CN109134660B

CN109134660B - Methods of targeting chimeric antigen receptors of Mesothelin in combination with expression of anti-PD1 antibodies and uses thereof

Info

Publication number: CN109134660B
Application number: CN201710459212.XA
Authority: CN
Inventors: 黄飞; 金涛; 王海鹰; 何凤; 史子啸
Original assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Priority date: 2017-06-16
Filing date: 2017-06-16
Publication date: 2022-07-01
Anticipated expiration: 2037-06-16
Also published as: CN109134660A

Abstract

The present invention relates to chimeric antigen receptors targeting meso-aPD1 and uses thereof. In particular, the invention provides a polynucleotide sequence selected from: (1) contains the coding sequence of anti-Meso single-chain antibody, the coding sequence of human CD8 alpha hinge region, the coding sequence of human CD8 transmembrane region, the coding sequence of human 41BB intracellular region and the coding sequence of anti-human PD1 single-chain antibody which are connected in sequence; 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 Mesothelin-aPD1 CAR-T cell prepared by the invention has strong killing function on specific tumor cells, and the CAR-T cell prepared by the invention can secrete anti-PD1 antibody to have the function of regulating an immunosuppressive microenvironment.

Description

Methods of targeting chimeric antigen receptors of Mesothelin in combination with expression of anti-PD1 antibodies and uses thereof

Technical Field

The invention belongs to the field of chimeric antigen receptors, and particularly relates to a chimeric antigen receptor targeting Mesothelin (SS1) -CD28-BBz-aPD1 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 peripheral normal pancreatic tissues have no Mesothelin 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 sarcomas and desmoplastic small round cell tumors, and with little or no expression of Mesothelin in breast carcinomas, thyroid carcinomas, renal cell carcinomas, bladder metastasizing cell carcinomas, melanoma and liver carcinomas.

The biological function of Mesothelin is not yet clear. Pastan et al constructed a Mesothelin gene mutant mouse that grew and propagated identically to a sibling wild-type mouse and did not have a 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 Mesothelin gene expression is regulated by important signaling pathways such as Wnt, which are continuously activated in ovarian and pancreatic cancers, resulting in increased Mesothelin expression. 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 the scFV fragment from 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.

Currently, there are 10 clinical trials of anti-Mesothelin CAR-T cell therapy registered in clinical trials. In a phase I clinical study conducted at the university of pennsylvania, patients had progressed further after receiving first-line treatment and tumor tissues expressed Mesothelin, which received T cell therapy with transient CAR mRNA. This Mesothelin-specific, second-generation CAR has a CD3 ξ and a 4-1BB costimulator domain. These Mesothelin-specific CAR T survives short, showed anti-tumor effects in two patients, and it was shown that Mesothelin could act as an antigen recognized by CAR T cells, and a means of transiently transforming mRNA was also feasible. Another phase I clinical study conducted at university of Pennsylvania used a lentivirus-transfected Mesothelin-specific CAR. This study, starting at 7 months 2014, was directed to chemotherapy-resistant malignant pancreatic cancer, epithelial ovarian cancer, and malignant epithelial pleural mesothelioma. In the early results of the study in 6 patients, 4 patients had stable disease after 28 days of CAR T cell infusion. CAR T cell infusion did not cause acute side effects, and the persistence of lentiviral transfected constructed CAR T cells was improved compared to mRNA transient.

PD1(programmed death 1) was originally obtained in apoptotic T cell hybridomas and was named the programmed death 1 receptor as it is associated with apoptosis. The PD1 receptor is expressed on the surface of T cells and primary B cells and plays a role in the differentiation and apoptosis of these cells. PD1 has two ligands, PD-L1(B7-H1) and PD-L2(B7-DC), belonging to the B7 family of proteins (blood.2009.114(8): p.1537-44.). PD-L1 protein is widely expressed in antigen presenting cells, activated T, B cells, macrophages, placental trophoblasts, myocardial endothelium and thymic cortical epithelial cells. PD-L1 interacts with the receptor PD1 on T cells and plays an important role in the negative regulation of immune responses. Normally, when the body encounters a foreign pathogen or an antigen invader, the antigen presenting cell captures the antigen, processes the antigen into an epitope which can be recognized by a T cell, binds to an MHC molecule and presents the outside of the cell for the recognition of the T cell. T cells are bound to MHC molecules of antigen presenting cells through TCR, and in addition, a costimulatory signal CD28 receptor is bound to B7-1(CD80) or B7-2(CD86) on the surface of the original T cells, the T cells receive a positive regulatory signal, the original T cells are activated into effector T cells, and an immune response is initiated. When continuous antigen stimulation is available, in order to avoid excessive response, the activated T cell surface expresses PD1, and the PD-L1 is combined with the surface of an antigen presenting cell to transmit negative regulation signals to the T cell, so that the T cell is reduced in proliferation or is apoptotic. The research finds that the expression of the PD-L1 protein can be detected in a plurality of human tumor tissues, the microenvironment at the tumor part can induce the expression of the PD-L1 on tumor cells, and the combination of the expressed PD-L1 and the PD1 on the surface of the T cells can inhibit the anti-tumor activity of the T cells, so that the tumor cells can escape from the monitoring and elimination of the immune system of the body, and the generation and the growth of tumors are facilitated.

