CN111100206A

CN111100206A - Chimeric antigen receptor method targeting mesothelin and CD19 double targets

Info

Publication number: CN111100206A
Application number: CN201811264059.6A
Authority: CN
Inventors: 王海鹰
Original assignee: Shanghai Hrain Biotechnology Co ltd
Current assignee: Shanghai Hrain Biotechnology Co ltd
Priority date: 2018-10-29
Filing date: 2018-10-29
Publication date: 2020-05-05

Abstract

The invention relates to a chimeric antigen receptor targeting mesothelin and CD19 dual targets and uses thereof. In particular, the invention provides a polynucleotide sequence selected from: (1) comprises a sequence of coding sequence of MASK, coding sequence of anti-mesothelin single-chain antibody, coding sequence of anti-CD 19 single-chain antibody, coding sequence of human IgG4 hinge region, coding sequence of human CD28 transmembrane region, coding sequence of human 41BB intracellular region, coding sequence of human CD3 zeta intracellular region, and optionally coding sequence of fragment containing extracellular domain III and extracellular domain IV of EGFR; and (2) the complement of the polynucleotide sequence of (1). The invention also provides a related fusion protein, a vector containing the coding sequence, and applications of the fusion protein, the coding sequence and the vector. The CART cell prepared by the invention has a tEGFR module which has the functions of in vivo tracing and safety switching.

Description

Chimeric antigen receptor method targeting mesothelin and CD19 double targets

Technical Field

The invention belongs to the field of cell therapy, and particularly relates to a chimeric antigen receptor targeting mesothelin and CD19 double targets and application thereof.

Background

Pancreatic cancer (Pancreatic cancer) is a clinically common malignancy of the digestive system, most commonly found in people over the age of 50. The incidence rate of the colon cancer has obvious regional difference, the incidence rate is gradually increased in recent years, the colon cancer is the 4 th common malignant tumor in European and American countries, the 2 nd cause of death of the digestive tract cancer is second to the colon cancer, the attack is hidden, early symptoms are not specific, and the surgical resection rate is low. In the process of tumorigenesis and development, the activation of a plurality of genes and the expression of products thereof play an important role, the precise molecular mechanism in the gene is not completely clear, and with the rapid development of modern images and endoscopic technologies, the clear diagnosis of pancreatic cancer with more obvious symptoms and signs and images is not difficult, but the best operation time is lost mostly at the moment, and the diagnosis of early patients is difficult. Therefore, a new therapeutic means is sought, and treatment of pancreatic cancer is urgent.

Mesothelin (Mesothelin) is also the focus of current research in the study of the invasive metastatic process of tumors. Chang et al cloned the antigen recognized by monoclonal antibody by using HeLa cell of cervical cancer in 1996, and found that the antigen exists in normal mesothelial cell, so the antigen is named Mesothelin. The main reason for the low survival rate and poor prognosis of tumor patients after operation is related to infiltration and metastasis of the tumor patients, and the research on the infiltration and metastasis mechanism of the tumor is the current hotspot and difficulty. The Mesothelin gene encodes a 69kDa precursor protein that is processed to form a 40kDa membrane-bound protein (i.e., Mesothelin) and an shed fragment of 3lkDa, termed the megakaryocyte stimulating factor MPF. Mesothelin is highly expressed in various tumor tissues, the Mesothelin mRNA and protein levels in the serum of ovarian cancer patients are highly expressed, and tissue section staining shows that 66 percent of non-mucinous ovarian cancers are Mesothelin positive; in the detection of pleural mesothelioma, 15 cases diagnosed with epithelial mesothelioma are all positive, and 4 cases diagnosed with sarcoma mesothelioma are all negative; argani et al report that in resected primary pancreatic cancer, immunohistochemical detection shows that 54 strong positives exist in 60 cases, while the peripheral normal pancreatic tissue has no Mesothel in reactivity; in other solid tumor assays, mesothelin immunoreactivity was found in frozen sections of squamous cell carcinomas of the neck, head, neck, vagina, lung and esophagus, with small expression of mesothelin in lung adenocarcinomas, endometrial carcinomas, borderline synovial sarcomas and desmoplastic small round cell tumors, and with little or no expression of mesothelin in breast carcinomas, thyroid carcinomas, renal cell carcinomas, bladder metastatic cell carcinomas, melanoma and liver carcinomas. The biological function of Mesothelin has not been clarified yet. Pastan et al constructed a Mesothel in mutant mouse that grew and propagated identically to a sibling wild-type mouse and showed no statistical difference in platelet counts; studies have shown that Mesothelin binding to CA 125 mediates cell adhesion, and thus researchers also believe that CA 125 and Mesothelin may play a significant role in metastatic spread of ovarian cancer; in addition, it has been shown that expression of the Mesothelin gene is regulated by important signaling pathways such as Wnt, which leads to increased expression of Mesothelin in ovarian and pancreatic cancers. Although. The function of Mesothelin and its carcinogenesis remain to be further clarified, but its distribution in normal tissues is limited and it is highly expressed in some tumor tissues, so Mesothelin can be targeted as a tumor-specific antibody therapy.

Chimeric Antigen Receptor-T cell (CAR-T) T cell refers to a T cell that is genetically modified to recognize a specific Antigen of interest in an MHC non-limiting manner and to continuously activate expanded T cells. The international cell therapy association (interna) in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors besides surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a tumor-associated antigen (TAA) binding region (usually derived from a scFv fragment of the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.

There are 10 current clinical trials of anti-Mesothelin CAR-T cell therapy registered with clinical trials, gov, mainly against malignancies such as pancreatic cancer, ovarian cancer, pleural tumor, lung cancer and breast cancer in phase I clinical studies conducted at the university of pennsylvania, patients are progressing after receiving first line therapy and tumor tissues express Mesothelin, they receive T cell therapy transiently transferring CARmRNA.

