID NO. 14 in the sequence listing; There is a linking peptide P2A between the downstream of the antigen receptor and the fluorescent protein tag, and its amino acid sequence is shown in SEQ ID NO. 13 in the sequence listing.

Preferably, the nucleic acid sequence of the dichimeric antigen receptor is shown in SEQ ID NO. 15 in the sequence listing.

位点特异性整合酶系统通过电转的方式将表达双嵌合抗原受体的基因插入到T淋巴细胞的基因组中，以制备dCAR-T细胞。A method for transfection and preparation of dCAR-T cells capable of expressing the above-mentioned dual chimeric antigen receptor: connecting the dual chimeric antigen receptor gene to an overexpression vector, using

The site-specific integrase system inserts the gene expressing the dual chimeric antigen receptor into the genome of T lymphocytes by electroporation to prepare dCAR-T cells.

In the present invention, the dual chimeric antigen receptor Signal peptide I-hMSLNscFv-CD8α-CD8TM-4/1BB-IRES-Signal peptide II-hCEAscFv-CD8α-CD8TM-CD3ζ-P2A-GFP, the first chimeric antigen receptor is composed of The light chain and heavy chain variable region MSLNscFv, human CD8α hinge region, human CD8 transmembrane region and intracellular 4/1BB costimulatory signal domain structure of MSLN monoclonal antibody are constructed in tandem; the second chimeric antigen receptor is composed of CEA The light chain and heavy chain variable region CEAscFv, human CD8α hinge region, human CD8 transmembrane region and intracellular CD3ζ structure of monoclonal antibody are constructed in tandem; upstream of the two chimeric antigen receptors each has a secretion signal peptide, which is used for The formation and localization of the CAR structure in the extracellular domain protein were directed, and an internal ribosome entry site (IRES) sequence was used to connect the two chimeric receptors; the end of the sequence was inserted with a GFP green protein fluorescent tag.

A dual chimeric antigen receptor dCAR-T cell, the dCAR-T cell can express the dual chimeric antigen receptor according to any one of claims 1-14. The in vivo and in vitro activities of dual-receptor dCAR-T cells are regulated by two tumor-associated antigens, CEA and MSLN, which can improve the safety and efficacy of tumor cell therapy and promote its application in tumor immunotherapy.

A method for controlling the dCAR-T cells prepared above with dual chimeric antigen receptors, using two tumor-related antigens CEA and MSLN to jointly control the activity of the dCAR-T cells.

The application of the above-mentioned dual chimeric antigen receptor dCAR-T cells in the field of preparation of tumor drugs for the treatment of overexpressed tumor antigens CEA and MSLN.

Further, the tumors include pancreatic cancer, lung cancer, gastric cancer, colon cancer, breast cancer, liver cancer, melanoma, cervical cancer and kidney cancer, prostate cancer, tongue squamous cell carcinoma, bladder cancer, ovarian cancer, and nasopharyngeal cancer.

Use of the dichimeric antigen receptor described in any one of the above in the preparation of a medicament for treating tumors.

A tumor cell therapy drug, comprising the above-mentioned dual chimeric antigen receptor, or the above-mentioned dCAR-T cell, and pharmaceutically acceptable excipients.

The first chimeric antigen receptor is respectively composed of the light chain and heavy chain variable regions hMSLNscFv of the anti-human MSLN monoclonal antibody, the extracellular hinge region, the transmembrane region and the intracellular region, and the costimulatory signal domain structure in series; The second chimeric antigen receptor is respectively composed of the light chain and heavy chain variable region hCEAscFv of the anti-human CEA monoclonal antibody, the extracellular hinge region, the transmembrane region, the intracellular region and the antigen recognition signal transduction domain in series; There is a secretory signal peptide upstream of each chimeric antigen receptor. In order to facilitate the detection of dCAR-T cells in vitro and in vivo, GFP green fluorescent protein was inserted at the end of its sequence.

3. Beneficial effects

Compared with the prior art, the beneficial effects of the present invention are:

(1) The present invention mainly introduces the construction of a novel dual-receptor CAR-T cell and a preliminary study on its activity. In general, conventional T cell activation requires stimulation under dual-signal conditions. The present invention expresses its dual signal pathway separately into two independent chimeric antigen receptor forms through genetic means. The activation (phosphorylation) of the signal transduction domain CD3ζ of the first signaling pathway is controlled by the first antigen to recognize the corresponding receptor; the activation (phosphorylation) of the co-stimulatory signal transduction domain 4/1BB of the second signaling pathway is controlled by the second Control of antigen recognition by corresponding receptors. This new model of CAR-T cells can only be fully activated under the premise of the presence of two antigens at the same time, thereby exerting the killing toxicity to tumor cells. A variety of tumor-associated antigens are usually expressed on the surface of tumor cells, and there are differences in the expression of tumor-associated antigens between paracancerous tissues and tumor tissues. Therefore, combining two different tumor-related antigens to regulate the constructed dCAR-T cells can effectively improve its safety.

(2) The present invention optimizes the codons according to the gene sequence information of hMSLNscFv and hCEAscFv, and at the same time according to human CD8α signal peptide gene, human CD8α hinge region gene, human CD8 transmembrane region and intracellular region gene, and human CD3ζ The gene sequence information of the intracellular region and 4/1BB costimulatory signal domain was obtained. Finally, the dual chimeric antigen receptor Signal peptideⅠ-hMSLNscFv-CD8α-CD8TM-4/1BB-IRES-Signal peptideⅡ-hCEAscFv-CD8α was synthesized by the method of whole gene synthesis. - Gene fragment of CD8TM-CD3ζ.

整合酶系统的过表达载体pCF-PGK-MCS(pCF-空载体)中，构成抗人MSLN和抗人CEA的双CAR表达质粒(pCF-dCAR质粒)，利用该质粒与整合酶质粒

通过电转的方式共转染T细胞，并采用优化的培养条件使T细胞表达双嵌合抗原受体，从而赋予T细胞更高的特异性和安全性。(3) Due to the long gene fragment, the conventional lentiviral transfection method is not satisfactory at present. Therefore, the study adopts the method of electrotransfection to integrate the target sequence into T cells. XbaI inserted into

In the overexpression vector pCF-PGK-MCS (pCF-empty vector) of the integrase system, an anti-human MSLN and anti-human CEA dual CAR expression plasmid (pCF-dCAR plasmid) is formed, using this plasmid and the integrase plasmid

T cells are co-transfected by electroporation, and optimized culture conditions are used to make T cells express dual chimeric antigen receptors, thereby endowing T cells with higher specificity and safety.

Functional verification of the constructed dCAR-T cells, including in vitro and in vivo experiments. The obtained dCAR-T cells were co-cultured with AsPC-1 overexpressing MSLN and CEA double-positive tumor cells in vitro. By detecting the activation and tumoricidal activities of dCAR-T cells, as well as the tumoricidal properties of dCAR-T cells in vivo, we explored the functional activity of such dCAR-T cells. In order to verify its specificity and safety, another negative control cell line was designed, that is, MSLN and CEA did not express tumor cells to confirm the specific killing effect of the constructed dCAR-T cells on tumor cells. It was finally confirmed that the constructed dCAR-T cells achieved their own activation and killing functions by targeting two TAAs on the surface of tumor cells. The design of this CAR structure can greatly improve the targeting specificity of T cells. Therefore, the safe application of CAR-T cells is finally realized, the damage to normal tissues is reduced, and the currently facing non-tumor toxicity is effectively solved. Therefore, the dual chimeric antigen receptor T cells of the present invention have good application prospects in the immunotherapy of tumors.