The PD1/PD-L1 pathway inhibitor can block the combination of PD1 and PD-L1, block negative regulation signals, enable T cells to recover activity, and further enhance immune response. The PD1/PDL1 pathway inhibitor mainly comprises anti-PD1 or anti-PD-L1 monoclonal antibody. The rate of Opdivo, executed in precious, was first approved in japan for the treatment of advanced melanoma in 7 months 2014, becoming the first globally approved PD1 inhibitor to be marketed. The PD1 inhibitor shows life cycle curative effect in phase III clinical experiments for the first time, and compared with the chemotherapeutic dacarbazine, the survival rate of 1 year is 73 percent to 42 percent, the response rate is 40 percent to 14 percent, and the adverse reaction is reduced. While Keytruda (pembrolizumab), Merck in the United states, successfully landed in the US market as the first PD-1 inhibitor as a result of an unconventional large phase I clinical trial involving 1000 patients 9 months 2014, was approved for the treatment of advanced melanoma patients who failed to surgical resection or had developed metastases and were unresponsive to other drugs (N Engl J Med.2013Jul 11; 369(2): 134-44.).

Although the compound combination has wide prospect, the current antitumor drug treatment window is generally narrow, the effect of the combined medication is still difficult to predict, and the exertion of the PD1 effect is seriously restricted. The rapid development of CAR-T cells provides a new opportunity for this. CAR-T cells are highly targeted and highly specific, and can proliferate rapidly in large numbers after stimulation by tumor antigens, and thus may be limited by inhibitory signals, thereby compromising their anti-tumor capacity. If the inhibitor of the surface of the T cell PD1 can be blocked, the CAR-T cell can be freed from the constraint, and the tumor killing effect can be fully exerted. Based on this, the strategy of combining CAR-T cells with blocking of PD1/PD-L1 signaling has rapidly received attention from researchers (oncoimmunology.2014Dec 21; 3(11): e 970027.).

A series of studies were conducted by professor Edmund Moon, university of Pennsylvania, and a team in his area on this combined application strategy. When the TCR-T cell with NY-ESO-1 as an antigen target is used for killing tumor cells, the anti-PD1 antibody is added, so that the phenomenon of T cell function decline can be obviously improved; accordingly, in the mouse model of subcutaneous transplanted tumor, the tumor clearance of TCR-T cells is limited, and the complete elimination of tumor can be achieved after the treatment of PD1 antibody (Clin Cancer Res.2016.22(2): p.436-47). Meanwhile, the team designs a new generation of CAR-T cells by using a genetic engineering technology, namely a transgenic receptor PD1CD28 is inserted into the CAR-T cells through a virus vector, the structure consists of an extracellular section of PD1 and a transmembrane section and an intracellular section of a costimulatory molecule CD28, and after PD1 and a tumor cell surface ligand PD-L1 are combined, a PD1/PD-L1 inhibition signal is converted into an activation signal, so that the power is increased for the functions of the CAR-T cells. The effect is successfully verified in preclinical animal model research, and compared with the T cell without inserted PD1CD28, the CAR-T cell loaded with PD1CD28 can generate stronger immune response to a mouse tumor model and increase the survival rate of mice.

The research initially shows the feasibility of the combined application strategy of the CAR-T cell and the blocking PD1/PD-L1 signal in tumor treatment, and on the basis, the optimal combination of the CAR-T cell and the blocking PD1/PD-L1 signal is realized by fully utilizing the mature gene modification technology developed at present. The patent of us takes the heavy chain and the light chain of the scFV of the Mesothelin antibody as the structure of CAR, and lays a good foundation for development of clinical experiments in the future.

Disclosure of Invention

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

(1) a polynucleotide sequence comprising the coding sequence of an anti-Mesothelin single-chain antibody, the coding sequence of a human CD8 alpha hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human CD28 intracellular region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3 zeta intracellular region, and the coding sequence of an anti-human PD1 single-chain antibody, which are linked in this order; and

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

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 amino acid sequence of the signal peptide is as set forth in amino acids 1-22 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-Mesothelin single chain antibody is as set forth in amino acids 23-128 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-Mesothelin single chain antibody is as shown in SEQ ID NO:1, amino acids 141-259. In one or more embodiments, the amino acid sequence of the human CD8 alpha hinge region is as set forth in SEQ ID NO 1 at amino acids 260-306. In one or more embodiments, the amino acid sequence of the transmembrane region of human CD8 is depicted as amino acids 307-328 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human CD28 is depicted as amino acids 329-369 of SEQ ID NO. 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human 41BB is as shown in amino acids 370-417 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 381-491 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of human P2A is depicted as amino acids 529-554 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the human IL-2 signal peptide is as set forth in amino acid 555-574 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-PD1 single-chain antibody is shown as amino acids 575-687 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-PD1 single-chain antibody is shown as amino acids 703 and 809 of SEQ ID NO. 1.