Through the development of clinical tests and the analysis of test results, researchers have deeper knowledge about the application defects of the MesothelinCAR-T cell treatment method, so that the research aiming at the problems can be further developed. Given that in the treatment of solid tumors, promoting maximal efficiency of CAR-T cells into tumor tissue is an important guarantee for therapeutic efficacy. According to the fact that pleural mesothelioma cells can secrete a large amount of chemokine CCL2, an anti-Mesothelin CAR-T cell which simultaneously expresses CCL2 receptor CCR2 is designed by Carl June research group at the university of Pennsylvania, and therefore the CAR-T cell is chemotactic to a tumor tissue to efficiently exert a killing effect through the action of CCL2/CCR 2. Compared with the effect of the CAR T cells infused in the thoracic cavity and systemically infused in malignant pleural tumors in the Sprono-Katelin cancer center, the intrathoracic infusion mode is found to ensure that the cells have strong persistence and the tumor is accumulated in a large amount, thereby playing a better anti-tumor role. The sialon-katelin cancer center is about to develop further clinical studies regarding the safety of this infusion modality.

One advantage of CAR-T cells is that they are active drugs, and once infused, physiological mechanisms regulate T cell balance, memory formation, and antigen-driven expansion. However, this treatment is not complete and T cells can miss the target and attack other tissues or expand too much beyond what is needed for treatment. Given that CAR-T cells have been included in the standard therapeutic range, it is very useful to design patient or drug-controlled turn-on or turn-off mechanisms to regulate the presence of CAR-T cells. For technical reasons, the shutdown mechanism is more easily applied to T cells. As one of them, the iCas9 system is under clinical study. When the cell expresses the iCas9, the small molecule compound can induce the iCas9 precursor molecule to form a dimer and activate an apoptosis pathway, thereby achieving the purpose of removing the cell. Small molecule AP1903 has been used to induce iCas9 dimers and clear T cells in graft versus host disease, demonstrating the feasibility of this approach (Clin Cancer Res.2016Apr15; 22(8): 1875-84.).

In addition, it is also possible to use clearing antibodies that have been used clinically to allow CAR-T cells to express proteins to which these antibodies are directed, such as tEGFR, and to clear the corresponding CAR-T cells by administration of antibody drugs after the therapeutic-related toxic response has developed or after the therapy has been completed (Sci Transl Med 2015; 7: 275ra 22.).

The invention uses humanized Mesothelin (SS1), SS1, isolated from a human Fab library against recombinant Mesothelin for the constructed Mesothelin and CD19 targeting dual target CAR. The scFv, Fab and IgG1 antibodies to SS1 specifically bind to human mesothelin and recombinant mesothelin, and have high affinity. SS1 was the first reported fully humanized monoclonal antibody targeting mesothelin, and has therapeutic and diagnostic effects on tumors. The introduction of the CD19 target point can increase the persistence of the CART cell, namely, after the double-target-point CART is infused into blood, the double-target-point CART is greatly expanded after being contacted with a CD19 target antigen, the CART cell is continuously stimulated by a CD19 antigen in the blood, so that more CART cells enter into a solid tumor, and the function of the mesothelin CART cell is indirectly increased. The invention modifies the CART cell, namely introduces a safety switch, namely a tEGFR structure, which can lead the CAR-T cell to be well traced in vivo, and more importantly, the structure can be used as the safety switch of the CAR-T cell: that is, when the action is not wanted, the Tumesitumab can be added, and the infused CAR-T cells aiming at the Mesothel in target can be safely and effectively controlled to play the action in vivo. The invention lays a good foundation for clinical experiments and clinical treatment. The CART cell prepared by the invention has a Mask sequence, and when the CART cell enters a solid tumor microenvironment, the Mask sequence can be cut off due to protease to expose the anti-mesothelin single-chain antibody, so that the anti-mesothelin single-chain antibody can play a role.

With the accumulation and continued sophistication of the experience of CAR-T cell therapy, there is increasing interest in its use in solid tumors. Under the environment, the pace is to be accelerated, and the CAR-T cell therapy is promoted to rapidly advance on the way of solid tumors by applying and developing clinical tests by utilizing the existing work foundation, research and development teams and medical teams.

Disclosure of Invention

In a first aspect, the present invention provides a polynucleotide sequence selected from the group consisting of:

(1) contains a MASK coding sequence, an anti-mesothelin single-chain antibody coding sequence, an anti-CD 19 single-chain antibody coding sequence, a human IgG4 hinge region coding sequence, a human CD28 transmembrane region coding sequence, a human 41BB intracellular region coding sequence, a human CD3 zeta intracellular region coding sequence, and an optional EGFR coding sequence of a fragment containing an extracellular domain III and an extracellular domain IV which are connected in sequence; and

(2) (1) the complement of the polynucleotide sequence.

In one or more embodiments, the coding sequence of the signal peptide preceding the coding sequence of the anti-mesothelin single chain antibody is as shown in the nucleotide sequence at positions 1-66 of SEQ ID NO 1. In one or more embodiments, the Mask signal peptide has a polynucleotide sequence as set forth in polynucleotides 67-129 of SEQ ID NO. 1. In one or more embodiments, the polynucleotide sequence of the splicing fragment is as set forth in the polynucleotide at position 130-207 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the anti-mesothelin single chain antibody is as shown in the nucleotide sequence at position 208-918 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the anti-CD 19 single-chain antibody is shown as the nucleotide sequence at positions 979-1713 of SEQ ID NO. 1. In one or more embodiments, the coding sequence for the hinge region of human IgG4 is as shown in nucleotide sequence 1714-1749 of SEQ ID NO 1. In one or more embodiments, the coding sequence for the transmembrane region of human CD28 is as shown in nucleotide sequence 1753-1833 of SEQ ID NO. 1. In one or more embodiments, the coding sequence of the intracellular region of human 41BB is as shown in the nucleotide sequence at position 1834-1959 of SEQ ID NO. 1. In one or more embodiments, the coding sequence for the intracellular region of human CD3 ζ is as set forth in nucleotide sequences at 1960-2295 of SEQ ID NO: 1. In one or more embodiments, the coding sequence of the fragment of EGFR is as shown in nucleotide sequence 2440-3444 of SEQ ID NO. 1.