Description of drawings

Figure 1 is a schematic diagram of the mechanism of action of the novel dual-receptor dCAR-T cells;

Figure 2 is a peak map of partial sequencing results of pCF-CARs overexpression plasmids;

Figure 3 is a schematic diagram of the gene structure of four CAR-T cells;

Figure 4 is a schematic diagram of four constructed CAR-T cells;

Figure 5 is a diagram of the culture state of CD4 ⁺ T cells after magnetic bead sorting;

Figure 6 is a diagram showing the culture state of CD8 ⁺ T cells after magnetic bead sorting;

Fig. 7 is a diagram showing the detection status of CD4 ⁺ T cells labeled by flow cytometry;

Figure 8 is a diagram showing the detection status of CD8 ⁺ T cells labeled by flow cytometry;

Fig. 9 is a screening diagram for the construction of CARs-T cells by electroporation;

Figure 10 is a flow cytometric identification of the expression levels of CEA and MSLN in wild-type AsPC-1 cells;

Figure 11 shows the expression levels of CEA and MSLN in wild-type PANC-1 cells identified by flow cytometry;

Figure 12 is a diagram showing the screening status of the antibiotic puromycin in AsPC-1 cells;

Figure 13 is a state diagram of the screening of the antibiotic puromycin in PANC-1 cells;

Figure 14 is the expression level of RFP in lentivirus-transfected recombinant tumor cells;

Figure 15 is a graph showing the detection of CD69 levels activated by CARs-T cells in vitro;

Figure 16 is a graph showing the detection of CD25 levels activated by CARs-T cells in vitro;

Figure 17 is a graph showing the detection of the cytotoxic release factor IL-2 level in CARs-T cells in vitro;

Figure 18 is a graph showing the detection of the cytotoxic release factor IFN-γ level in CARs-T cells in vitro;

Fig. 19 is a graph of in vitro cytotoxic LDH detection of CARs-T cells;

Figure 20 is a graph of in vitro cytotoxic LDH detection of CARs-T cells;

Figure 21 is a graph of in vitro cytotoxic LDH detection of CARs-T cells;

Figure 22 is a schematic diagram of the detection of cytokine IL-2 levels in nude mice;

Figure 23 is a schematic diagram of the detection of cytokine IFNγ levels in nude mice;

Figure 24 is a schematic diagram of tumor volume in nude mice over time;

Figure 25 is a schematic diagram of the detection of cytokine IL-2 levels in nude mice;

Figure 26 is a schematic diagram of the detection of cytokine IFNγ levels in nude mice;

Figure 27 is a schematic diagram of the detection of cytokine IL-6 levels in nude mice;

Figure 28 is a schematic diagram of the detection of cytokine TNFα levels in nude mice;

Figure 29 is a graph showing the expression levels of various solid tumor CEA antigens;

Figure 30 is a graph showing the expression levels of various solid tumor MSLN antigens;

FIG. 31 is a graph of in vivo tumor volume detection of various solid tumors.

Detailed ways

The present invention will be further described below with reference to specific embodiments.

Example 1

Determination of the full gene sequence of the dual CAR

1.1 Obtain the gene sequences of hMSLNscFv and hCEAscFv, and analyze and optimize their gene sequences to ensure that the encoded amino acid sequence is more suitable for high-efficiency expression in human T lymphocytes. For the gene sequence information of hMSLNscFv, please refer to SEQUENCE LISTING (SEQ ID NO. 3 in the sequence table); for the gene sequence information of hCEAscFv, please refer to SEQUENCE LISTING (SEQ ID NO. 5 in the sequence table).

1.2 Obtain the gene sequences of human CD8α signal peptide gene, human CD8α hinge region gene, human CD8 transmembrane region and intracellular region genes, and human CD3ζ and 4/1BB signal domains.

1.3 The above gene sequences are sequenced according to human CD8α signal peptide, hMSLNscFv, human CD8α hinge region, human CD8 transmembrane region and intracellular region, human 4/1BB costimulatory signal domain, IRES sequence, human CD8α signal peptide, hCEAscFv, human CD8α The hinge region, human CD8 transmembrane region and intracellular region, and the gene sequence of human CD3ζ intracellular region are connected, and Kozak sequence is introduced in the most upstream, and the complete dCAR gene sequence information is finally constructed: Signal peptide I-hMSLNscFv-CD8α-CD8TM- 4/1BB-IRES-Signal peptide II-hCEAscFv-CD8α-CD8TM-CD3ζ-P2A-GFP. The structure of dCAR-T cells and the mechanism by which they interact with target cells are shown in Figure 1.

Example 2

Construction of expression plasmids

位点特异性重组酶系统的过表达载体pFC-MCS，最终构建重组质粒pFC-dCAR，引入GFP荧光标签是为了便于对构建dCAR-T细胞进行检测和跟踪。2.1 The optimized complete double chimeric receptor gene sequence was fully synthesized, and cloned into the corresponding enzyme cleavage sites AflII and XbaI.

The overexpression vector pFC-MCS of the site-specific recombinase system finally constructs the recombinant plasmid pFC-dCAR, and the GFP fluorescent tag is introduced to facilitate the detection and tracking of the constructed dCAR-T cells.

2.2 Sequence the recombinant plasmid, and compare the sequencing result with the designed CAR gene sequence. The result confirms that the obtained synthetic sequence is correct and that the target gene fragment has been connected to the corresponding overexpression vector.

Part of the sequencing results are shown in Figure 2.

Example 3

Control group genetic design

In order to fully verify the efficacy and safety of the designed dual-receptor CAR-T cells, multiple control groups were designed in the experiment. Namely two negative control structures: CAR1, Signal peptide-hCEAscFv-CD8α-CD8TM-CD3ζ-IRES-GFP and CAR2, Signal peptide-hMSLNscFv-CD8α-CD8TM-4/1BB-IRES-GFP; one positive control structure: CAR3, Signal peptide-hCEAscFv-CD8α-CD8TM-4/1BB-CD3ζ-IRES-GFP. The sequence information of each element is shown in Example 1, the schematic diagrams of the gene sequence elements of the three control groups are shown in Figure 3, and the schematic diagrams of various types of CAR-T cells constructed are shown in Figure 4.

Example 4

Mass extraction of overexpression plasmids

The constructed recombinant plasmids pCF-dCAR, pCF-CAR1, pCF-CAR2 and pCF-CAR3 were transformed into DH5α competent E. coli to obtain recombinant strains, and then cultured in LB liquid medium containing ampicillin sodium (Amp ⁺ ) antibiotics , and extract recombinant plasmid and integrase plasmid (Omega endotoxin-free plasmid extraction kit) by alkaline lysis method for transfection. Specific implementation steps:

4.1 Plasmid transformation and expansion culture

1) Take out the competent DH5α Escherichia coli (100 μL) frozen at -80°C and thaw it on ice. Immediately add 5 μL of the plasmid to be transformed (concentration is about 40 ng/μL) before it is completely thawed, and mix gently. After homogenization, place on ice for 30min;

2) Accurately heat shock in a constant temperature water bath at 42°C for 90s, then quickly put it on ice for 5min;

3) Add 900 μL of LB liquid medium preheated to room temperature and without antibiotics, shake and activate for 60 min at 37° C. and 150 rpm to recover the bacteria and express the resistance gene;

4) Centrifuge the above-mentioned bacterial liquid under the condition of 4000rpm for 30s, remove the supernatant of about 900 μL, and evenly coat the remaining bacterial liquid on the LB plate (Amp, 50mg/L) after shaking up;

5) After sealing the coated plate (with a parafilm), stand for 15 minutes at 37°C (the bacterial liquid is completely absorbed), and finally invert at a constant temperature of 37°C for 16h;

6) Pick a single colony in the above plate and inoculate it into 1 mL of liquid LB medium (Amp ⁺ , 50 mg/L), 37 ° C, 200 rpm shaking culture for 12 h, and store at 4 ° C;

7) The recombinant bacteria obtained above were inoculated into 50 mL of fresh and preheated LB liquid medium (Amp ⁺ , 50 mg/L) according to the ratio of 1:50, incubated at a constant temperature overnight (37° C., 200 rpm), and stored at 4° C. for Prepare a large amount of plasmid extraction.

4.2 Mass extraction of plasmids

1) by the above-mentioned obtained bacterial liquid (centrifuged for multiple times), after 10000rpm centrifugation for 1min, try to remove as much as possible;

2) Slowly add 250 μL of solution I (the specified amount of RNase A has been added) to the cells obtained by centrifugation, and shake vigorously to fully dissolve the cells;

3) Slowly add 250 μL of solution II to the above lysed bacteria solution, shake the centrifuge tube up and down 6 times until the solution is clear;

4) Take 125 μL of pre-cooled solution N3 and add it to the above-mentioned centrifuge tube, invert it up and down 6 times (white flocculent precipitate appears) and then centrifuge at high speed for 10 minutes at 12000 rpm;

5) After centrifugation, carefully pipette the centrifugation supernatant into a new 1.5mL centrifuge tube (about 600μL);

6) Add an equal volume of ETR binding buffer (600 μL) to the supernatant, shake the centrifuge tube up and down 10 times, and incubate at room temperature for 5 min;

7) Slowly move 700 μL of the above solution to the adsorption column HiBind, then centrifuge for 1 min (8000 rpm) and discard the filtrate, repeat once, and pass all the above obtained solution through the column;

8) 500 μL of ETR cleaning solution was drawn into the above adsorption column, and the filtrate was discarded after centrifugation for 1 min (8000 rpm);

9) Add 500 μL of EHB buffer to the adsorption column, centrifuge for 1 min (8000 rpm) and discard the filtrate;

10) Pipette 700 μL of DNA washing solution into the adsorption column (with absolute ethanol added), centrifuge for 1 min (8000 rpm) and discard the filtrate. This step is repeated once, and the plasmid is washed again;

11) Put the above-mentioned adsorption column into the recovery tube, centrifuge at the maximum speed for 2min (13000rpm), and remove the residual solution to the greatest extent;

12) Reposition the adsorption column in a new centrifuge tube, slowly add 50 μL of ddH ₂ O from the center of the column, dissolve for 1 min at room temperature, and then centrifuge at high speed for 1 min (13000 rpm), and store the resulting product at -20°C for long-term storage.