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 nucleotide sequence 1-66 of SEQ ID NO. 2. In one or more embodiments, the light chain variable region encoding sequence of the anti-Mesothelin single chain antibody is as set forth in nucleotide sequences at positions 67-384 of SEQ ID NO. 2. In one or more embodiments, the coding sequence of the heavy chain variable region of the anti-Mesothelin single-chain antibody is shown as the nucleotide sequence at position 421-777 in SEQ ID NO. 2. In one or more embodiments, the coding sequence for the human CD8 α hinge region is as shown in nucleotide sequence 778-918 of SEQ ID NO. 2. In one or more embodiments, the coding sequence for the transmembrane region of human CD8 is as shown in nucleotide sequence 919-984 of SEQ ID NO 2. In one or more embodiments, the coding sequence for the intracellular domain of human CD28 is as shown in nucleotide sequence 985-1107 of SEQ ID NO. 2. In one or more embodiments, the coding sequence of the intracellular region of human 41BB is as shown in nucleotide sequence 1108-1251 of SEQ ID NO 2. In one or more embodiments, the coding sequence for the intracellular region of human CD3 ζ is as set forth in nucleotide sequence 1252-1584 of SEQ ID NO. 2. In one or more embodiments, the coding sequence of human P2A is as shown in nucleotide sequence 1585-1662 of SEQ ID NO 2. In one or more embodiments, the coding sequence for the human IL-2 signal peptide is as set forth in nucleotide sequence 1663-1722 of SEQ ID NO. In one or more embodiments, the coding sequence of the heavy chain variable region of the anti-PD1 single-chain antibody is shown as the nucleotide sequence at position 1723-2061 of SEQ ID NO. 2. In one or more embodiments, the coding sequence of the light chain variable region of the anti-PD1 single-chain antibody is shown in the nucleotide sequence 2107-2427 of SEQ ID NO. 2.

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

(1) a fusion protein comprising an anti-Mesothelin single-chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human CD28 intracellular region, a human 41BB intracellular region, a human CD3 ζ intracellular region, and an anti-human PD1 single-chain antibody connected in this order; 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, the anti-Mesothelin single chain antibody is anti-Mesothelin monoclonal antibody SS 1.

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 amino acid sequence of the signal peptide is as set forth in amino acids 1-22 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-Mesothelin single chain antibody is as set forth in amino acids 23-128 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-Mesothelin single chain antibody is as shown in SEQ ID NO:1, amino acids 141-259. In one or more embodiments, the amino acid sequence of the human CD8 α hinge region is depicted as amino acids 260-306 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the transmembrane region of human CD8 is depicted as amino acids 307-328 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human CD28 is depicted as amino acids 329-369 of SEQ ID NO. 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human 41BB is as shown in amino acids 370-417 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the intracellular domain of human CD3 ζ is as set forth in amino acids 381-491 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of human P2A is depicted as amino acids 529-554 of SEQ ID NO 1. In one or more embodiments, the amino acid sequence of the intracellular domain of the human IL-2 signal peptide is as set forth in amino acids 555-574 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the heavy chain variable region of the anti-PD1 single-chain antibody is shown as amino acids 575-687 of SEQ ID NO: 1. In one or more embodiments, the amino acid sequence of the light chain variable region of the anti-PD1 single-chain antibody is shown as amino acids 703 and 809 of SEQ ID NO. 1.

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, a polynucleotide sequence 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.

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, 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.

Drawings

FIG. 1 is a schematic representation of RV-Mesothelin-aPD1-CAR retroviral expression vectors. SP: a signal peptide; VL: a light chain variable region; and Lk: joint (G)₄S)₃(ii) a VH: a heavy chain variable region; h: a CD8 a hinge region; TM: the CD8 transmembrane domain.

FIG. 2 is a partial sequencing peak plot of the RV-Mesothelin CAR-aPD1 retrovirus expression plasmid

FIG. 3 shows the efficiency of Mesothelin-aPD1 CAR-T expression by a flow cytometer after retroviral infection of T cells for 72 hours

FIG. 4293 results of anti-Human Fab staining after 30min incubation of T-PD1 cells with Mesothelin-28BBz-aPD1 virus

FIG. 5 is a 5 day preparation of Mesothelin-tEGFR CAR-T/Mesothelin-aPD1 CAR-T cells co-cultured with target cells for 5 hours CD107a expression

FIG. 6 is the expression of CAR-T surface PD1 after 5 days of preparation of Mesothelin-tEGFR CAR-T/Mesothelin-aPD1 CAR-T and 24 hours of co-culture with target cells

FIG. 7 shows the killing effect on tumor cells after 5 days of preparation of Mesothelin-tEGFR CAR-T/Mesothelin-aPD1 CAR-T cells co-cultured with target cells for 16 hours

Detailed Description

The present invention provides a Chimeric Antigen Receptor (CAR) targeting Mesothelin. The CAR comprises fragments of anti-Mesothelin single chain antibody, human CD8 a hinge region, human CD8 transmembrane region, human CD28 intracellular region, human 41BB intracellular region, human CD3 ζ intracellular region, and anti-human PD1 single chain antibody, linked in sequence.