In a second aspect, the invention provides a fusion protein selected from the group consisting of:

(1) a coding sequence comprising a fragment of the MASK coding sequence, an anti-mesothelin single chain antibody, an anti-CD 19 single chain antibody, a human IgG4 hinge region, a human CD28 transmembrane region, a human 41BB intracellular region, and a human CD3 zeta intracellular region, optionally EGFR, comprising extracellular domain III and extracellular domain IV; and

(2) a fusion protein derived from (1) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (1) and retaining the activity of activated T cells;

preferably, said anti-mesothelin monoclonal antibody SS 1;

preferably, the anti-CD 19 monoclonal antibody FMC 63.

In one or more embodiments, the polynucleotide sequence further comprises a coding sequence for a signal peptide prior to the coding sequence for the anti-mesothelin single chain antibody. In one or more embodiments, the signal peptide has an amino acid sequence as set forth in amino acids 1-22 of SEQ ID NO. 2. In one or more embodiments, the Mask signal peptide is encoded as shown in amino acid sequence 23-43 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the anti-mesothelin single chain antibody is as set forth in amino acids 70-306 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the anti-CD 19 single chain antibody is shown as amino acids 327 and 571 of SEQ ID NO: 2. In one or more embodiments, the amino acid sequence of the hinge region of human IgG4 is depicted as amino acids 572-583 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the transmembrane region of human CD28 is depicted as amino acid 585 of position 611 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the intracellular domain of human 41BB is as shown in amino acids 612-653 of SEQ ID NO 2. In one or more embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 654-765 of SEQ ID NO. 2. In one or more embodiments, the fragment of EGFR contains or consists of the extracellular domain III, the extracellular domain IV, and the transmembrane region of EGFR. In one or more embodiments, the fragment of EGFR comprises or consists of the amino acid sequence at position 310-646 of human EGFR. In one or more embodiments, the amino acid sequence of the fragment of EGFR is as set forth in amino acids 814-1148 of SEQ ID NO 2.

In a third aspect, the invention provides a nucleic acid construct comprising a polynucleotide sequence as described herein.

In one or more embodiments, the nucleic acid construct is a vector. In one or more embodiments, the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR, pis packaging signal, a cleavage site, woodchuck hepatitis virus post-transcriptional regulatory elements, polynucleotide sequences described herein, and optionally a selectable marker.

In a fourth aspect, the invention provides a retrovirus containing a nucleic acid construct as described herein, preferably containing the vector, more preferably containing the retroviral vector.

In a fifth aspect, the invention provides a genetically modified T cell comprising a polynucleotide sequence as described herein, or comprising a nucleic acid construct as described herein, or infected with a retrovirus as described herein, or stably expressing a fusion protein as described herein and optionally a fragment of EGFR comprising extracellular domain III, extracellular domain IV and optionally a transmembrane region.

In a sixth aspect, the invention provides a pharmaceutical composition comprising a genetically modified T cell as described herein.

In a seventh aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct or retrovirus as described herein in the preparation of an activated T cell.

In an eighth aspect, the invention provides the use of a polynucleotide sequence, fusion protein, nucleic acid construct, retrovirus, or genetically modified T cell as described herein, or a pharmaceutical composition thereof, in the manufacture of a medicament for the treatment of a mesothelin-mediated disease.

In one or more embodiments, the mesothelin-mediated disease is ovarian cancer, pleural mesothelioma, pancreatic cancer, and squamous carcinoma of the cervix, head, neck, vagina, lung, and esophagus. In one or more embodiments, the mesothelin-mediated disease is malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer.

Drawings

FIG. 1: MASK-Meso-CD19-CAR-tEGFR retroviral expression vector (RV-MASK-Meso-CD19-CAR-tEGFR) schematic.

Detailed Description

The present invention provides a Chimeric Antigen Receptor (CAR) targeting humanized mesothelin. The CAR comprises a MASK coding sequence, an anti-mesothelin single-chain antibody coding sequence, an anti-CD 19 single-chain antibody coding sequence, a human IgG4 hinge region coding sequence, a human CD28 transmembrane region coding sequence, a human 41BB intracellular region coding sequence, a human CD3 zeta intracellular region coding sequence, and an optional EGFR coding sequence of a fragment containing an extracellular domain III and an extracellular domain IV which are connected in sequence. .

The anti-mesothelin single chain antibody suitable for use in the present invention may be derived from a variety of anti-mesothelin monoclonal antibodies known in the art.

Thus, in certain embodiments, an anti-mesothelin single chain antibody suitable for use in the present invention contains a sequence that specifically recognizes human mesothelin. Optionally, the light chain variable region and the heavy chain variable region may be linked together by a linker sequence. Such single chain antibodies that may be exemplified include, but are not limited to, YP218Fv-PE38, YP223, SS 1. In certain embodiments, the monoclonal antibody is SS 1.

The fusion proteins of the present invention, such as the light chain variable region and the heavy chain variable region of an anti-mesothelin single chain antibody, the light chain variable region and the heavy chain variable region of an anti-CD 19 single chain antibody, the human IgG4 hinge region, the human CD28 transmembrane region, the human 41BB intracellular region and the human CD3 zeta intracellular region, can be directly linked to each other or can be linked by a linker sequence. The linker sequence may be one known in the art to be suitable for use with antibodies, for example, a G and S containing linker sequence. Typically, the linker contains one or more motifs which repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent in the linker sequence with no intervening amino acid residues between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motifs. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is usually 2 to 20, such as 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (a), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), and the like.

It will be appreciated that in gene cloning procedures it is often necessary to design appropriate cleavage sites which will introduce one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, facilitate expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of a host cell, or facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-terminus or the carboxy-terminus of the fusion protein of the invention (i.e., the CAR) may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty 1. These tags can be used to purify proteins.