The DNA concentrations of the two plasmids obtained were determined by the nucleic acid quantifier NanoDrop2000. The results are shown in Table 1.

Table 1 Extracted plasmid DNA concentration

Example 5

Isolation of Human Peripheral Blood Mononuclear Cells

In this example, peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cell, PBMC) are mainly isolated from peripheral blood of healthy blood donors, and Ficoll reagent is used for density gradient centrifugation. Specific implementation steps:

1) Use an anticoagulation tube (containing anticoagulant) to collect 4 mL of peripheral blood from fresh healthy blood donors, shake well for use;

2) adding an equal volume of phosphate buffered saline PBS to the above-mentioned blood to dilute the blood;

3) Add 2 mL of Ficoll reagent for lymphocyte separation solution respectively;

4) Use a pipette to suck the diluted blood, add it slowly along the tube wall, place the diluted blood on the top of the stratified liquid, keep the interface between the two clear, and add 4 mL of diluted blood to each centrifuge tube;

5) Centrifuge at 2000rpm for 20min;

6) After centrifugation, suck the mononuclear cell layer and transfer it into a new centrifuge tube;

7) Slowly add 5 mL of PBS buffer, and after mixing, centrifuge at 1500 rpm for 10 min, aspirate and discard the supernatant, and repeat this step;

8) Resuspend the cells with 2 mL of RPMI1640 medium containing 10% fetal bovine serum;

9) Using an automatic cell counter (Invitrogen) to count and determine the viability of the isolated cells;

10) The remaining cell suspension is stored in liquid nitrogen to prepare for the subsequent magnetic bead sorting.

Counting results showed that the concentration of PBMC obtained was 1.49×10 ⁷ /mL; the number of viable cells was about 96.8%, indicating that the extracted cells had high activity and could meet the needs of magnetic bead sorting.

Example 6

Sorting of human T lymphocyte subsets CD4 and CD8

In this example, the Milteni magnetic bead separation method was used to further magnetic bead separation of the obtained PBMC to obtain two T cell subtypes, CD4 and CD8, which provided preparations for further research on the active function of dual chimeric receptor T cells. The preparation method of the sorting buffer is as follows: prepare 10% bovine serum albumin (BSA) with PBS (pH7.2) buffer and prepare 0.5mol/L EDTA with deionized water Disodium salt (EDTA) stock solution; take 0.4 mL of EDTA stock solution and 5 mL of BSA stock solution in a 100 mL graduated cylinder, and finally add PBS (pH 7.2) buffer to 100 mL; filter with 0.22 μm filter membrane under sterile conditions, Store at 4°C for later use.

The specific implementation steps of magnetic bead sorting:

1) The isolated PBMC cells were recovered, the cell density was 5×10 ⁵ /mL, the medium was RPMI1640 medium containing 10% fetal bovine serum, and the growth factor human interleukin-2 (Interleukin-2, IL-2) was added. Incubate for 48h at 37°C, 5% _CO2 ;

2) Accurately count the cells after the culture, take the cell culture medium with a total cell number of 1×10 ⁷ and collect the cells under the condition of 300×g, 10min, and finally fully resuspend the cells with the sorting buffer;

3) Add CD4 or CD8 magnetic microbeads for labeling, mix well and let stand for 15min;

4) Add 1 mL of sorting buffer after magnetic labeling, and centrifuge at 300 × g for 10 min after mixing, completely discard the supernatant, and then resuspend the cells;

5) Select the MS sorting column, place it in the sorting device (magnetic field), and add 500 μL of sorting buffer to rinse the column before loading;

6) Add all the above resuspended cell suspension to the rinsed MS sorting column, and rinse the sorting column with 500 μL of sorting buffer;

7) Detect the two T cell subsets of CD4 and CD8 obtained by sorting.

The sorted CD4 ⁺ T cells were detected with PE-labeled human anti-CD4 flow antibody, and CD8 ⁺ T cells were detected with APC-labeled human anti-CD8 flow antibody.

FIG. 5 is a diagram of the culture state of CD4 ⁺ T cells after magnetic bead sorting; FIG. 6 is a diagram of the culture state of CD8 ⁺ T cells after magnetic bead sorting, and the cells are uniform in size after sorting. Figures 7 and 8 are the morphological detection diagrams of single cells labeled with CD4 ⁺ T and CD8 ⁺ T antibodies by flow cytometry, respectively. The channel Ch01 is the brightfield channel of the flow cytometer, the channel Ch06 is the side scatter channel, the channel Ch03 is the PE fluorescence channel, and the channel Ch11 is the APC fluorescence channel. The results showed that the antigens mainly labeled by the flow antibody were located in the cell membrane, and the size of CD4 ⁺ T and CD8 ⁺ T cells was about 8-10 μm from the figure.

Example 7

Preparation of Chimeric Antigen Receptor T Cells by Transfection

This example describes in detail the construction methods of various chimeric antigen receptor T cells in the study. The PhiC31 site-specific integrase system was used to insert the dual chimeric antigen receptor dCAR gene into the genome of mammalian T lymphocytes. Thus, CARs-T cells (including CD4 ⁺ T and CD8 ⁺ T cells) are prepared. The intracellular recombination system mediated by PhiC31 integrase has the advantage of unidirectional integration. Compared with other recombination systems, it can safely and efficiently integrate exogenous large fragments of genes into the target genome. It can be used in a variety of higher plants. It has the characteristics of long-term and high-efficiency expression of exogenous genes and functions in animal cells. Using the electroporation apparatus and electroporation kit of Lonza company, the specific implementation steps of the electroporation test: 1) respectively recover the CD4 ⁺ T and CD8 ⁺ T cells obtained by magnetic bead sorting, adjust the cell density to 5×10 ⁵ /mL, and grow the T cells. The medium of 10% fetal calf serum-containing RPMI1640 medium was incubated at 37°C, 5% CO ₂ for 24h; 2) CD3/CD28 magnetic beads were added for activation, and the medium was incubated at 37°C, 5% CO ₂ for 24h; 3) The Discard the supernatant by centrifugation, and resuspend the cells with 100 μL of 4D-Nucleofector electroporation solution; 3) Discard the supernatant by centrifugation, and resuspend the cells with 100 μL of 4D-Nucleofector electroporation solution; 4) Add 2 μg of electroporation plasmid mixture , select F1-115 program for electroporation; 5) Transfer the mixed cell suspension to a 12-well plate to continue culturing, after 6 hours of culture, replace the fresh medium to continue culturing; 6) After 42 hours, finally observe the transfected cells through a fluorescence microscope cell state.

The transfection results are shown in Figure 9. The results show that the cells have been successfully transfected, and the transfection efficiency is about 35%, which meets the needs of later experiments.

Example 8

Construction of target cells

Pancreatic cancer is a malignant tumor of the digestive tract with a high degree of malignancy, which is difficult to diagnose and treat. Although medical technology has greatly improved in the past 20 years, there are still many problems in the diagnosis and treatment of pancreatic cancer. The rate is not high, the operative mortality rate is still high, and the cure rate is very low. In recent years, with the continuous development of bioinformatics and tumor tissue chips, it is found that most pancreatic cancer cells highly express two tumor antigens, MSLN and CEA, and the dual-receptor CAR-T cells designed by the present invention can specifically kill Tumor cells that overexpress both antigens. In theory, targeting two tumor-associated antigens is much safer than CAR-T cell therapy designed with only one antigen, and can effectively reduce targeted non-tumor toxicity. In the present invention, a pancreatic cancer cell AsPC-1 that overexpresses two tumor antigens is selected, and a negative cell PANC-1 (which does not express these two antigens) is selected at the same time.