The anti-Mesothelin single chain antibody suitable for use in the present invention may be derived from various anti-Mesothelin (SS1) monoclonal antibodies known in the art.

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. Preferably, these monoclonal antibodies specifically recognize the amino acid segment 296 through 390 th positions of mesothelin.

Thus, in certain embodiments, an anti-mesothelin single chain antibody suitable for use in the present invention comprises the light chain variable region and the heavy chain variable region of a monoclonal antibody that specifically recognizes the 296-390 th amino acid segment of 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 and SS 1. In certain embodiments, the monoclonal antibody is SS 1.

The variable regions of the light chain and the heavy chain forming the fusion protein of the present invention, such as the anti-mesothelin single chain antibody, the human CD8 α hinge region, the human CD8 transmembrane region, the human CD28 intracellular region, 41BB and the human CD3 ζ intracellular region, etc., may be directly linked to each other, or may be linked via 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 can 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 represented by the amino acid sequence at positions 22-491 of SEQ ID NO.1, a CAR represented by the amino acid sequence at positions 22-809 of SEQ ID NO.1, a CAR represented by the amino acid sequence at positions 1-491 of SEQ ID NO.1, or a mutant of a CAR represented by SEQ ID NO. 1. 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: an amino acid sequence as shown in positions 22-491 of SEQ ID NO 1, an amino acid sequence as shown in positions 22-809 of SEQ ID NO 1, an amino acid sequence as shown in positions 1-491 of SEQ ID NO 1 or an amino acid sequence as shown in SEQ ID NO 1 which has one or several mutations (insertions, deletions or substitutions) while still retaining the biological activity of the CAR. The number of mutations usually means within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, conservative substitutions with amino acids of similar or similar properties are not typically used in the art to alter the function of a protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). Thus, substitution of one or more sites with another amino acid residue from the same side chain species in the polypeptide of the invention will not substantially affect its activity.

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 nucleotides 67-1107 of SEQ ID NO. 2, or as set forth in nucleotides 1-1107 of SEQ ID NO. 2.

In addition, the coding sequence for the signal peptide may be linked to the coding sequence for the intracellular domain of human CD3 ζ in a CAR of the invention via the coding sequence for the P2A polypeptide. In one or more embodiments, the amino acid sequence of the P2A peptide is set forth in amino acids 529-554 of SEQ ID NO 1.

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 protein (CAR). The nucleic acid construct may be manipulated prior to insertion into the vector depending on the 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.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the 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. Further, inducible promoters are also contemplated. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter during periods of expression and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a 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.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

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 the 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.

T cells suitable for use in the present invention may be of various types from various sources. For example, T cells may be derived from PBMCs of B cell malignancy patients.

In certain embodiments, after T cells are obtained, activation may be stimulated with an appropriate amount (e.g., 30-80 ng/ml, such as 50ng/ml) of CD3 antibody prior to culturing in an appropriate amount (e.g., 30-80 IU/ml, such as 50IU/ml) of IL2 medium for use.

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.

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 described herein and optionally anti-PD1, 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.

Herein, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

"patient," "subject," "individual," and the like are used interchangeably herein and refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

The present invention employs the gene sequence of an anti-Mesothelin antibody (specifically a scFV derived from SS1), a gene fragment of the present whole-gene synthetic chimeric antigen receptor Mesothelin scFV-CD8H & TM-CD28-41BB-CD3 ζ -PD1, inserted into a retroviral vector MSCV, an empty vector MSCV, which can be used for recombinant introduction of a nucleic acid sequence of interest, i.e. a nucleic acid sequence encoding a CAR. The recombinant plasmid packages the virus in 293T cells, infects T cells, and causes the T cells to express the chimeric antigen receptor. In one embodiment of the invention, the transformation method to achieve chimeric antigen receptor gene modified T lymphocytes is based on a retroviral transformation method. The method has the advantages of high transformation efficiency, stable expression of exogenous genes, and capability of shortening the time for in vitro culture of T lymphocytes to reach clinical level number. On the surface of the transgenic T lymphocyte, the transformed nucleic acid is expressed by transcription and translation. The CAR-expressing retrovirus obtained by the invention is used for preparing CAR-T cells by a Retronectin method, the CAR-T cells after 3 days are prepared, the infection efficiency of the CAR is detected by flow type detection, the expression of the PD1 antibody of the supernatant is detected, the CAR-T cells are cultured with Mesothelin positive tumor cells (SKOV3 and SKOV3-Mesothelin) in vitro for 5 hours after 5 days are prepared, the expression of CD107a and PD1 is detected, and the CAR-T cells are cultured with the Mesothelin positive tumor cells (SKOV3 and SKOV3-Mesothelin) in vitro for 16 hours after 5 days are prepared, and the specific killing effect (cytotoxicity) of the CAR-T cells on the tumor cells is detected. Therefore, the Mesothelin CAR-aPD1 can be applied to the treatment of mesothelioma, pancreatic cancer, ovarian cancer and the like.

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.