The invention also includes a CAR shown as amino acid sequence 23-1148 of SEQ ID NO. 2, a CAR shown as amino acid sequence 1-1148 of SEQ ID NO. 2, or a mutant of the CAR shown as SEQ ID NO. 2. These mutants include: an amino acid sequence that has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retains the biological activity (e.g., activating T cells) of the CAR. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI.

Mutants also include amino acid sequences as shown in positions 23-1148 of SEQ ID NO. 2, amino acid sequences as shown in positions 1-1148 of SEQ ID NO. 2, or amino acid sequences as shown in SEQ ID NO. 2 having one or several mutations (insertions, deletions or substitutions) while still retaining the biological activity of the CAR, the several mutations generally refer to within 1-10, such as 1-8, 1-5, or 1-3 substitutions preferably conservative substitutions, for example, in the art, conservative substitutions with amino acids of similar or similar nature do not generally alter the function of the protein or polypeptide "functionally similar or similar amino acids" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with polar side chains (e.g., glycine, asparagine, threonine, phenylalanine, tyrosine, tryptophan.

The present invention includes polynucleotide sequences encoding the fusion proteins of the present invention. The polynucleotide sequences of the invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e., nucleotide sequences which encode the same amino acid sequence but differ in nucleotide sequence.

The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. For example, in certain embodiments, the polynucleotide sequence encoding the fusion protein described herein is as set forth in SEQ ID NO.1, nucleotides 67-3444, or as set forth in SEQ ID NO.1, nucleotides 1-3444.

In certain embodiments, the polynucleotide sequences of the invention further comprise nucleotide sequences encoding fragments of EGFR.

The EGFR suitable for use in the present invention may be an EGFR known in the art, e.g., from human. EGFR contains N-terminal extracellular domains I and II, extracellular domain III, extracellular domain IV, transmembrane, juxtamembrane domain and tyrosine kinase domain. The present invention preferably uses a truncated EGFR ("tfegfr", i.e., a fragment of EGFR as described herein), particularly a truncated EGFR that does not include its intracellular regions (membrane proximal domain and tyrosine kinase domain). In certain embodiments, EGFR that does not include an intracellular region may be further truncated to include no extracellular domains I and II. Thus, in certain embodiments, the EGFR used in the present invention contains or consists of the extracellular domain III, the extracellular domain IV and the transmembrane region of EGFR. In certain embodiments, the tEGFR comprises or consists of the amino acid sequence at positions 310 and 646 of the human EGFR, wherein the amino acid sequence at positions 310 and 480 is the extracellular domain III of the human EGFR, the amino acid sequence at positions 481 and 620 is the extracellular domain IV of the human EGFR, and the amino acid sequence at positions 621 and 646 is the transmembrane region of the human EGFR.

The invention also relates to nucleic acid constructs comprising the polynucleotide sequences described herein, and one or more control sequences operably linked to these sequences. The polynucleotide sequences of the invention can be manipulated in a variety of ways to ensure expression of the fusion proteins (CAR and/or tfegfr). The nucleic acid construct may be manipulated prior to insertion into the vector, depending on the type of expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

In certain embodiments, the nucleic acid construct is a vector. Expression of a polynucleotide sequence of the invention is typically achieved by operably linking the polynucleotide sequence to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The polynucleotide sequences of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold spring harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

For example, in certain embodiments, the invention uses a retroviral vector that contains a replication initiation site, a 3 'LTR, a 5' LTR, pis packaging signal, a cleavage site, woodchuck hepatitis virus post-transcriptional regulatory elements, polynucleotide sequences described herein, and optionally a selectable marker. The woodchuck hepatitis virus post-transcriptional regulatory element can enhance the stability of viral transcripts.

Another example of a suitable promoter is the extended growth factor-1 α (EF-1 α). however, other constitutive promoter sequences can also be used, including but not limited to the simian virus 40(SV40) early promoter, mouse breast cancer virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoLV promoter, the avian leukemia virus promoter, the EB virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein.

Methods for introducing and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell by any method known in the art, for example, mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly retroviral vectors, which have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Thus, in certain embodiments, the invention also provides a retrovirus for activating T cells, the virus comprising a retroviral vector as described herein and corresponding packaging genes, such as gag, pol and vsvg.

Thus, in certain embodiments, the invention provides a genetically modified T cell comprising a polynucleotide sequence as described herein, or comprising a retroviral vector as described herein, or infected with a retrovirus as described herein, or prepared by a method as described herein, or stably expressing a fusion protein as described herein and optionally a tfegfr.

The CAR-T cells of the invention can undergo robust in vivo T cell expansion and sustained at high levels in the blood and bone marrow for extended amounts of time, and form specific memory T cells. Without wishing to be bound by any particular theory, the CAR-T cells of the invention can differentiate into a central memory-like state in vivo upon encountering and subsequently depleting target cells expressing a surrogate antigen.

The invention also includes a class of cell therapies in which T cells are genetically modified to express a CAR and optionally a tfegfr as described herein, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing tumor cells of the recipient. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

The anti-tumor immune response elicited by the CAR-T cells can be an active or passive immune response. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-T cells induce an immune response specific for the antigen-binding portion in the CAR.

Thus, the diseases that can be treated with the CARs, their coding sequences, nucleic acid constructs, expression vectors, viruses, and CAR-T cells of the invention are preferably mesothelin-mediated diseases.

In particular, herein, "mesothelin-mediated diseases" include, inter alia, various types of ovarian cancer, pleural mesothelioma (e.g., epithelial mesothelioma), pancreatic cancer, and squamous carcinoma of the cervix, head, neck, vagina, lung, and esophagus. In certain embodiments, the mesothelin-mediated disease is malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as relevant cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise CAR-T cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease.