8.1 Expression of MSLN and CEA antigens on the surface of wild-type tumor cells

The selected pancreatic cancer cell AsPC-1 was purchased from ATCC (CRL-1682), and the medium was 1640 (containing 10% FBS), and the selected pancreatic cancer cell PANC-1 was purchased from ATCC (CRL-1469), and the medium was DMEM ( with 10% FBS). The expression levels of MSLN and CEA on the surface of the above two wild-type pancreatic cancer tumor cells were detected by flow antibody PE-MSLN and FITC-CEA, and the corresponding isotype control antibodies PE-IgG2aκ and FITC-IgG1κ were selected at the same time. The specific implementation steps are as follows:

1. The above-mentioned cells cultured overnight were digested into single cells by trypsinization, and 1×10 ⁶ cells were taken into a flow tube after counting.

2. Add 2 mL of PBS, centrifuge at 800×g for 5 min, discard the supernatant, repeat once, and finally resuspend the cells with 100 μL of PBS.

3. Two tubes of each type of cells, one tube was added with flow direct-labeled antibodies (PE-MSLN and FITC-CEA), the other tube was added with corresponding isotype control antibodies (PE-IgG2aκ and FITC-IgG1κ), and incubated at 4°C for 1 h.

4. After incubation, add PBS, centrifuge, and discard the supernatant.

5. On an ice bath, add 300 μL of PBS to the tube to resuspend the cells, filter the cell suspension with a filter, and perform flow cytometry analysis.

See Figure 10 and Figure 11 for the results of flow cytometry (the abscissas in the figure all represent the high expression of CEA cells, and the ordinates all represent the high expression of MSLN cells), the results show: AsPC-1 pancreatic cancer cells have high expression levels Both tumor antigens, CEA and MSLN, were expressed; in contrast, PANC-1 pancreatic cancer cells did not express either antigen.

8.2 Construction and identification of recombinant pancreatic cancer cells

In this experiment, in order to facilitate the study of the interaction between effector target cells in vitro and in vivo, we labeled the effector target cells with fluorescent proteins of different colors. The red fluorescent protein RFP was overexpressed in target cells AsPC-1 and PANC-1 by lentiviral transfection. First, the killing curves of puromycin for these two tumor cells were obtained, and the specific implementation steps were as follows:

1) Resuscitate target cells AsPC-1 and PANC-1, adjust the cell state to logarithmic growth phase, inoculate 5×10 ⁴ tumor cells per well, inoculate 7 wells in total, and add 500 μL of complete culture to each well.

2) After overnight culture, take out the cell well plate, leave one well without adding puromycin, and add different concentrations of puromycin to the other wells respectively.

3) The plate was placed in a 37°C, 5% CO2 incubator to continue culturing, and the lowest concentration that could completely kill all cells on the fifth day was taken as the optimum concentration of puromycin for the selection of stable cell lines.

After the determination of the killing curve, the final screening concentration of puromycin for AsPC-1 and PANC-1 tumor cells was 1 μg/mL. See Figures 12 and 13 for cell state diagrams of the Puromycin antibiotic screening curve (d in the figure refers to the number of days).

Then, the packaged lentiviral particle Lenti-RFP is used for transfection, and the specific implementation steps are as follows:

1) Seed into a 6-well plate at a density of 3×10 ⁵ cells/well, each tumor cell was seeded into 2 wells, and 2 mL of the corresponding complete medium (RPMI1640, 10% FBS) was added to each well. The 6-well plate was placed in a 37°C, 5% CO2 incubator for overnight incubation.

2) Take out a tube of RFP-overexpressing lentiviral particles and place it in a 37°C water bath to thaw quickly.

3) Add polybrene and thawed lentiviral particles and mix gently.

4) Place the 6-well plate in a horizontal centrifuge and centrifuge at 800×g for 1 h. After being placed in the incubator for 24 hours, the medium containing the virus supernatant was removed from the plate, and fresh medium without antibiotics was added to continue the culture for 48 hours.

5) Take out the well plate from the incubator, add a medium containing puromycin antibiotic, and the concentrations of puromycin and hygromycin used are the concentrations obtained by screening in Examples 3.4 and 3.5, respectively.

6) The cells in the control wells without virus infection were used as controls, and cultured continuously for 5 days until the cells in the control group died completely, and the cells survived in the virus-infected wells were successfully constructed stable cell lines.

7) Western blot was used to detect the expression of fluorescent protein RFP in stable cell lines.

The detection results are shown in Figure 14. The results show that both AsPC-1 and PANC-1 cells after lentivirus transfection overexpress the fluorescent protein RFP.

Example 9

dCAR-T cell activation assay in vitro

This example details the research on the in vitro activation test of DAC T cells. The effector cells were selected as CARs-T cells engineered with CD4 ⁺ T cells. The detected indicators were the expression levels of T cell surface activation markers CD69 and CD25 and the levels of cytokines IL-2 and IFNγ produced during the activation process. In this experiment, the selected target cells are AsPC-1 tumor cells transformed in the laboratory, and the transformed AsPC-1 cells were selected mainly because they can overexpress MSLN and CEA tumor-related antigens. At the same time, PANC-1 tumor cells were selected as negative control (without MSLN and CEA). A total of 6 groups are designed (three duplicate holes are designed for each group), and the specific plans are:

Group 1: The effector cells are first-generation CAR-T cells (CζCAR-T: chimeric receptors formed by fusion of immunoglobulin scFv and CD3ζ intracellular domains, engineered T cells), and the target cells are engineered AsPC- 1 tumor cell (AsPC-1-RFP);

Group 2: The effector cells are MSLN-specific CAR-T cells with a co-stimulatory signal of 4/1BB (MBB CAR-T::CAR has only one stimulatory molecule 4/1BB in structure), and the target cells are engineered AsPC -1 tumor cells (AsPC-1-RFP);

Group 3: The effector cells are dual chimeric antigen receptor T cells (CζMBB CAR-T) specific for MSLN and CEA, and the target cells are engineered AsPC-1 tumor cells (AsPC-1-RFP);

Group 4: The effector cells are the traditional second-generation CAR-T cells specific to CEA (CζBB CAR-T: the second-generation CAR, which is a chimeric receptor formed by the fusion of immunoglobulin scFv and CD3ζ intracellular domain, and adding a Costimulatory molecule 4/1BB), the target cells are engineered AsPC-1 tumor cells (AsPC-1-RFP);

Group 5: The effector cells are T cells (No CAR), and the target cells are engineered AsPC-1 tumor cells (AsPC-1-RFP);

Group 6: The effector cells are CζMBB CAR-T cells, and the target cells are engineered PANC-1 tumor cells (PANC-1-RFP);

Specific implementation steps:

1) T cells and tumor cells were added to the U-shaped 96-well plate for plating, and the co-culture medium was selected as RPMI1640 medium;

2) After adding methyl gibberellic acid acetomethyl for overnight culture (8h), centrifuge at 2000×g for 5min, and collect cells and supernatant respectively;

3) The collected cells were directly labeled with flow-through antibodies CD69 and CD25, and the number of activated T cells was detected by flow-through. At the same time, the contents of IL-2 and IFNγ in the collected supernatant were detected by ELISA.

The detection results of CAR-T cell surface activator CD69 are shown in Figure 15, and the detection results of CD25 are shown in Figure 16. Data processing is based on the results of CAR-T cells in the positive control group. The values in other experimental groups are the ratios of the measured CD69 or CD25 numbers in the group to the positive experimental group. The closer the ratio is to 1.0, the higher the activation level of T cells in the experimental group. The closer it is to the baseline group, the closer the ratio is to 0, indicating that the T cell activation level of the experimental group is significantly different from the baseline group. The results show that there are obvious differences between the dual-receptor dCAR-T experimental group (CζMBB CAR-T) and other experimental groups, that is, the constructed dCAR-T cells can be fully activated only when they recognize both MSLN and CEA antigens at the same time. The double-negative tumor cell experimental group and other single-receptor CAR-T experimental groups were basically unable to activate. It is worth noting that in the CEA-specific single-receptor CAR-T experimental group (CζBBCAR-T), the results showed a certain number of activated CAR-T cells, which may be due to the fact that when only CEA antigen was recognized, CAR-T cells The T signaling pathway is the same as the first-generation CAR-T, with transient activation and killing functions.