The NT cells used in the examples were untransfected T cells of the same origin as in example 4, and used as control cells. K562 cells and cells from ATCC cell bank that were negative expressing Mesothelin were used as control cells. SKOV3 cells and cells from the ATCC cell bank that positively expressed Mesothelin were used as control cells. SKOV3-Mesothelin cell (SKOV 3-Meso) is a SKOV3 stable cell strain which is made to highly express Mesothelin by genetic engineering means.

Example 1: determination of Mesothelin CAR-aPD1 Gene sequence

The gene sequence information of the heavy chain and light chain variable regions of the anti-Mesothelin antibody (SS1) is searched from an NCBI website database, and the sequences are subjected to codon optimization on a website https:// sg.idtdna.com/site, so that the expression of human cells is more suitable under the condition of unchanged coding amino acid sequences.

For each amino acid and gene sequence information see SEQENCE LISTING (SEQUNCE ID NO.1-2)

And (3) sequentially connecting the sequences, and introducing different enzyme cutting sites at the connection positions of the sequences to form complete MesothelinCAR-aPD1 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 with T4 ligase (NEB) into the NotI-EcoRI site of retroviral rv (mscv) vector, 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-synthetic Meso CAR-aPD1 sequence to verify the correct sequence. The sequencing primer is as follows:

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. FIG. 2 shows a partial sequencing peak plot of the retroviral expression plasmid.

Example 3: retroviral packaging

1. Day 1 293T cells should be less than 20 passages, but overgrown. Plating the cells in 0.6 x 10^6cells/ml, adding 10ml of DMEM medium into a 10cm dish, fully mixing the cells, and culturing at 37 ℃ 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 retrointerbone, 10ug of Gag-pol, 6.25ug of VSVg, CaCl for each plasmid₂250ul，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-packed and stored at-80 ℃, and preheated fresh DMEM medium was continuously added.

Example 4: retroviral infection of human T cells

1. Separating with Ficcol separation solution (tertiary sea of Tianjin) to obtain relatively pure CD3+ T cells, and adjusting cell density to 1 × 10 with medium containing 5% AB serum X-VIVO (LONZA)⁶and/mL. Inoculating 1ml of cells into anti-human 50ng/ml CD3 antibody (Beijing Holland) and 50ng/ml CD28 antibody (Beijing Holland), adding 100IU/ml interleukin 2 (Beijing double Lut), stimulating and culturing for 48 hours, and infecting viruses;

every other day after T cell activation culture, the non-tissue treated plates were coated with 250. mu.l/well of a 24-well plate by Retronectin (Takara) diluted with PBS to a final concentration of 15. mu.g/ml. Protected from light and kept at 4 ℃ overnight for use.

And 3, after the T cells are activated and cultured for two days, taking out 2 coated 24-well plates, sucking and removing the coating solution, adding HBSS containing 2% BSA, and sealing at room temperature for 30 min. The volume of blocking solution was 500. mu.l per well, and the blocking solution was aspirated and the plate washed twice with HBSS containing 2.5% HEPES.

4. Adding the virus solution into each well, adding 2ml of virus solution into each well, centrifuging at 32 ℃ for 2h at 2000 g.

5. The supernatant was discarded, and activated T cells were added to each well of a 24-well plate at 1X 10⁶The volume is 1ml, and the culture medium is T cell culture medium added with IL-2200 IU/ml. Centrifuge at 30 ℃ for 10min at 1000 g.

6. After centrifugation, the plates were incubated at 37 ℃ in a 5% CO2 incubator.

7. 24h after infection, the cell suspension was aspirated and centrifuged at 1200rpm, 4 ℃ for 7 min.

8. After the cells are infected, the density of the cells is observed every day, and a T cell culture solution containing IL-2100 IU/ml is supplemented at a proper time to maintain the density of the T cells at 5 x 10⁵Cells were expanded at around/ml.

Example 5: flow cytometry for detecting proportion of infected T lymphocytes and expression of surface CAR protein

And respectively centrifuging to collect CAR-T cells and NT cells (control group) 72 hours after infection, washing with PBS 1 time, discarding supernatant, adding corresponding antibody, washing with PBS 30min in the dark, resuspending, and finally detecting with a flow cytometer. CAR + was detected by Anti-mouse IgG F (ab') antibody (Jackson Immunoresearch) and APC-Anti EGFR antibody.

The results of this example show in FIG. 3 that the expression efficiency of Mesothelin-aPD1 CAR + reached 58.31% and that of Mesothelin-tEGFR CAR + reached 65.61% 72 hours after infection of T cells with the retrovirus prepared in example 3.

Example 6: expression of secreted PD1 antibody in flow-assay virus

The Mesothelin-tEGFR/Mesothelin CAR-aPD1 viruses were incubated with 293T-PD1 (PD 1 overexpressing cells from this company) for 30min, and then stained with anti-human Fab for 30min before detection on the machine.

The results of this example are shown in FIG. 4, and the expression rate of secreted anti-PD1 detected by the flow-type results is 96.3%.