When referring to an "immunologically effective amount", "an anti-tumor effective amount", "a tumor-inhibiting effective amount", or a "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis, and individual differences in the condition of the patient (subject). It can be generally pointed out that: pharmaceutical compositions comprising T cells described herein can be in the range of 10⁴To 10⁹Dosage of individual cells/kg body weight, preferably 10⁵To 10⁶Dosage of individual cells/kg body weight. The T cell composition may also be administered multiple times at these doses. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., Rosenberg et al, New Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof can be combined with other therapies known in the art. Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressive agents. For example, treatment may be in conjunction with radiation or chemotherapeutic agents known in the art for the treatment of mesothelin-mediated diseases.

The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Example 1: determination of Mask-Meso-CD19-CAR-tEGFR Gene sequence

anti-Meso antibody heavy chain and light chain variable region gene sequence information (SS1) is searched from an NCBI website database, anti-CD 19 antibody heavy chain and light chain variable region gene sequence information (FMC63) sequences are subjected to codon optimization on a website http:// sg.idtdna.com/site, and the fact that the encoding amino acid sequence is not changed is guaranteed to be more suitable for human cell expression.

The nucleotide and amino acid sequence information of each gene is shown in (SEQINCE ID NO.1-2)

The sequences are connected in sequence, different enzyme cutting sites are introduced at the joints of the sequences to form complete Mask-Meso-CD 19-IgG4-CD28-41BB-tEGFR gene sequence information.

Example 2: construction of viral vectors comprising the nucleic acid sequence of the CAR molecule

The nucleotide sequence of the CAR molecule prepared in example 1 was double digested with NotI (NEB) and EcoRI (NEB), ligated into the NotI-EcoRI site of the retroviral RV vector with T4 ligase (NEB), transformed into competent e.coli (DH5 α), the recombinant plasmid was sent to marine biotechnology limited for sequencing, and the sequencing results were aligned with the quasi-synthesized Meso-CD 19-IgG4-CD28-41 BB-tffr sequence to verify whether the sequence was correct.

Sense sequence AGCATCGTTCTGTGTTGTCTC (SEQUNCE ID NO.3)

Antisense sequence TGTTTGTCTTGTGGCAATACAC (SEQUNCE ID NO.4)

After the sequencing is correct, plasmids are extracted and purified by using a plasmid purification kit of Qiagen company, and 293T cells are transfected by a plasmid calcium phosphate method for purifying the plasmids to carry out a retrovirus packaging experiment.

The plasmid map constructed in this example is shown in FIG. 1.

Example 3: retroviral packaging

1. Day 1 293T cells should be less than 20 passages, but overgrown. Plating with 0.6 x 10 cells/ml, adding 10ml DMEM medium to 10cm dish, mixing well, culturing at 37 degrees overnight.

2. On day 2, 293T cells are transfected to a confluence of about 90% (usually, plating for about 14-18 h); plasmid complexes were prepared with 12.5ug of each plasmid, 12.5ug of Retro backbone (MSCV), 10ug of Gag-pol, 6.25ug of VSVg, CaCl₂250ul，H₂O is 1ml, and the total volume is 1.25 ml; in another tube, an equal volume of HBS to plasmid complex was added, and the plasmid complex was vortexed for 20 seconds. The mixture was gently added to 293T dishes, incubated at 37 ℃ for 4h, medium removed, washed once with PBS, and re-added with pre-warmed fresh medium.

3. Day 4: after transfection for 48h, the supernatant was collected, filtered through a 0.45um filter, split-charged and stored at-80 ℃, and preheated fresh DMEM medium was added continuously.

Sequence listing

<110> Shanghai Hengrunheng Dasheng Biotech Co., Ltd

<120> chimeric antigen receptor method targeting both mesothelin and CD19 targets

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>3444

<212>DNA

<213> Artificial sequence (Homo sapiens)

<400>1

atggacttcc aggtgcagat ttttagtttt cttttgatct ccgccagcgt gataatgtca 60

cgaggacaag ggcagtctgg acaatgtatc tcaccccgcg gttgccctga tggtccatac 120

gtcatgtatg gctcaagcgg aggaagtggc ggttccggag gatccggcct gagcggcaga 180

tctgacaacc acggatccag cggtaccgat atagagctca cccagagtcc cgcaatcatg 240

tcagcctctc ccggcgaaaa agtgaccatg acctgtagtg cttccagttc tgttagttat 300

atgcactggt atcaacagaa gtccgggaca agtcctaaac gctggattta tgacacttcc 360

aaactggctt ctggagtgcc tgggcggttc agcgggagcg gttccggtaa ctcttacagc 420

ctgaccatct cttcagtcga agctgaagac gatgccacgt attattgcca gcaatggagt 480

aagcacccac tgacatttgg gtgcgggacc aagcttgaaa taaagggtgg cggcagcggg 540

ggcggaagcg gcgggggaag ccaggtgcaa cttcagcaat caggtcccga gttggaaaag 600

ccgggagcca gcgttaagat ctcatgcaaa gctagcggct actctttcac aggatatacc 660

atgaattggg tcaagcaaag ccatggaaaa tgtttggaat ggatcggact gattaccccc 720

tacaacgggg ccagctccta caatcagaaa tttaggggta aggccactct cacagtggat 780

aaaagctcaa gtactgccta tatggacctg cttagtctga cctcagagga tagtgccgtg 840

tacttttgtg ccagaggcgg ttacgacggg cgagggtttg actactgggg gcaggggacg 900

acggttactg tgtctagtgg tggaggcggc agtggcggag gtgggagcgg agggggcggt 960

tccggtggcg ggggatctga ggtgaagctg caggaaagcg gccctggcct ggtggccccc 1020

agccagagcc tgagcgtgac ctgcaccgtg agcggcgtga gcctgcccga ctacggcgtg 1080

agctggatcc ggcagccccc caggaagggc ctggaatggc tgggcgtgat ctggggcagc 1140

gagaccacct actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacagc 1200

aagagccagg tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactac 1260

tgcgccaagc actactacta cggcggcagc tacgccatgg actactgggg ccagggcacc 1320

agcgtgaccg tgagcagcgg cagcacctcc ggcagcggca agcctggcag cggcgagggc 1380

agcaccaagg gcgacatcca gatgacccag accacctcca gcctgagcgc cagcctgggc 1440

gaccgggtga ccatcagctg ccgggccagc caggacatca gcaagtacct gaactggtat 1500

cagcagaagc ccgacggcac cgtcaagctg ctgatctacc acaccagccg gctgcacagc 1560

ggcgtgccca gccggtttag cggcagcggc tccggcaccg actacagcct gaccatctcc 1620

aacctggaac aggaagatat cgccacctac ttttgccagc agggcaacac actgccctac 1680

acctttggcg gcggaacaaa gctggaaatc accgagagca agtacggacc gccctgcccc 1740

ccttgcccta tgttctgggt gctggtggtg gtcggaggcg tgctggcctg ctacagcctg 1800

ctggtcaccg tggccttcat catcttttgg gtgaaacggg gcagaaagaa actcctgtat 1860

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1920

tgccgatttc cagaagaaga agaaggagga tgtgaactgc gggtgaagtt cagcagaagc 1980

gccgacgccc ctgcctacca gcagggccag aatcagctgt acaacgagct gaacctgggc 2040

agaagggaag agtacgacgt cctggataag cggagaggcc gggaccctga gatgggcggc 2100

aagcctcggc ggaagaaccc ccaggaaggc ctgtataacg aactgcagaa agacaagatg 2160

gccgaggcct acagcgagat cggcatgaag ggcgagcgga ggcggggcaa gggccacgac 2220

ggcctgtatc agggcctgtc caccgccacc aaggatacct acgacgccct gcacatgcag 2280

gccctgcccc caaggcgagc taaacgaggc tcaggcgcga cgaactttag tttgctgaag 2340

caagctgggg atgtagagga aaatccgggt cccatgttgc tccttgtgac gagcctcctg 2400

ctctgcgagc tgccccatcc agccttcctc ctcatcccgc ggaaggtgtg caatggcata 2460

ggcattggcg agtttaaaga ttctctgagc ataaatgcta cgaatattaa gcatttcaag 2520

aattgtactt ctattagtgg cgacctccat attcttccgg ttgccttcag gggtgactct 2580

ttcacccaca cacctccatt ggatccacaa gaacttgaca tcctgaagac ggttaaagag 2640

attacaggct tcctccttat ccaagcgtgg cccgagaaca gaacggactt gcacgccttt 2700

gagaacctcg aaataatacg gggtcggacg aagcaacacg gccaatttag ccttgcggtt 2760

gttagtctga acattacttc tctcggcctt cgctctttga aagaaatcag cgacggagat 2820

gtcatcatta gtggaaacaa gaacctgtgc tacgcgaaca caatcaactg gaagaagctc 2880

ttcggtactt caggccaaaa gacaaagatt attagtaaca gaggagagaa tagctgtaag 2940

gctaccggac aagtttgtca cgccttgtgt agtccagagg gttgctgggg accggaacca 3000

agggattgcg tcagttgccg gaacgtgagt cgcggacgcg agtgtgtgga taagtgcaat 3060

cttctggaag gggaaccgcg agagtttgta gaaaattccg aatgtataca gtgtcatccc 3120

gagtgtcttc cacaagcaat gaatatcaca tgtacaggga ggggtcctga taactgtatc 3180

caatgtgcac actacataga tggtcctcac tgtgtaaaga cgtgccccgc cggagtaatg 3240

ggtgaaaaca acaccctcgt gtggaagtac gccgatgccg ggcatgtctg tcatttgtgt 3300

catcccaact gcacatatgg ctgtaccggt cctggattgg agggctgtcc aacaaacggg 3360

ccgaaaatac cgagtatcgc aacaggcatg gtgggagcac ttttgcttct cctcgttgtc 3420

gccctgggca tcggcttgtt catg 3444

<210>2

<211>1148

<212>PRT

<213> Artificial sequence (Homo sapiens)

<400>2

Met Ala Pro Gly Val Gly Ile Pro Ser Pro Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Ala Gly Gly Gly Gly Ser Gly Gly Cys Ile Ser Pro

20 25 30

Ala Gly Cys Pro Ala Gly Pro Thr Val Met Thr Gly Ser Ser Gly Gly

3540 45

Ser Gly Gly Ser Gly Gly Ser Gly Leu Ser Gly Ala Ser Ala Ala His

50 55 60

Gly Ser Ser Gly Thr Ala Ile Gly Leu Thr Gly Ser Pro Ala Ile Met

65 70 75 80

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

85 90 95

Ser Val Ser Thr Met His Thr Thr Gly Gly Leu Ser Gly Thr Ser Pro

100 105 110

Leu Ala Thr Ile Thr Ala Thr Ser Leu Leu Ala Ser Gly Val Pro Gly

115 120 125

Ala Pro Ser Gly Ser Gly Ser Gly Ala Ser Thr Ser Leu Thr Ile Ser

130 135 140

Ser Val Gly Ala Gly Ala Ala Ala Thr Thr Thr Cys Gly Gly Thr Ser

145 150 155 160

Leu His Pro Leu Thr Pro Gly Cys Gly Thr Leu Leu Gly Ile Leu Gly

165 170 175

Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Val Gly Leu Gly

180 185 190

Gly Ser Gly Pro Gly Leu Gly Leu Pro Gly Ala Ser Val Leu Ile Ser

195200 205

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

210 215 220

Leu Gly Ser His Gly Leu Cys Leu Gly Thr Ile Gly Leu Ile Thr Pro

225 230 235 240

Thr Ala Gly Ala Ser Ser Thr Ala Gly Leu Pro Ala Gly Leu Ala Thr

245 250 255

Leu Thr Val Ala Leu Ser Ser Ser Thr Ala Thr Met Ala Leu Leu Ser

260 265 270

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

275 280 285

Ala Gly Ala Gly Pro Ala Thr Thr Gly Gly Gly Thr Thr Val Thr Val

290 295 300

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

305 310 315 320

Ser Gly Gly Gly Gly Ser Gly Val Leu Leu Gly Gly Ser Gly Pro Gly

325 330 335

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

340 345 350

Val Ser Leu Pro Ala Thr Gly Val Ser Thr Ile Ala Gly Pro Pro Ala

355 360365

Leu Gly Leu Gly Thr Leu Gly Val Ile Thr Gly Ser Gly Thr Thr Thr

370 375 380

Thr Ala Ser Ala Leu Leu Ser Ala Leu Thr Ile Ile Leu Ala Ala Ser

385 390 395 400

Leu Ser Gly Val Pro Leu Leu Met Ala Ser Leu Gly Thr Ala Ala Thr

405 410 415

Ala Ile Thr Thr Cys Ala Leu His Thr Thr Thr Gly Gly Ser Thr Ala

420 425 430

Met Ala Thr Thr Gly Gly Gly Thr Ser Val Thr Val Ser Ser Gly Ser

435 440 445

Thr Ser Gly Ser Gly Leu Pro Gly Ser Gly Gly Gly Ser Thr Leu Gly

450 455 460

Ala Ile Gly Met Thr Gly Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

465 470 475 480

Ala Ala Val Thr Ile Ser Cys Ala Ala Ser Gly Ala Ile Ser Leu Thr

485 490 495

Leu Ala Thr Thr Gly Gly Leu Pro Ala Gly Thr Val Leu Leu Leu Ile

500 505 510

Thr His Thr Ser Ala Leu His Ser Gly Val Pro Ser Ala Pro Ser Gly

515 520525

Ser Gly Ser Gly Thr Ala Thr Ser Leu Thr Ile Ser Ala Leu Gly Gly

530 535 540

Gly Ala Ile Ala Thr Thr Pro Cys Gly Gly Gly Ala Thr Leu Pro Thr

545 550 555 560

Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Thr Gly Ser Leu Thr Gly

565 570 575

Pro Pro Cys Pro Pro Cys Pro Met Pro Thr Val Leu Val Val Val Gly

580 585 590

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

595 600 605

Pro Thr Val Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly

610 615 620

Pro Pro Met Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser

625 630 635 640

Cys Ala Pro Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu

645 650 655

Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Gly Gly Gly Gly Ala Gly

660 665 670

Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu

675 680 685

Ala Leu Ala Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Ala Ala

690 695 700

Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met

705 710 715 720

Ala Gly Ala Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly

725 730 735

Leu Gly His Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala

740 745 750

Thr Thr Ala Ala Leu His Met Gly Ala Leu Pro Pro Ala Ala Ala Leu

755 760 765

Ala Gly Ser Gly Ala Thr Ala Pro Ser Leu Leu Leu Gly Ala Gly Ala

770 775 780

Val Gly Gly Ala Pro Gly Pro Met Leu Leu Leu Val Thr Ser Leu Leu

785 790 795 800

Leu Cys Gly Leu Pro His Pro Ala Pro Leu Leu Ile Pro Ala Leu Val

805 810 815

Cys Ala Gly Ile Gly Ile Gly Gly Pro Leu Ala Ser Leu Ser Ile Ala

820 825 830

Ala Thr Ala Ile Leu His Pro Leu Ala Cys Thr Ser Ile Ser Gly Ala

835 840 845

Leu His Ile Leu Pro Val Ala Pro Ala Gly Ala Ser Pro Thr His Thr

850 855 860

Pro Pro Leu Ala Pro Gly Gly Leu Ala Ile Leu Leu Thr Val Leu Gly

865 870 875 880

Ile Thr Gly Pro Leu Leu Ile Gly Ala Thr Pro Gly Ala Ala Thr Ala

885 890 895

Leu His Ala Pro Gly Ala Leu Gly Ile Ile Ala Gly Ala Thr Leu Gly

900 905 910

His Gly Gly Pro Ser Leu Ala Val Val Ser Leu Ala Ile Thr Ser Leu

915 920 925

Gly Leu Ala Ser Leu Leu Gly Ile Ser Ala Gly Ala Val Ile Ile Ser

930 935 940

Gly Ala Leu Ala Leu Cys Thr Ala Ala Thr Ile Ala Thr Leu Leu Leu

945 950 955 960

Pro Gly Thr Ser Gly Gly Leu Thr Leu Ile Ile Ser Ala Ala Gly Gly

965 970 975

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

980 985 990

Gly Gly Cys Thr Gly Pro Gly Pro Ala Ala Cys Val Ser Cys Ala Ala

995 1000 1005

Val Ser Ala Gly Ala Gly Cys Val Ala Leu Cys Ala Leu Leu Gly Gly

1010 1015 1020

Gly Pro Ala Gly Pro Val Gly Ala Ser Gly Cys Ile Gly Cys His Pro

1025 1030 1035 1040

Gly Cys Leu Pro Gly Ala Met Ala Ile Thr Cys Thr Gly Ala Gly Pro

1045 1050 1055

Ala Ala Cys Ile Gly Cys Ala His Thr Ile Ala Gly Pro His Cys Val

1060 1065 1070

Leu Thr Cys Pro Ala Gly Val Met Gly Gly Ala Ala Thr Leu Val Thr

1075 1080 1085

Leu Thr Ala Ala Ala Gly His Val Cys His Leu Cys His Pro Ala Cys

1090 1095 1100

Thr Thr Gly Cys Thr Gly Pro Gly Leu Gly Gly Cys Pro Thr Ala Gly

1105 1110 1115 1120

Pro Leu Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

1125 1130 1135

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Pro Met

1140 1145

<210>3

<211>21

<212>DNA

<213> Artificial sequence (Homo sapiens)

<400>3

agcatcgttc tgtgttgtct c 21

<210>4

<211>22

<212>DNA

<213> Artificial sequence (Homo sapiens)

<400>4

tgtttgtctt gtggcaatac ac 22

Claims

1. An mCAR structure, characterized by:

(1) a masking peptide structure;

(2) an antigen-specific recognition region;

(3) a transmembrane region;

(4) one or more costimulator regions;

(5) an intracellular signaling region.

2. A masking peptide of claim 1 having the sequence QGQSGQCISPRGCPDGPYVMY.

3. The antigen recognition region of claim 1 which is an scFv fragment.

4. The scFv fragment of claim 3 comprising scFv for Mesothelin and scFv for CD 19.

5. The masked peptide structure of claim 1 and the specific recognition region are linked to two linking fragments (linker) via a proteolytic cleavage site.

6. A polynucleotide sequence selected from the group consisting of:

(2) (1) the complement of the polynucleotide sequence.

7. The polynucleotide sequence of claim 6,

the polynucleotide sequence also comprises a coding sequence of a signal peptide in front of the coding sequence of the anti-mesothelin single-chain antibody, and preferably, the polynucleotide sequence of the signal peptide is shown as the 1 st-66 th polynucleotide in SEQ ID NO 1; and/or

The polynucleotide sequence of the Mask signal peptide is shown as the 67 th-129 th polynucleotide of SEQ ID NO. 1; and/or

The polynucleotide sequence of the splicing connecting segment is shown as the polynucleotide at the 130-th and 207-th positions of SEQ ID NO. 1;

and/or

The polynucleotide sequence of the anti-mesothelin single-chain antibody is shown as the polynucleotide at position 208-918 of SEQ ID NO. 1; and/or

The polynucleotide sequence of the anti-CD 19 single-chain antibody is shown in the SEQ ID NO.1 polynucleotide at position 979-1713; and/or

The polynucleotide sequence of the human IgG4 hinge region is shown in SEQ ID NO.1, 1714-1749 polynucleotide; and/or

The polynucleotide sequence of the transmembrane region of the human CD28 is shown as the polynucleotide at the 1753-position 1833 of SEQ ID NO. 1; and/or

The polynucleotide sequence of the human 41BB intracellular region is shown in the polynucleotide at the 1834-1959-th site of SEQ ID NO. 1; and/or

The polynucleotide sequence of the intracellular region of human CD3 zeta is shown in the polynucleotide at the 1960-2295 th site of SEQ ID NO. 1; and/or

The EGFR fragment contains or consists of the extracellular domain III, the extracellular domain IV and the transmembrane region of the EGFR; preferably, the fragment comprises or consists of the polynucleotide sequence at position 310-646 of human EGFR; more preferably, the polynucleotide sequence of said fragment is as shown in SEQ ID NO 1, polynucleotide 2440-3444.

8. A fusion protein selected from the group consisting of:

preferably, the anti-mesothelin monoclonal antibody is SS 1.

9. The fusion protein of claim 8, wherein the fusion protein has one or more of the following characteristics:

the fusion protein also comprises a signal peptide at the N end of the anti-mesothelin single-chain antibody, preferably, the amino acid sequence of the signal peptide is shown as amino acids 1-22 of SEQ ID NO. 2;

the coding sequence of the Mask signal peptide is shown as amino acid sequence 23-43 of SEQ ID NO 2;

the amino acid sequence of the anti-mesothelin single-chain antibody is shown as amino acids 70-306 of SEQ ID NO 2;

the amino acid sequence of the anti-CD 19 single-chain antibody is shown as the 327 st and 571 st amino acids of SEQ ID NO 2;

the amino acid sequence of the hinge region of the human IgG4 is shown as the amino acid at the 572 th and 583 th positions of SEQ ID NO. 2;

the amino acid sequence of the transmembrane region of the human CD28 is shown as the amino acid 585-611 of SEQ ID NO 2;

the amino acid sequence of the intracellular region of the human 41BB is shown as the amino acid 612-653 of SEQ ID NO 2;

the amino acid sequence of the intracellular region of the human CD3 zeta is shown as the amino acid at the 654-765 position of SEQ ID NO. 2; and

the EGFR fragment contains or consists of the extracellular domain III, the extracellular domain IV and the transmembrane region of the EGFR; preferably, the fragment comprises or consists of the amino acid sequence at positions 310-646 of human EGFR; more preferably, the amino acid sequence of said fragment is as shown in amino acids 814-1148 of SEQ ID NO 2.

10. A nucleic acid construct comprising the polynucleotide sequence of any one of claims 6-7;

preferably, the nucleic acid construct is a vector;

more preferably, the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR, pis packaging signal, a cleavage site, woodchuck hepatitis virus post-transcriptional regulatory elements, and a polynucleotide sequence according to any one of claims 1-2.

11. A retrovirus containing the nucleic acid construct of claim 10, preferably containing the vector, more preferably containing the retroviral vector.

12. A genetically modified T-cell or a pharmaceutical composition comprising a genetically modified T-cell, wherein said cell comprises a polynucleotide sequence according to any one of claims 6 to 7, or comprises a nucleic acid construct according to claim 10, or is infected with a retrovirus according to claim 11, or stably expresses a fusion protein according to claim 8 and optionally an extracellular domain III, extracellular domain IV fragment-containing portion of EGFR.

13. Use of a polynucleotide sequence according to any one of claims 6 to 7, a fusion protein according to any one of claims 8 to 9, a nucleic acid construct according to claim 10 or a retrovirus according to claim 11 in the preparation of an activated T cell.

14. Use of the polynucleotide sequence of any one of claims 6-7, the fusion protein of any one of claims 8-9, the nucleic acid construct of claim 10, the retrovirus of claim 11, or the genetically modified T-cell of claim 12, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating a mesothelin-mediated disease;

preferably, the mesothelin-mediated disease is ovarian cancer, pleural mesothelioma, pancreatic cancer, and squamous carcinoma of the cervix, head, neck, vagina, lung and esophagus, preferably malignant pleural mesothelioma, pancreatic cancer, ovarian cancer and lung cancer.