The results of the analysis of IL-2 in the cell supernatant are shown in Figure 17. The level of IL-2 produced in the double-receptor dCAR-T cell experimental group was 38.64 pg/mL, which was much higher than the levels in other experimental groups, indicating that only the dCAR-T cells in the double-positive antigen tumor cell experimental group were completely activation. At the same time, a certain level of IL-2 was also produced in the CEA single-receptor CAR-T cell experimental group. This is because, after recognizing the antigen CEA, the single-receptor CAR-T cells specific to CEA can also be temporarily activated. But this activation is low and persistent.

The results of the analysis of IFNγ in the cell supernatant are shown in FIG. 18 . The double-receptor dCAR-T cell (CζMBB CAR-T) experimental group produced IFNγ at a level of 21.31 pg/mL, which was significantly higher than that of other experimental groups, indicating that only in the presence of double-positive tumor cells was dCAR-T cells completely ground activated. At the same time, a certain level of IFNγ was also produced in the CEA single-receptor CAR-T cell (CζBB CAR-T) experimental group, about 9.02 pg/mL. This test result is consistent with IL-2, that is, the constructed dCAR-T cells can be permanently activated only when they are simultaneously recognized by both MSLN and CEA antigens.

Example 10

In vitro tumor killing test of dCAR-T cells

This example verifies the cytotoxic activity of the constructed CARs-T cells against tumor cells in vitro, and the selected effector cells are CD8 ⁺ T cells. A total of six experimental groups were designed, with three replicate wells in each group, and the experimental design of each group was the same as that in Example 9. The cytotoxicity of CARs-T cells and the levels of cytokines released during the process were mainly examined.

10.1 Basic Principles of LDH

还原成橙色的甲臜产物，

甲臜产物的吸光度与LDH的水平呈正比，利用此原理可以测定靶细胞的死亡或受损的数量，从而最终测定出CAR-T细胞的细胞毒性。具体实验过程中要测定以下几项指标，包括效应细胞自发LDH释放，校正效应细胞自发释放出来的LDH；靶细胞自发LDH释放，校正靶细胞自发释放出来的LDH；靶细胞最大LDH释放，计算时需要该对照以确定100％的LDH释放；体积校正对照，校正由于加入裂解液(10X)引起的体积变化；培养基背景对照，校正由培养基中血清产生的LDH活性以及酚红造成的背景吸收。最终通过下面公式来计算靶细胞的死亡率(CARs-T细胞的细胞毒性)：The method used in this test to detect the in vitro antitumor activity of CARs-T cells is the LDH detection method. Lactate dehydrogenase (LDH) is a common oxidoreductase widely present in the cytoplasm. When the cell membrane is damaged, LDH will be released into the culture medium. mortality rate. The principle of this method is that the released LDH catalyzes the formation of acetone and NADH from lactic acid, and NADH can convert the water-soluble tetrazolium salt through the electron carrier.

reduced to an orange formazan product,

The absorbance of the formazan product is proportional to the level of LDH. Using this principle, the number of dead or damaged target cells can be determined, and finally the cytotoxicity of CAR-T cells can be determined. In the specific experimental process, the following indicators should be measured, including the spontaneous release of LDH from effector cells, which corrects the LDH spontaneously released by effector cells; the spontaneous release of LDH from target cells, which corrects the LDH spontaneously released by target cells; the maximum LDH release from target cells, which is calculated when This control is required to determine 100% LDH release; volume correction control, which corrects for volume changes due to the addition of lysate (10X); medium background control, which corrects for LDH activity due to serum in the medium and background absorption due to phenol red . The target cell death rate (CARs-T cell cytotoxicity) was finally calculated by the following formula:

LDH detection

Specific implementation steps:

1) In a U-shaped 96-well plate, add effector cells and target cells, select the co-culture medium as RPMI1640 (10% fetal bovine serum), add human IL-2, and culture in a 37°C CO ₂ incubator for 24h ;

2) After adding Lysis Buffer to the wells with high control, incubate in a 37°C CO ₂ incubator for 30min.

3) Centrifuge the 96-well plate at 250×g for 2 min after culturing, and carefully aspirate 100 μL of the supernatant from each well into a new 96-well plate.

4) After adding 100 μL of Working Solution to each well, incubate for 30 min in the dark at room temperature.

5) Add 50 μL of Stop Solution to each well.

6) Finally, measure the absorbance at 490 nm with a microplate reader.

The results of the in vitro tumoricidal activity of CARs-T cells detected by the LDH method are shown in Figure 19. The results show that the tumor cell survival rate of the dual-receptor dCAR-T cell (CζMBB CAR-T) experimental group was only 14.67%. far lower than other experimental groups. In the double-negative tumor cell experimental group and the MSLN-specific single-receptor CAR-T cells (MBB CAR-T: MSLN-specific CAR-T cells with a costimulatory signal of 4/1BB), the tumor cell survival rate was Both were approximately 100%, similar to the cytotoxicity of wild-type T cells, that is, CAR-T did not exert cytotoxicity in both cases. On the contrary, the target cell survival rate in the experimental group of CEA-specific single-receptor CAR-T cells (CEA-CAR-T: traditional second-generation CAR-T cells specific to CEA) was about 75.33%, at which time CARs -T cells exert a limited degree of cytotoxicity. The experimental results show that the constructed dual-receptor dCAR-T cells can exert a greater degree of cytotoxicity only under the premise of two tumor antigens, MSLN and CEA. Under the condition of only one antigen, the killing activity of dCAR-T cells against tumor cells is very low or even non-existent. It shows that the constructed dCAR-T cells have obvious cytotoxic specificity, which will greatly improve the safety of CAR-T cells.

In addition, we also constructed single-receptor CAR-T cells targeting non-tumor-associated antigens and dual-receptor CAR-T cells targeting non-tumor-associated antigens, which are traditional second-generation CAR-T cells specific for MSLN ( MSLN-CAR-T), traditional second-generation CAR-T cells specific to CEA (CEA-CAR-T), traditional second-generation CAR-T cells specific to G2D (G2D-CAR-T), first chimeric antigen Dual chimeric receptor CAR-T cells with receptor targeting MSLN and second chimeric antigen receptor targeting G2D (dCAR1: dual chimeric antigen receptor T cells specific for MSLN and G2D), and the first chimeric Dual chimeric receptor CAR-T cells with antigen receptor targeting G2D and a second antigen receptor targeting CEA (dCAR2: dual chimeric antigen receptor T cells specific for CEA and G2D). See Figure 20-21 for details. The experimental results show that for tumor cells overexpressing MSLN and CEA but not expressing G2D, CAR1 and CAR2 dual receptor CAR-T cells have no killing activity on target cells, indicating that our constructed dual receptor CAR-T cells have no killing activity against target cells. Receptor CAR-T cells have good activity and safety against tumors overexpressing MSLN and CEA.

10.3 Cytokine levels released by CAR-T cells in vitro

Because effector T cells release a large amount of cytokines, such as IL-2 and IFN-γ, while killing tumors. The cytotoxic capacity of effector cells can also be calculated by measuring the release levels of IL-2 and IFN-γ in cell culture supernatants. Specific implementation steps:

1) Experimental group design: In a U-shaped 96-well plate, add effector cells and target cells, select RPMI1640 (10% fetal bovine serum) as the co-culture medium, add human IL-2, and set up three duplicate wells;

2) Incubate for 48h at 37°C, 5% CO ₂ ;

4) After the culture, put the 96-well plate on a horizontal centrifuge at 2000 × g for 10 min;

5) The supernatant in each well was slowly taken out, and the levels of cytokines IL-2 and IFN-γ were detected by ELISA.

See Figures 22 and 23 for the detection results of cytokine release levels. The results of IL-2 cytokine detection showed that the double-receptor dCAR-T cell experimental group (CζMBB CAR-T) had the highest IL-2 release concentration, which was far greater than other experimental groups, which indicated that in the double-positive tumor cell experimental group, dCAR -T cells exerted a greater degree of tumoricidal function. Similarly, in the detection level of IFN-γ cytokines, it can be seen that the double-receptor dCAR-T cell experimental group (CζMBB CAR-T) released the largest amount of IFN-γ. The test results are consistent.