Example 7: detection of CD107a expression following coculture of CAR-T cells with target cells

1. Adding CAR-T/NT cells 2 x 10 to each well of a V-bottom 96-well plate⁵Sum target cells (SKOV3 and SKOV 3-Meso)/control cells (K562)2 x 10⁵Each cell was resuspended in 100ul of IL-2-free X-VIVO complete medium, BD GolgiStop (containing monesin, 1. mu.l BD GolgiStop per 1ml of medium) was added to each well, 2ul of CD107a antibody (1:50) was added to each well, incubated at 37 ℃ for 5 hours, and the cells were collected.

2. The samples were centrifuged to remove the medium, washed once with PBS, 400g, and centrifuged at 4 ℃ for 5 minutes. The supernatant was discarded, and appropriate amounts of specific surface antibodies CAR, CD3, CD4, CD8 were added to each tube, resuspended in a volume of 100ul, and incubated on ice for 30 minutes in the absence of light.

3. Cells were washed 1 time with 3mL PBS per tube and centrifuged at 400g for 5 min. The supernatant was carefully aspirated.

4. Appropriate amount of PBS was resuspended and CAR, CD3, CD4, CD8, CD107a were detected by flow cytometry.

The results of this example are shown in fig. 5. Figure 5 shows that Mesothelin-tfegfr CAR-T cells and Mesothelin-aPD1 CAR-T cells, after co-culturing with target cells, achieve approximately 60% expression of Mesothelin-tfegfr CAR-T in co-culture with two target cells CD107a, and approximately 70% expression of Mesothelin-aPD1 CAR-T cells CD107 a.

Example 8: detection of surface PD1 expression following coculture of CAR-T cells with target cells

1. Adding CAR-T/NT cells 2 x 10 to each well of a V-bottom 96-well plate⁵And (3) neutralizing target cells (SKOV3 and SKOV3-Meso), setting a group with K562 cells as a negative control, re-suspending to 100ul of an X-VIVO complete culture medium containing IL-2, incubating for 24 hours and 48 hours at 37 ℃, and collecting the cells.

2. The samples were centrifuged to remove the medium, washed once with PBS, 400g, and centrifuged at 4 ℃ for 5 minutes. The supernatant was discarded, and appropriate amounts of specific surface antibodies CD3, CAR, PD1 were added to each tube, resuspended in a volume of 100ul, and incubated on ice for 30min in the dark.

4. Appropriate amount of PBS was resuspended, CD3 and PD1 were detected by flow cytometry, and the expression of PD1 in CD3+ cell population was analyzed.

The results of this example are shown in figure 6. FIG. 6 shows that both CAR-T and target cells were co-cultured with a significant increase in surface PD 1.

Example 9: detection of tumor-specific cell killing after Co-culture of CAR-T cells with target cells

The K562 cells (negative control cells) were resuspended in serum-free medium (1640) at a cell concentration of 1X 10⁶Perml, the fluorescent dye BMQC (2,3,6,7-tetrahydro-9-bromomethyl-1H,5Hquinolizino (9,1-gh) coumarins) was added to a final concentration of 5. mu.M.

2. Mixing, and incubating at 37 deg.C for 30 min.

3. Centrifugation was carried out at 1500rpm for 5min at room temperature, the supernatant was discarded and the cells resuspended in cytotoxic medium (phenol red-free 1640+ 5% AB serum) and incubated for 60min at 37 ℃.

4. Fresh cytotoxic Medium cells were washed twice and resuspended in fresh cytotoxic Medium at a density of 1X 10⁶/ml。

5. Target cells (SKOV3 and SKOV3-Meso) were suspended in PBS containing 0.1% BSA at a concentration of 1X 10⁶/ml。

6. The fluorescent dye CFSE (fluorescent dye) (CFSE) was added to a final concentration of 1. mu.M.

7. Mixing, and incubating at 37 deg.C for 10 min.

8. After the incubation was completed, FBS in an equal volume to the cell suspension was added and incubated at room temperature for 2min to terminate the labeling reaction.

9. Cells were washed and resuspended in fresh cytotoxic medium at a density of 1X 10⁶/ml。

10. Effector T cells were washed and suspended in cytotoxic medium at a concentration of 5X 10⁶/ml。

11. In all experiments, the cytotoxicity of CAR-T cells was compared to that of uninfected negative control effector T cells (NTs).

CAR-T and NT, according to effector cell: the target cells were cultured in 5ml sterile test tubes (BD Biosciences) at a ratio of 5:1, 1:1, with two wells per set. In each co-culture group, 50,000 (50. mu.l) target cells and 50,000 (50. mu.l) negative control cells were present. A panel was set up to contain only target cells and negative control cells.

13. The co-cultured cells were incubated at 37 ℃ for 16 h.

14. After incubation was complete, cells were washed with PBS and immediately followed by rapid addition of 7-AAD (7-aminoactomycin D) at the concentrations recommended by the instructions and incubation on ice for 30 min.

15. The Flow-type detection is directly carried out without cleaning, and the data is analyzed by Flow Jo.

16. Assay the proportion of viable U266 target cells and viable K562 negative control cells after co-culture of T cells and target cells was determined using 7AAD negative viable cell gating.