Example 11

In vivo tumor killing test of dCAR-T cells

First, 6-8 week old female nude mice BALB/c were selected and randomly divided into six groups. Groups 1 to 5 were the experimental groups with syngeneic tumor cells AsPC-1, and group 6 was the negative control group of tumor cells. PANC-1. Logarithmic growth of various tumor cells was collected, and 5×10 ⁶ tumor cells were inoculated into nude mice by subcutaneous transplantation. After 7 days of inoculation, corresponding effector cells (1×10 ⁷ ) were sequentially inoculated into the tail vein. Specific inoculation scheme: group 1 nude mice were inoculated with engineered AsPC-1 tumor cells and unmodified T cells; group 2 nude mice were inoculated with modified AsPC-1 tumor cells and CζCAR-T cells; group 3 nude mice were inoculated with modified AsPC-1 tumor cells AsPC-1 tumor cells and MBB CAR-T cells; group four nude mice were inoculated with engineered AsPC-1 tumor cells and CζMBBCAR-T cells; group five nude mice were inoculated with engineered AsPC-1 tumor cells and CζBB CAR-T cells Cells; Group three nude mice were inoculated with engineered PANC-1 tumor cells and CζMBB CAR-T cells. The day when the tumor cells were inoculated was recorded as the 0th day, and the 7th day was injected with 1×10 ⁷ CARs-T cells. The effector cell CζBB CAR-T injected into nude mice in group five is a second-generation CAR-T cell specific for CEA, which serves as a positive control for detecting dCAR-T cytotoxicity. Detection protocol: The tumor volume of each group of nude mice was measured every two days from the 7th day; 50 μL of nude mouse blood was collected on the 7th day, 14th day, 21st day and 28th day, respectively, for serum detection. Cytokines IL-2, IFN-γ, IL-6 and TNFα in .

The trend lines of tumor volume in nude mice of all experimental groups over time are shown in Figure 24. It can be seen from the figure that the tumor volume of double-positive tumor cells AsPC-1 can be effectively suppressed only in the presence of dual-receptor dCAR-T cells. Under these conditions, significant cytotoxicity can be exerted. In addition, we found that the tumor volume of the syngeneic tumor cell AsPC-1 was also suppressed to a certain extent in the experimental group of single receptor CAR-T cells specific for CEA, indicating that the CζCAR-T cells in the syngeneic tumor cell AsPC-1 It also has a small amount of cytotoxicity in the presence of . The analysis showed that the structure of CζCAR-T cells is the structure of a first-generation CAR-T cell, therefore, it can exert transient cytotoxicity when it recognizes the CEA antigen. The growth trend of tumor volume in other groups before and after CARs-T cell injection did not change.

The detection of cytokine release levels is shown in Figures 25-28. CARs-T cells will significantly release cytokines IL-2, IFN-γ, IL-6 and TNFα in the process of killing tumors. The antitumor activity of CARs-T cells in vivo was characterized by detecting the release levels of these cytokines in nude mice. The changes in the release levels of the four cytokines in vivo with time are basically the same, and the results show that the constructed dual-receptor dCAR-T cells can kill the syngeneic tumor cell AsPC-1 and release significant levels of cytokines at the same time. The release levels of cytokines were consistent with those of the positive control group (CζBB CAR-T cells). The curve of cytokines changing with time was detected in the positive control group, indicating that various cytokines released by dCAR-T cells in the process of killing tumor cells gradually increased, especially IL-6 increased the most, which is consistent with the clinical application of CAR. -Consistent with the phenomenon of cytokine release syndrome that occurs with T cell therapy. At the same time, we found that in the dual-receptor dCAR-T cell treatment group, the release trend of the four cytokines was very similar to the positive CAR-T cell treatment group, but the level of release would be significantly lower than that in the positive control group, which can be well resolved Toxic side effects of cytokine release syndrome faced during CAR-T cell therapy. In addition, we found that a small amount of cytokines were released in the CζCAR-T cell treatment group, which was consistent with the corresponding trend of tumor volume. Has mild and transient cytotoxicity. No significant cytotoxicity was found for CAR-T cells in other single-receptor experimental groups, especially for PANC-1 cells in the tumor cell negative control group, neither significant regression of tumor volume nor significant cytokine release was found. In the double-positive antigen experimental group, the levels of IL-2 and IFN-γ were the highest and continuously increasing, but the increasing trend of these cytokines gradually decreased in the later period, which may be due to the activity of CARs-T cells infused into nude mice. Gradually decrease the reason. In summary, the constructed dCAR-T cells can be fully activated in vivo and have persistent cytotoxicity only when they recognize both MSLN and CEA antigens in vivo, thereby exerting more efficient anti-tumor activity. In other experiments Neither group could achieve long-term inhibition of tumor growth. The in vivo test confirmed that the design of this dual-receptor CAR structure would endow T cells with strong tumor specificity, which could solve the toxicity of targeting non-tumor, and significantly improve the safety of its application.

Example 12

Application of dual chimeric antigen receptor T cells in solid tumors

According to the above-mentioned in vitro and in vivo test results of dCAR-T cells, in order to further verify the therapeutic effect of this dual-receptor CAR-T cell in solid tumors and the applicable tumor types, the selected solid tumor cells were verified to have high expression of carcinoembryonic antigen. and mesothelin. With reference to relevant literature and corresponding clinical trial data, the indications for this dual-receptor CAR-T cell therapy are mainly pancreatic cancer AsPC-1, lung cancer A549, gastric cancer MG803, colon cancer SW480, breast cancer MCF7, liver cancer SMCC7721, melanoma A375, cervical cancer Hela, kidney cancer A498, prostate cancer PC-3, tongue squamous cell carcinoma Cal-27, bladder cancer BT-B, ovarian cancer OV90 and nasopharyngeal carcinoma 5-8F cells. First, the levels of carcinoembryonic antigen and mesothelin expressed on the surface of these solid tumor cells were respectively detected by flow-through antibodies. For a summary of the detection results of these solid tumors, please refer to Figures 29-31. The test results showed that the selected tumor cells highly expressed carcinoembryonic antigen and mesothelin, which were in line with the tumor antigens required by the designed dual-receptor CAR-T cells. The above test results show that the dual-receptor dCAR-T cells in the present invention are suitable for a variety of solid tumor types.

The cytotoxicity of dual-receptor dCAR-T cells on the selected solid tumors was tested by in vivo experiments. Female nude mice 6-8 weeks old BALB/c were selected and randomly divided into nine groups, respectively inoculated with the above nine tumor cells (1×10 ⁶ ) subcutaneously. 2×10 ⁶ dCAR-T cells were injected intravenously. Detection scheme: The tumor volume of each group of nude mice was measured on the 21st day. The tumor volume changes of various tumor cells are shown in FIG. 29 . Through the detection of tumor volume in nude mice, the results showed that after the infusion of dCAR-T cells in nude mice, the tumor volume in the above selected solid tumors showed a significant decrease trend, which indicated that dCAR-T cells were effective for the above selected solid tumors. have high cytotoxicity. Therefore, the dual-receptor dCAR-T cells designed in the present invention have better cytotoxicity to solid tumors that highly express carcinoembryonic antigen CEA and mesothelin MSLN antigen, and the level of released cytokines is reduced. The designed dual-receptor dCAR-T cells are controlled by two tumor-associated antigens at the same time. Therefore, the CAR-T cells in this design mode can reduce the damage to normal tissues, thereby further improving the CAR-T cells. Application security.

sequence listing

<110> China Pharmaceutical University

<120> A dual chimeric antigen receptor, T cell and its construction method and application

<140> 201910469880X

<141> 2019-05-31

<150> 2018107081807

<151> 2018-07-02

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ctcactcagt ctccagcaat catgtctgca tctccagggg agaaggtcac catgacctgc 540

agtgccagct caagtgtaag ttacatgcac tggtaccagc agaagtcagg cacctccccc 600

aaaagatgga tttatgacac atccaaactg gcttctggag tcccaggtcg cttcagtggc 660

agtgggtctg gaaactctta ctctctcaca atcagcagcg tggaggctga agatgatgca 720

acttattact gccagcagtg gagtaagcac cctctcacgt acggtgctgg gacaaagttg 780

gaaatcaaaa gcagcaccac taccccagca ccgaggccac ccaccccggc tcctaccatc 840

gcctcccagc ctctgtccct gcgtccggag gcatgtagac ccgcagctgg tggggccgtg 900

catacccggg gtcttgactt cgcctgcgat atctacattt gggcccctct ggctggtact 960

tgcggggtcc tgctgctttc actcgtgatc actctttact gtaagcgcgg tcggaagaag 1020

ctgctgtaca tctttaagca acccttcatg aggcctgtgc agactactca agaggaggac 1080

ggctgttcat gccggttccc agaggaggag gaaggcggct gcgaactgta agaattctgc 1140

agatatccgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata 1200

aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt 1260

gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct 1320

cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 1380

ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga 1440

caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 1500

ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt 1560

attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg 1620

gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc 1680

gaaccacggg gacgtggttt tcctttgaaa aacacgatga taagcttgcc acaacccaca 1740

aggagacgac cttccgcggc cgcatggccc tccctgtcac cgccctgctg cttccgctgg 1800

ctcttctgct ccacgccgct cggcccctgc aggctagtgg tggtggtggt tctggtggtg 1860

gtggttctgg tggtggtggt tctgctagcc aagttaaact ggaacagtcc ggtgctgaag 1920

ttgtcaaacc aggtgcttcc gtgaagttgt cctgtaaagc gtctggtttt aacatcaagg 1980

attcgtatat gcattggttg agacaagggc caggacaaag attggaatgg attggctgga 2040

ttgatccaga gaatggtgat actgagtacg ctcctaaatt tcagggaaag gctactttta 2100

ctaccgacac ttccgctaat accgcatact tgggcttatc ttccttgaga ccagaggaca 2160

ctgccgtata ctactgcaac gaagggacac caactggtcc ttactatttc gactactggg 2220

gacaaggtac cttagttact gtctctagcg gtggcggagg ttcaggcggt ggagggtctg 2280

gaggtggcgg tagtgaaaat gtgctgaccc aatctccaag ctccatgtct gtttctgttg 2340

gcgatagagt aaccatcgct tgtagcgcat cctctagtgt cccatatatg cactggcttc 2400

aacagaagcc aggtaaaagc ccaaagttgt ggatttattt gacatccaac ttggcttctg 2460

gagtcccttc aaggttttct ggttccggct caggaaccga ttatagtttg actattagct 2520

cagtgcagcc agaggatgct gcaacctact attgccagca aaggtcctca tatccactga 2580

ctttcggggg tggaacgaag ttggaaatca aggctgcagc cggatccacc actaccccag 2640

caccgaggcc acccaccccg gctcctacca tcgcctccca gcctctgtcc ctgcgtccgg 2700

aggcatgtag acccgcagct ggtggggccg tgcatacccg gggtcttgac ttcgcctgcg 2760

atatctacat ttgggcccct ctggctggta cttgcggggt cctgctgctt tcactcgtga 2820

tcactcttta ctgtcgcgtg aaattcagcc gcagcgcaga tgctccagcc taccagcagg 2880

ggcagaacca gctctacaac gaactcaatc ttggtcggag agaggagtac gacgtgctgg 2940

acaagcggag aggacgggac ccagaaatgg gcgggaagcc gcgcagaaag aatccccaag 3000

agggcctgta caacgagctc caaaaggata agatggcaga agcctatagc gagattggta 3060

tgaaagggga acgcagaaga ggcaaaggcc acgacggact gtaccaggga ctcagcaccg 3120

ccaccaagga cacctatgac gctcttcaca tgcaggccct gccgcctcgg 3170

<210> 2

<211> 244

<212> PRT

<213> Artificial sequences

<400> 2

Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro

1 5 10 15

Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr

20 25 30

Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu

35 40 45

Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn Gln

50 55 60

Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr

65 70 75 80

Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr

85 90 95

Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Ser Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro

130 135 140

Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser

145 150 155 160

Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Gly

165 170 175

Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly

180 185 190

Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu

195 200 205

Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys Gln

210 215 220

Gln Trp Ser Lys His Pro Leu Thr Tyr Gly Ala Gly Thr Lys Leu Glu

225 230 235 240

Ile Lys Ser Ser

<210> 3

<211> 732

<212> DNA

<213> Artificial sequences

<400> 3

ggatcccagg tacaactgca gcagtctggg cctgagctgg agaagcctgg cgcttcagtg 60

aagatatcct gcaaggcttc tggttactca ttcactggct acaccatgaa ctgggtgaag 120

cagagccatg gaaagagcct tgagtggatt ggacttatta ctccttacaa tggtgcttct 180

agctacaacc agaagttcag gggcaaggcc acattaactg tagacaagtc atccagcaca 240

gcctacatgg acctcctcag tctgacatct gaagactctg cagtctattt ctgtgcaagg 300

ggggttacg acgggagggg ttttgactac tggggccaag ggaccacggt caccgtctcc 360

tcaggtggag gcggttcagg cggcggtggc tctagcggtg gtggatcgga catcgagctc 420

actcagtctc cagcaatcat gtctgcatct ccaggggaga aggtcaccat gacctgcagt 480

gccagctcaa gtgtaagtta catgcactgg taccagcaga agtcaggcac ctcccccaaa 540

agatggattt atgacacatc caaactggct tctggagtcc caggtcgctt cagtggcagt 600

gggtctggaa actcttactc tctcacaatc agcagcgtgg aggctgaaga tgatgcaact 660

tattactgcc agcagtggag taagcaccct ctcacgtacg gtgctgggac aaagttggaa 720

atcaaaagca gc 732

<210> 4

<211> 267

<212> PRT

<213> Artificial sequences

<400> 4

Leu Gln Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Ala Ser Gln Val Lys Leu Glu Gln Ser Gly Ala Glu Val

20 25 30

Val Lys Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Phe

35 40 45

Asn Ile Lys Asp Ser Tyr Met His Trp Leu Arg Gln Gly Pro Gly Gln

50 55 60

Arg Leu Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu

65 70 75 80

Tyr Ala Pro Lys Phe Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser

85 90 95

Ala Asn Thr Ala Tyr Leu Gly Leu Ser Ser Leu Arg Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asn Val Leu

145 150 155 160

Thr Gln Ser Pro Ser Ser Met Ser Val Ser Val Gly Asp Arg Val Thr

165 170 175

Ile Ala Cys Ser Ala Ser Ser Ser Val Pro Tyr Met His Trp Leu Gln

180 185 190

Gln Lys Pro Gly Lys Ser Pro Lys Leu Trp Ile Tyr Leu Thr Ser Asn

195 200 205

Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Asp Tyr Ser Leu Thr Ile Ser Ser Val Gln Pro Glu Asp Ala Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly Gly Gly

245 250 255

Thr Lys Leu Glu Ile Lys Ala Ala Ala Gly Ser

260 265

<210> 5

<211> 801

<212> DNA

<213> Artificial sequences

<400> 5

ctgcaggcta gtggtggtgg tggttctggt ggtggtggtt ctggtggtgg tggttctgct 60

agccaagtta aactggaaca gtccggtgct gaagttgtca aaccaggtgc ttccgtgaag 120

ttgtcctgta aagcgtctgg ttttaacatc aaggattcgt atatgcattg gttgagacaa 180

gggccaggac aaagattgga atggattggc tggattgatc cagagaatgg tgatactgag 240

tacgctccta aatttcaggg aaaggctact tttactaccg acacttccgc taataccgca 300

tacttgggct tatcttcctt gagaccagag gacactgccg tatactactg caacgaaggg 360

acaccaactg gtccttacta tttcgactac tggggacaag gtaccttagt tactgtctct 420

agcggtggcg gaggttcagg cggtggaggg tctggaggtg gcggtagtga aaatgtgctg 480

acccaatctc caagctccat gtctgtttct gttggcgata gagtaaccat cgcttgtagc 540

gcatcctcta gtgtcccata tatgcactgg cttcaacaga agccaggtaa aagcccaaag 600

ttgtggattt atttgacatc caacttggct tctggagtcc cttcaaggtt ttctggttcc 660

ggctcaggaa ccgattatag tttgactatt agctcagtgc agccagagga tgctgcaacc 720

tactattgcc agcaaaggtc ctcatatcca ctgactttcg ggggtggaac gaagttggaa 780

atcaaggctg cagccggatc c 801

<210> 6

<211> 111

<212> PRT

<213> Artificial sequences

<400> 6

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

65 70 75 80

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

85 90 95

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

100 105 110

<210> 7

<211> 333

<212> DNA

<213> Artificial sequences

<400> 7

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatcta catttgggcc cctctggctg gtacttgcgg ggtcctgctg 180

ctttcactcg tgatcactct ttactgtaag cgcggtcgga agaagctgct gtacatcttt 240

aagcaaccct tcatgaggcc tgtgcagact actcaagagg aggacggctg ttcatgccgg 300

ttcccagagg aggaggaagg cggctgcgaa ctg 333

<210> 8

<211> 181

<212> PRT

<213> Artificial sequences

<400> 8

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

65 70 75 80

Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

85 90 95

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

100 105 110

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

115 120 125

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

130 135 140

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

145 150 155 160

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

165 170 175

Ala Leu Pro Pro Arg

180

<210> 9

<211> 543

<212> DNA

<213> Artificial sequences

<400> 9

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatcta catttgggcc cctctggctg gtacttgcgg ggtcctgctg 180

ctttcactcg tgatcactct ttactgtcgc gtgaaattca gccgcagcgc agatgctcca 240

gcctaccagc aggggcagaa ccagctctac aacgaactca atcttggtcg gagagaggag 300

tacgacgtgc tggacaagcg gagaggacgg gacccagaaa tgggcgggaa gccgcgcaga 360

aagaatcccc aagagggcct gtacaacgag ctccaaaagg ataagatggc agaagcctat 420

agcgagattg gtatgaaagg ggaacgcaga agaggcaaag gccacgacgg actgtaccag 480

ggactcagca ccgccaccaa ggacacctat gacgctcttc acatgcaggc cctgccgcct 540

cgg 543

<210> 10

<211> 21

<212> PRT

<213> Artificial sequences

<400> 10

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 11

<211> 63

<212> DNA

<213> Artificial sequences

<400> 11

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 12

<211> 21

<212> PRT

<213> Artificial sequences

<400> 12

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 13

<211> 63

<212> DNA

<213> Artificial sequences

<400> 13

atggccctcc ctgtcaccgc cctgctgctt ccgctggctc ttctgctcca cgccgctcgg 60

ccc 63

<210> 14

<211> 608

<212> DNA

<213> Artificial sequences

<400> 14

cgcccctctc cctcccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 60

tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 120

cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 180

ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 240

caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 300

ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 360

cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 420

aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 480

tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 540

ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac 600

gaccttcc 608

Claims (21)

1. A bipartite antigen receptor, characterized by: the double chimeric antigen receptor consists of a first chimeric antigen receptor aiming at MSLN and a second chimeric antigen receptor aiming at CEA.

2. The bipartite antigen receptor of claim 1, wherein: the first chimeric antigen receptor comprises light chain and heavy chain variable regions hMSLNscFv and a signaling domain of a human MSLN monoclonal antibody; the MSLNscFv sequence is SEQ ID NO.1 in a sequence list or a variant with 80 percent of sequence similarity with the MSLNscFv sequence; the signal conduction structure area is composed of a hinge area, a transmembrane area and an intracellular signal area which are connected in series.

3. The bipartite antigen receptor of claim 2, wherein: the MSLNscFv of the light chain and heavy chain variable regions of the MSLN monoclonal antibody is provided with a linker between the light chain and the heavy chain, the linker can be different amino acid sequences, and preferably is GlyGlyGlySerGlyGlyGly Ser SerGlyGlyGly Ser connecting peptide.

4. The bipartite antigen receptor of claim 2, wherein: the hinge region of the first chimeric antigen receptor is a CD8 a hinge; the transmembrane region is a human CD3 transmembrane region, a human CD8 transmembrane region or a human CD28 transmembrane region; the signaling intracellular region consists of the costimulatory signaling domain CD137(4/1 BB).

5. The bipartite antigen receptor of claim 2, wherein: the upstream of the first chimeric antigen receptor contains a signal peptide, the amino acid sequence of the signal peptide is shown as SEQ ID NO.2 in a sequence list, and the corresponding nucleotide sequence is shown as SEQ ID NO.3 in the sequence list.

6. The bipartite antigen receptor of claim 2 or 4, wherein: the amino acid sequence of the signal conduction structure of the first chimeric antigen receptor is shown as SEQ ID NO.4 in the sequence list, and the corresponding nucleotide sequence is shown as SEQ ID NO.5 in the sequence list.

7. The bipartite antigen receptor of claim 1, wherein: the second chimeric antigen receptor comprises light chain and heavy chain variable regions hCEASV and a signal conduction structural domain of the human CEA monoclonal antibody; the CEAscFv sequence is SEQ ID NO.6 in a sequence list or a variant with 80% sequence similarity; the signal conduction structure area is composed of a hinge area, a transmembrane area and an intracellular signal area which are connected in series.

8. The bipartite antigen receptor of claim 7, wherein: the CEAscFv of the light chain and heavy chain variable regions of the CEA monoclonal antibody is provided with a linker between the light chain and the heavy chain, and the linker can be different amino acid sequences, and is preferably GlyGlyGlyGlySerGlyGlyGly Ser SerGlyGlyGly Ser connecting peptide.

9. The bipartite antigen receptor of claim 7, wherein: the hinge region of the second chimeric antigen receptor is a CD8 a hinge; the transmembrane region is a human CD3 transmembrane region, a human CD8 transmembrane region or a human CD28 transmembrane region; the signaling intracellular domain consists of the antigen recognition signaling domain CD3 ζ.

10. The bipartite antigen receptor of claim 7, wherein: the upstream of the second chimeric antigen receptor contains a signal peptide, the amino acid sequence of the signal peptide is shown as SEQ ID NO.7 in the sequence list, and the corresponding nucleotide sequence is shown as SEQ ID NO.8 in the sequence list.

11. A bipartite antigen receptor according to claim 7 or 9, wherein: the amino acid sequence of the signal conduction structure of the second chimeric antigen receptor is shown as SEQ ID NO.9 in the sequence list, and the corresponding nucleotide sequence is shown as SEQ ID NO.10 in the sequence list.

12. The bipartite antigen receptor of claim 1, wherein: the upstream of the gene sequence of the double-chimeric antigen receptor contains a Kozak sequence, and the nucleotide sequence of the Kozak sequence is shown as SEQ ID NO.11 in a sequence list; the connecting sequence IRES is contained between the first chimeric antigen receptor and the second chimeric antigen receptor, and the nucleotide sequence of the connecting sequence IRES is shown as SEQ ID NO.12 in the sequence list.

13. The bipartite antigen receptor of claim 1, wherein: the downstream of the first chimeric antigen receptor and the second chimeric antigen receptor contains a section of fluorescent protein label GFP, and the amino acid sequence of the fluorescent protein label GFP is shown as SEQ ID NO.14 in a sequence list; a section of connecting peptide P2A is contained between the downstream of the double-chimeric antigen receptor and the fluorescent protein label, and the amino acid sequence of the connecting peptide is shown as SEQ ID NO.13 in a sequence list.

14. The bipartite antigen receptor of claim 1, wherein: the nucleic acid sequence of the double-chimeric antigen receptor is shown as SEQ ID NO.15 in a sequence list.

15. A method of transfecting dCAR-T cells capable of expressing the bipartite antigen receptor of claim 1 or 13, comprising: linking the double chimeric antigen receptor gene to an over-expression vector by

The site-specific integrase system inserts a gene expressing a double chimeric antigen receptor into the genome of a T lymphocyte by means of electroporation to produce dCAR-T cells.

16. A bi-chimeric antigen receptor T cell, characterized by: the T cell is capable of expressing the bipartite antigen receptor of any one of claims 1-14.

17. A method of controlling the chimeric antigen receptor T cells prepared according to claim 15, wherein: the activity of the double-chimeric antigen receptor T cells is controlled by combining tumor-associated antigens CEA and MSLN.

18. Use of the bipartite antigen receptor T cell according to claim 17 for the preparation of a medicament for the treatment of tumors overexpressing the tumor antigens CEA and MSLN.

19. The use of the bipartite antigen receptor T cells according to claim 18 for the preparation of a medicament for the treatment of tumors overexpressing the tumor antigens CEA and MSLN, characterized in that: the tumor comprises pancreatic cancer, lung cancer, gastric cancer, colon cancer, breast cancer, liver cancer, melanoma, cervical cancer, renal cancer, prostatic cancer, tongue squamous cell carcinoma, bladder cancer, ovarian cancer, and nasopharyngeal carcinoma.

20. Use of a bipartite antigen receptor according to any one of claims 1-14 for the preparation of a medicament for the treatment of a tumour.

21. A therapeutic agent for tumor cells, comprising the bipartite chimeric antigen receptor of any one of claims 1-14 or the bipartite chimeric antigen receptor T-cell of claim 16, and a pharmaceutically acceptable excipient.

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