17. For each group of co-cultured T cells and target cells

18. The% cytotoxic killer cells is 100-the calibrated target cell survival%, i.e. (number of live target cells without effector cells-number of live target cells with effector cells)/number of live control cells.

The results of this example are shown in figure 7. Figure 6 shows that Meso-aPD1 CAR-T kills around 20% of both target cells at an effective target ratio of E: T ═ 5: 1.

Sequence listing

<110> Shanghai Hengrunheng Dasheng Biotech Co., Ltd

<120> method for targeting chimeric antigen receptor of Mesothelin and expressing anti-PD1 antibody in combination and use thereof

<170> PatentIn version 3.3

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agcctggtga tcacactgta ctgcaggtcc aaaagatcta ggctgctgca ttctgattac 1020

atgaacatga cgccgcgccg ccctggtcca accagaaagc attatcagcc ctatgcaccc 1080

cctagagact ttgccgccta tcgttcgaag ttcagtgtcg tgaagagagg ccggaagaag 1140

ctgctgtaca tcttcaagca gcctttcatg aggcccgtgc agactaccca ggaggaagat 1200

ggatgcagct gtagattccc tgaagaggag gaaggaggct gtgagctgag agtgaagttc 1260

tcccgaagcg cagatgcccc agcctatcag cagggacaga atcagctgta caacgagctg 1320

aacctgggaa gacgggagga atacgatgtg ctggacaaaa ggcggggcag agatcctgag 1380

atgggcggca aaccaagacg gaagaacccc caggaaggtc tgtataatga gctgcagaaa 1440

gacaagatgg ctgaggccta ctcagaaatc gggatgaagg gcgaaagaag gagaggaaaa 1500

ggccacgacg gactgtacca ggggctgagt acagcaacaa aagacaccta tgacgctctg 1560

cacatgcagg ctctgccacc aagaagagct aagcgcggct caggcgcgac caacttttct 1620

ctgcttaagc aggcaggcga cgtggaagag aaccccgggc ccatgtacag aatgcagctg 1680

ttgtcttgta ttgccctttc tctcgccctc gtaacaaatt cacaagtcca attggtggag 1740

tctggcggtg gggtagttca gcccggccga tccctgcgcc tggactgcaa agcttctgga 1800

attacgttct caaactccgg aatgcactgg gtgcggcaag cacctgggaa agggctggag 1860

tgggttgcgg tgatttggta cgatggctct aagaggtact acgcagacag cgttaaaggc 1920

agatttacta tatcccgaga taactctaaa aatacgctct tcctccaaat gaatagcctg 1980

agggcagaag acacagccgt ttactattgt gctaccaatg atgattactg gggacagggc 2040

accctggtta ccgtaagttc cggcggtggt ggaagtggag gagggggatc cggaggcggg 2100

ggttctgaga tcgtcctgac ccagtctcca gccactctct ccctgtctcc aggcgagcgc 2160

gctacactga gttgtagagc ttcccagtcc gtgagcagct atctggcctg gtatcagcag 2220

aagcctgggc aggctccacg gttgctgatt tatgacgcct ccaaccgcgc gactgggata 2280

ccagctaggt tttccggatc aggcagcggc actgatttta cactgaccat ctcatctctc 2340

gagccggaag atttcgccgt ttactattgt caacagagtt caaactggcc acggacattc 2400

ggtcagggga ccaaggttga aattaag 2427

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<223> primer

<400> 3

agcatcgttc tgtgttgtct c 21

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence

<223> primer

<400> 4

tgtttgtctt gtggcaatac ac 22

Claims

1. A polynucleotide whose sequence is selected from the group consisting of:

(1) a polynucleotide sequence comprising the coding sequence of an anti-Mesothelin single-chain antibody, the coding sequence of a human CD8 a hinge region, the coding sequence of a human CD8 transmembrane region, the coding sequence of a human CD28 intracellular region, the coding sequence of a human 41BB intracellular region, the coding sequence of a human CD3 ζ intracellular region, and the coding sequence of an anti-human PD1 single-chain antibody, which are linked in this order; and

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

the amino acid sequence of the light chain variable region of the anti-Mesothelin single-chain antibody is shown as amino acids 23-128 of SEQ ID NO 1;

the amino acid sequence of the heavy chain variable region of the anti-Mesothelin single-chain antibody is shown as the amino acid 141-259 position of SEQ ID NO: 1;

the amino acid sequence of the human CD8 alpha hinge region is shown as the amino acids at the 260-position 306 of SEQ ID NO. 1;

the amino acid sequence of the transmembrane region of the human CD8 is shown as the amino acid at the 307-328 position of SEQ ID NO. 1;

the amino acid sequence of the intracellular region of the human CD28 is shown as the amino acid at the 329-369 th position of SEQ ID NO. 1;

the amino acid sequence of the human 41BB intracellular domain is shown as amino acids 370-417 of SEQ ID NO 1;

the amino acid sequence of the intracellular region of human CD3 zeta is shown as the 418-528 amino acid of SEQ ID NO. 1;

the amino acid sequence of the heavy chain variable region of the anti-human PD1 single-chain antibody is shown as the amino acid at the 575 th-687 th site of SEQ ID NO 1;

the amino acid sequence of the light chain variable region of the anti-human PD1 single-chain antibody is shown as the amino acid 703-809 of SEQ ID NO. 1.

2. The polynucleotide of claim 1, wherein the sequence of said polynucleotide further comprises a coding sequence for a signal peptide prior to the coding sequence for said anti-Mesothelin single chain antibody.

3. The polynucleotide of claim 2, wherein the amino acid sequence of said signal peptide is as set forth in amino acids 1-22 of SEQ ID No. 1.

4. The polynucleotide of claim 2,

the coding sequence of the signal peptide before the coding sequence of the anti-Mesothelin single-chain antibody is shown as the nucleotide sequence from 1 st to 66 th positions in SEQ ID NO. 2; and/or

The coding sequence of the light chain variable region of the anti-Mesothelin single-chain antibody is shown as the nucleotide sequence of 67 th to 384 th positions of SEQ ID NO. 2; and/or

The coding sequence of the heavy chain variable region of the anti-Mesothelin single-chain antibody is shown as the nucleotide sequence at the 421-777 th site of SEQ ID NO. 2; and/or

The coding sequence of the human CD8 alpha hinge region is shown as the 778-918 th nucleotide sequence of SEQ ID NO. 2; and/or

The coding sequence of the transmembrane region of the human CD8 is shown as the nucleotide sequence at the 919-984 position of SEQ ID NO. 2; and/or

The coding sequence of the intracellular region of human CD28 is shown as the nucleotide sequence of position 985-1107 of SEQ ID NO 2; and/or

The coding sequence of the intracellular region of the human 41BB is shown as the nucleotide sequence at position 1108-1251 of SEQ ID NO. 2; and/or

The coding sequence of the intracellular region of human CD3 zeta is shown as the 1252-1584 th nucleotide sequence of SEQ ID NO. 2; and/or

The coding sequence of the heavy chain variable region of the anti-human PD1 single-chain antibody is shown as the nucleotide sequence of 1723-bit 2061 of SEQ ID NO 2; and/or

The coding sequence of the light chain variable region of the anti-human PD1 single-chain antibody is shown as the nucleotide sequence at 2107-2427 of SEQ ID NO. 2.

5. A fusion protein comprising an anti-Mesothelin single-chain antibody, a human CD8 α hinge region, a human CD8 transmembrane region, a human CD28 intracellular region, a human 41BB intracellular region, a human CD3 ζ intracellular region, and an anti-human PD1 single-chain antibody, which are linked in this order;

the amino acid sequence of the human CD8 alpha hinge region is shown as the amino acid at the 260-position 306 of SEQ ID NO: 1;

the amino acid sequence of the intracellular region of the human CD28 is shown as the amino acid at the 329-369 th site of SEQ ID NO. 1;

the amino acid sequence of the intracellular region of the human 41BB is shown as the amino acids at the 370-th and 417-th positions of SEQ ID NO. 1;

6. The fusion protein of claim 5, wherein the fusion protein has the following characteristics: the fusion protein further comprises a signal peptide at the N-terminus of the anti-Mesothelin single chain antibody.

7. The fusion protein of claim 6, wherein the signal peptide has the amino acid sequence shown as amino acids 1-22 of SEQ ID NO 1.

8. A nucleic acid construct comprising the polynucleotide sequence of claim 4.

9. The nucleic acid construct of claim 8, wherein said nucleic acid construct is a vector.

10. The nucleic acid construct of claim 9, wherein the nucleic acid construct is a retroviral vector comprising a replication initiation site, a 3 'LTR, a 5' LTR, and the polynucleotide sequence of claim 4.

11. A retrovirus containing the nucleic acid construct of claim 8.

12. The retrovirus of claim 11, wherein the nucleic acid construct is a vector.

13. The retrovirus of claim 12, wherein the nucleic acid construct is a retroviral vector.

14. A genetically modified T-cell or a pharmaceutical composition comprising a genetically modified T-cell, wherein the cell comprises a polynucleotide according to claim 4, or comprises a nucleic acid construct according to any one of claims 8 to 10, or is infected with a retrovirus according to any one of claims 11 to 13.

15. Use of a polynucleotide sequence as claimed in claim 4, a fusion protein as claimed in any one of claims 5 to 6, a nucleic acid construct as claimed in any one of claims 8 to 10 or a retrovirus as claimed in any one of claims 11 to 13 in the preparation of an activated T cell.

16. Use of a polynucleotide sequence as defined in claim 4, a fusion protein as defined in any one of claims 5 to 6, a nucleic acid construct as defined in any one of claims 8 to 10, a retrovirus according to any one of claims 11 to 13, or a genetically modified T cell according to claim 14 or a pharmaceutical composition thereof in the manufacture of a medicament for the treatment of a Mesothelin-mediated disease.

17. The use of claim 16, wherein the Mesothelin-mediated disease is ovarian cancer, pleural mesothelioma, pancreatic cancer, and squamous carcinoma of the cervix, head, neck, vagina, lung and esophagus.

18. The use of claim 16, wherein the Mesothelin-mediated disease is malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer.