CN116178567A

CN116178567A - Chimeric antigen receptor targeting TGF beta RII and application thereof

Info

Publication number: CN116178567A
Application number: CN202211336473.XA
Authority: CN
Inventors: 王永生; 陈玥; 李丹
Original assignee: West China Precision Medicine Industrial Technology Institute; West China Hospital of Sichuan University
Current assignee: West China Precision Medicine Industrial Technology Institute; West China Hospital of Sichuan University
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2023-05-30
Also published as: WO2024087267A1

Abstract

The invention provides a chimeric antigen receptor which can recognize and bind TGF beta RII, and the chimeric antigen receptor which can recognize and bind tumor specific antigen or tumor related antigen are combined to construct a double-target chimeric antigen receptor modified immune cell, so that the chimeric antigen receptor has excellent anti-tumor effect and wide application prospect.

Description

Chimeric antigen receptor targeting TGF beta RII and application thereof

Technical Field

The invention belongs to the field of tumor treatment, and particularly relates to a chimeric antigen receptor targeting TGF beta RII and application thereof.

Background

Normally, the immune system recognizes and eliminates tumor cells in the tumor microenvironment, but for survival and growth, tumor cells can adopt different strategies, so that the immune system of the human body is inhibited and can not normally kill the tumor cells, thereby surviving at each stage of the anti-tumor immune response. Tumor immunotherapy is a therapeutic method for controlling and eliminating tumors by restoring the normal anti-tumor immune response of the body. Tumor immunotherapy includes monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapies, and the like. In recent years, tumor immunotherapy was evaluated in 2013 by journal of science as the most important scientific breakthrough in the year due to its excellent therapeutic effect and innovativeness, wherein chimeric antigen receptor-modified T cell (Chimeric antigen receptor T cells, CAR-T) therapy and CTLA-4, PD-1/PD-L1 antibody therapy are considered as three major advances in tumor immunotherapy.

The CAR-T therapy is to construct CAR-T by using antigen-antibody fragments and combining intracellular activation and proliferation signals of T cells, so that the T cells directly obtain the specific recognition capability of the antibody and become effector T cells which are not dependent on HLA (human leukocyte antigen) limitation. The designed CAR-T cells can be cultured and grown in a laboratory, and billions of amplified CAR-T cells are injected into a patient, and the injected T cells can proliferate in the patient, kill tumor cells with corresponding specific antigens, survive for a long time and form immune memory. There have also been studies on expression of chimeric antigen receptors in immune cells other than T cells, such as monocytes, natural killer cells, neutrophils, etc., so that these immune cells also specifically recognize tumor cells and perform the function of immunotherapy.

The focus of CAR-modified immune cells on therapies to achieve immunotherapy of tumors is the design of Chimeric Antigen Receptors (CARs). CARs are composed of three functional domains, an extracellular domain, a transmembrane domain, and an intracellular domain, respectively. In general, the extracellular domain is a single chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding an antigen, or the scFv is composed of a Hinge region (Hinge) that serves as a linkage. Intracellular domains include costimulatory domains and signaling domains. The transmembrane domain then links the extracellular domain to the intracellular domain of the CAR and anchors the receptor to the T cell membrane. Among them, scFv is a key to antigen recognition targeting, and is an important factor affecting therapeutic effects.

To date, there have been many reports of CAR-T targeting tumor specific antigens or tumor associated antigens, for example ROR1 is a transmembrane protein in the RTK (receptor tyrosine kinase) family. During embryonic development, it is involved in the development of the central nervous system, heart, lung and bone tissue. Postpartum, but with small expression on adipose tissue, pancreas, lung and developing B cells (pre-B cells, immature B cells), is not expressed in most normal tissues. However, there are expressions in many blood and solid malignancies. At present, the research shows that ROR1 has high expression with different degrees in malignant tumors such as chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin lymphoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, melanoma and the like. And in ovarian cancer, triple-negative breast cancer, the extent of ROR1 expression is associated with a poor prognosis. ROR1 tends to be expressed in poorly differentiated tumors, and cancer cells expressing ROR1 show a greater ability to invade metastasis and recurrence, and express marker genes associated with Epithelial-mesenchymal transition (EMT). Therefore, ROR1 CAR-T developed based on ROR1 targets has been studied more recently, and shows an effective targeted killing effect on tumor cells.

Unfortunately, chimeric antigen receptors and immune cells expressing chimeric antigen receptors have been slow to progress in clinical applications in solid tumors to date, with only limited therapeutic responses. One of the main reasons is the presence of a large number of immune cell-inhibiting cytokines in the tumor microenvironment, which prevent the immune cells expressing the chimeric antigen receptor from reaching and maintaining a high number level.

Among the many cytokines that suppress immune cells, transforming growth factor beta (TGF- β) is produced by tumor cells, regulatory T cells (regulatory T cells, treg), myelogenous suppressor cells (myeloid-derived suppressor cells, MDSC), and the like, and is expressed at high levels in most solid tumor lesions such as ovarian cancer, lung cancer, colorectal cancer, and the like. TGF-beta directly inhibits T cell activity by binding to TGF-beta receptors TGF-beta RI and TGF-beta RII. TGF- β binds to dimerized TGF- βRII and recruits TGF- βRI to form hetero-tetramers, thereby causing phosphorylation of intracellular SMAD2 and SMAD 3. Phosphorylated SMADs induces inhibitory transcription programs leading to reduced cytokine production, reduced cytotoxicity and T cell expansion upon inhibition of antigen binding. Furthermore, TGF- β can promote T cell differentiation into Treg, which in turn can produce TGF- β and further promote immunosuppression and tumor tolerance, so that immunosuppression of TGF- β is considered to be one of the main causes of failure of anti-tumor activity.

Therefore, on the basis of the existing tumor-targeted CAR-T, how to effectively avoid the influence of cytokines such as TGF-beta and the like inhibiting immune cells by adopting a reasonable means for further improving the curative effect of the CAR-T is still to be further explored.

Disclosure of Invention

The invention aims to provide a chimeric antigen receptor targeting TGF-beta RII and a double-target chimeric antigen receptor formed by combining the chimeric antigen receptor with a traditional tumor-targeted chimeric antigen receptor.

The present invention provides a chimeric antigen receptor which is a chimeric antigen receptor capable of recognizing and binding TGF-beta RII.

Further, the chimeric antigen receptor comprises an antigen recognition domain, a transmembrane domain, and an intracellular domain; the antigen recognition domain is a single chain antibody to tgfbetarii.

Further, the amino acid sequence of the single-chain antibody of TGF-beta RII is shown as SEQ ID NO. 19.

Further, the transmembrane domain is any one or more of CD28 transmembrane segment, CD8, cd3ζ, CD134, CD137, ICOS, DAP10, CD 27;

the intracellular domain comprises a costimulatory signaling domain that is any one or more of the CD28 intracellular segments, 4-1BB, ICOS, CD27, OX40, myD88, CD 40.

Further, the transmembrane domain is CD8, and the amino acid sequence is shown as SEQ ID NO. 21; the intracellular domain is 4-1BB, and the amino acid sequence is shown as SEQ ID NO. 23.

Further, the chimeric antigen receptor further comprises a signal peptide 2, and preferably, the amino acid sequence of the signal peptide 2 is shown as SEQ ID NO. 15.

Furthermore, the chimeric antigen receptor further comprises a protein tag sequence, preferably a MYC tag sequence, and the amino acid sequence is shown as SEQ ID NO. 17.

Still further, the chimeric antigen receptor comprises the following fragments, linked in sequence: signal peptide 2, protein tag sequence, single chain antibody of TGF beta RII, CD8 transmembrane domain, 4-1BB costimulatory signal domain;

preferably, the chimeric antigen receptor amino acid sequence is shown in SEQ ID NO. 30.

The invention also provides a double-target chimeric antigen receptor, which comprises a chimeric antigen receptor 1 and a chimeric antigen receptor 2;

wherein chimeric antigen receptor 1 is a chimeric antigen receptor capable of recognizing and binding a tumor-specific antigen or a tumor-associated antigen; chimeric antigen receptor 2 is a chimeric antigen receptor according to any one of claims 1 to 7.

Further, the double-target chimeric antigen receptor is a combination of a chimeric antigen receptor 1 and a chimeric antigen receptor 2, or is formed by connecting the chimeric antigen receptor 1 and the chimeric antigen receptor 2.

Furthermore, the double-target chimeric antigen receptor is formed by connecting a chimeric antigen receptor 1 and a chimeric antigen receptor 2 through polypeptide fragments; preferably, the polypeptide fragment is a self-cleaving peptide fragment, more preferably a self-cleaving peptide P2A fragment, having the amino acid sequence shown in SEQ ID NO. 13.

Further, the chimeric antigen receptor 1 includes an antigen recognition domain, a transmembrane domain, and an intracellular domain; the antigen recognition domain is a single chain antibody capable of recognizing and binding to a tumor-specific antigen or a tumor-associated antigen;

preferably, the tumor-specific antigen or tumor-associated antigen is any one or more of CD19, CD20, MUC1, ROR1, EGFR, EGFRvIII, HER2, ERBB3, ERBB4, VEGFR1, VEGFR2, epCAM, CD44, IGFR.

Further, the single-chain antibody is a ROR1 single-chain antibody; preferably, the amino acid sequence of the single chain antibody of ROR1 is shown as SEQ ID NO. 3.

Still further, the transmembrane domain is any one or more of CD28 transmembrane segment, CD8, cd3ζ, CD134, CD137, ICOS, DAP10, CD 27;

the intracellular domain comprises a costimulatory signaling domain and a signaling domain, the costimulatory signaling domain being any one or more of the CD28 intracellular segment, 4-1BB, ICOS, CD27, OX40, myD88, CD 40; the signal transduction domain is cd3ζ or fceri.

Further, the transmembrane domain is a CD28 transmembrane segment, and the amino acid sequence is shown as SEQ ID NO. 7; the co-stimulatory signal domain is a CD28 intracellular segment, and the amino acid sequence is shown as SEQ ID NO. 9; the signal transduction domain is CD3 zeta, and the amino acid sequence is shown as SEQ ID NO. 11.

Further, the chimeric antigen receptor 1 further comprises a signal peptide 1 and a hinge region; preferably, the amino acid sequence of the signal peptide 1 is shown as SEQ ID NO.1, and the amino acid sequence of the hinge region is shown as SEQ ID NO. 5.

Still further, the chimeric antigen receptor 1 comprises the following fragments, which are sequentially linked: signal peptide 1, single chain antibody of ROR1, hinge region, CD28 transmembrane domain, CD28 intracellular segment costimulatory signal domain, cd3ζ signal transduction domain;

preferably, the amino acid sequence of the chimeric antigen receptor 1 is shown in SEQ ID NO. 29.

Further, the amino acid sequence of the double-target chimeric antigen receptor is shown as SEQ ID NO. 26.

The invention also provides a gene which codes for the chimeric antigen receptor or codes for the double-target chimeric antigen receptor; preferably, the nucleotide sequence of the gene for encoding the chimeric antigen receptor is shown as SEQ ID NO.27, and the nucleotide sequence of the gene for encoding the double-target chimeric antigen receptor is shown as SEQ ID NO. 25.

The invention also provides a vector which contains the gene; the vector is a plasmid or a virus.

The invention also provides a host cell which expresses the chimeric antigen receptor or expresses the chimeric antigen receptor with double targets, preferably a host cell containing the vector.

Further, the host cell is an immune response cell, preferably at least one of a T cell, a monocyte, a natural killer cell, or a neutrophil, and more preferably a T cell.

The invention also provides application of the chimeric antigen receptor, the double-target chimeric antigen receptor, the gene, the vector or the host cell in preparing medicines for preventing and/or treating tumors.

The invention has the beneficial effects that: the present invention provides a chimeric antigen receptor that targets tgfbetarii. On one hand, the anti-tumor targeting peptide can bind with TGF beta RII of tumor cells to enhance the recognition targeting effect of immune cells on tumors, on the other hand, the anti-tumor targeting peptide can also target the TGF beta RII of immune cells to realize the blocking of the TGF beta RII of the immune cells, and prevent the TGF beta from binding with TGF beta receptors of the immune cells so as to relieve the inhibition effect of the TGF beta on the immune cells, and meanwhile, the chimeric antigen receptor sequence of the targeting TGF-beta RII does not contain CD3 zeta activation signals and does not activate T cells to execute cytotoxicity.

Furthermore, the chimeric antigen receptor of the targeting TGF beta RII is used in combination with the chimeric antigen receptor of the traditional tumor targeting, so that the immune cells modified by the chimeric antigen receptor of the double target spots are constructed, the tumor specific targeting can be realized through the chimeric antigen receptor of the tumor targeting, the tumor is killed, and the inhibition of the cytokine TGF beta on the immune cells can be lightened while the tumor recognition targeting effect is enhanced by the effect of the chimeric antigen receptor of the targeting TGF beta RII, so that the synergistic anti-tumor effect is realized.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a diagram showing the structural composition of chimeric antigen receptor functional units in the viral vectors of the present invention. Wherein: a is a chimeric antigen receptor of a single-target ROR1, and B is a double-target chimeric antigen receptor.

Figure 2 shows the expression of a CAR of the invention on T cells.

FIG. 3 shows the expression of tumor cell surface antigens.

FIG. 4 shows IFN-gamma release levels after co-culture of CAR-T with tumor cells.

FIG. 5 shows IL-2 release levels after co-culture of CAR-T with tumor cells.

FIG. 6 shows TNF- α release levels after co-culture of CAR-T with tumor cells.

FIG. 7 is a cellular assay for IFN- γ secretion after co-culture of CAR-T with tumor cells.

FIG. 8 is a T cell granzyme B release assay after in vitro killing of tumor cells by CAR-T cells.

FIG. 9 shows T cell proliferation after co-culturing CAR-T cells with tumor cells.

FIG. 10 shows the detection of intracellular pSMAD2/3 signaling pathways of T cells after in vitro killing of tumor cells by CAR-T cells.

FIG. 11 shows the growth inhibition of SKOV3 ovarian cancer peritoneal disseminated model by CAR-T cells in vivo.

Detailed Description

The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.

EXAMPLE 1 Synthesis of double-target chimeric antigen receptor full-Length Gene, construction of recombinant plasmid vector was completed

The expression cassette of the dual-target chimeric antigen receptor prepared in this example was first prepared. The expression frame is sequentially from 5 ends to 3 ends: the signal peptide 1-Anti-ROR1 scFv-range-CD 28 transmembrane segment-CD 28 intracellular segment-CD 3 zeta-P2A-signal peptide 2-MYC protein tag-Anti-TGF-beta RII scFv-CD8 transmembrane domain-4-1 BB intracellular segment.

Wherein the sequence of each segment is as follows:

amino acid sequence of signal peptide 1 of chimeric antigen receptor 1 (SEQ ID No. 1):

MLLLVTSLLLCELPHPAFLLIP

coding nucleotide sequence (SEQ ID NO. 2):

ATGCTGCTCCTGGTGACCTCCCTCCTGCTGTGCGAGCTGCCCCACCCCGCCTTCCTGCTGATTCCC

amino acid sequence of single chain antibody of ROR1 (Anti-ROR 1 scFv) (SEQ ID No. 3):

QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGGGSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG

coding nucleotide sequence (SEQ ID NO. 4):

CAAGAGCAGCTGGTGGAGAGCGGGGGCAGACTGGTGACCCCCGGCGGCAGCCTGACCCTGAGCTGTAAGGCTAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTGAGACAAGCCCCTGGGAAGGGCCTGGAATGGATCGCCACCATCTACCCTAGCAGCGGCAAGACCTACTACGCTACCTGGGTGAACGGCAGATTCACCATCTCCTCCGACAACGCTCAGAACACCGTGGACCTGCAGATGAACAGCCTGACCGCCGCCGACCGGGCCACCTACTTCTGCGCTAGAGACAGCTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGGCCCGGCACCCTCGTGACAATTAGCAGCGGCGGGGGCGGCAGCGGCGGGGGCGGCAGCGGGGGGGGGGGCTCCGAGCTGGTCCTGACACAGAGCCCTAGCGTGAGCGCCGCTCTGGGCAGCCCCGCCAAGATCACCTGCACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCAGCTGCAAGGCGAGGCCCCTAGATATCTGATGCAAGTGCAGAGCGACGGCAGCTACACCAAGAGACCCGGCGTGCCCGACCGGTTCAGCGGCTCCTCCTCCGGCGCCGACAGATACCTCATCATCCCTAGCGTGCAAGCCGACGACGAGGCCGACTACTACTGCGGCGCCGACTACATCGGCGGCTACGTGTTTGGCGGGGGCACACAGCTGACCGTGACCGGC

amino acid sequence (SEQ ID NO. 5) of Hinge region (Hinge): ESKYGPPCPPCP

Coding nucleotide sequence (SEQ ID NO. 6):

GAGAGCAAATACGGCCCCCCCTGCCCCCCCTGTCCT

amino acid sequence of CD28 transmembrane segment (SEQ ID No. 7):

MFWVLVVVGGVLACYSLLVTVAFIIFWV

coding nucleotide sequence (SEQ ID NO. 8):

ATGTTCTGGGTGCTGGTGGTCGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG

amino acid sequence of the CD28 intracellular segment (SEQ ID No. 9): RSKRSRGCHSDYMNMTPRRPG PTRKHYQPYA PPRDFAAYRS

The coding nucleotide sequence (SEQ ID NO. 10):

AGAAGCAAGAGAAGCAGAGGCTGCCACAGCGACTACATGAACATGACCCCTAGAAGACCCGGCCCCACAAGAAAGCACTATCAGCCCTACGCCCCCCCTAGAGACTTCGCCGCCTACAGAAGC

amino acid sequence of CD3 ζ (SEQ ID No. 11):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

the coding nucleotide sequence (SEQ ID NO. 12):

AGAGTGAAGTTCAGCAGAAGCGCCGACGCCCCCGCCTATCAGCAAGGGCAGAATCAGCTGTACAATGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCTAGAAGAAAGAACCCCCAAGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGACGGAGAGGCAAGGGCCACGACGGCCTGTACCAAGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAAGCCCTGCCCCCTAGA

amino acid sequence of P2A (SEQ ID No. 13): GSGATNFSLLKQAGDVEENPGP

Coding nucleotide sequence (SEQ ID NO. 14):

GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGGGATGTGGAAGAAAACCCCGGCCCC

amino acid sequence (SEQ ID No. 15) of CD8 signal peptide (signal peptide 2):

MALPVTALLLPLALLLHAARP

the coding nucleotide sequence (SEQ ID NO. 16):

ATGGCCCTGCCCGTGACCGCCCTGCTCCTGCCCCTGGCCCTGCTCCTGCATGCTGCTAGACCC

amino acid sequence of MYC tag (SEQ ID No. 17): EQKLISEEDL

The coding nucleotide sequence (SEQ ID NO. 18):

GAGCAGAAGCTGATCAGCGAGGAGGACCTG

amino acid sequence of Anti-TGF-beta RII-scFv (SEQ ID NO. 19):

QLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDEAVYYCQQRSNWPPTFGQGTKVEIK

the coding nucleotide sequence (SEQ ID NO. 20):

CAGCTCCAAGTGCAAGAGAGCGGCCCTGGGCTGGTGAAGCCTAGCGAGACCCTGAGCCTCACATGCACCGTGAGCGGCGGCAGCATCAGCAACAGCTACTTCAGCTGGGGCTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATTGGCAGCTTCTACTACGGCGAGAAAACCTACTACAACCCTAGCCTGAAGAGCAGAGCCACCATCAGCATCGACACAAGCAAGAGCCAATTCAGCCTGAAGCTGAGCAGCGTCACAGCCGCCGACACCGCCGTGTATTACTGCCCTAGAGGCCCCACCATGATCAGAGGCGTGATCGATAGCTGGGGGCAAGGCACACTGGTGACCGTCAGCAGCGGCGGCGGGGGGAGCGGGGGGGGCGGCAGCGGCGGGGGCGGCTCCGAAATTGTCCTGACACAGAGCCCTGCCACACTGTCCCTCAGCCCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCTAGCCAAAGCGTGAGAAGCTACCTGGCCTGGTACCAACAAAAGCCCGGCCAAGCCCCTAGACTGCTGATCTACGACGCTAGCAACAGAGCCACCGGCATCCCCGCTAGATTCTCCGGCAGCGGGAGCGGCACCGACTTCACCCTCACCATCAGCAGCCTCGAGCCCGAGGACGAGGCCGTGTACTACTGTCAGCAGAGAAGCAACTGGCCCCCCACCTTCGGCCAAGGCACCAAGGTGGAGATTAAG

amino acid sequence of CD8 transmembrane domain (SEQ ID No. 21):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

the coding nucleotide sequence (SEQ ID NO. 22):

ACCACAACCCCTGCCCCTAGACCCCCTACACCTGCCCCCACCATTGCTAGCCAACCCCTCAGCCTCAGACCCGAAGCCTGTAGACCTGCTGCCGGCGGCGCTGTGCACACAAGAGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTCCTGAGCCTGGTGATCACCCTGTACTGC

amino acid sequence of the intracellular portion of 4-1BB (SEQ ID NO. 23):

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

the coding nucleotide sequence (SEQ ID NO. 24):

AAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAAGAGGAGGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGCGAGCTG

complete nucleotide sequence encoding the double-target chimeric antigen receptor (SEQ ID NO. 25):

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wherein the italic part is the nucleotide sequence encoding chimeric antigen receptor 1 (SEQ ID NO. 26), the underlined part is the nucleotide sequence encoding the polypeptide linking chimeric antigen receptor 1 and chimeric antigen receptor 2 (SEQ ID NO. 14), and the remaining part is the nucleotide sequence encoding chimeric antigen receptor 2 (SEQ ID NO. 27)

Amino acid sequence of the complete double-target chimeric antigen receptor (SEQ ID NO. 28):

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wherein the italic part is the amino acid sequence of chimeric antigen receptor 1 (SEQ ID NO. 29), the underlined part is the amino acid sequence of the polypeptide linking chimeric antigen receptor 1 and chimeric antigen receptor 2 (SEQ ID NO. 13), and the remaining part is the amino acid sequence of chimeric antigen receptor 2 (SEQ ID NO. 30).

The nucleotide sequence (SEQ ID NO. 25) encoding the complete double-target chimeric antigen receptor was synthesized and inserted into the EcoRI-BamH1 site of the lentiviral pWPXLd vector, which was preceded by the EF-1 alpha promoter, transformed into E.coli competent cells, after correct sequencing, the plasmid was extracted and purified using the Qiagen plasmid purification kit, the purification procedure being as described in the kit instructions, and the high quality plasmid of the recombinant expression vector was obtained, the inserted fragment of interest having the structure as described in FIG. 1B.

Example 2 transformation of cells with recombinant vectors

1. Culturing and passaging 293T cells:

opening the biosafety cabinet, wiping the table surface with 75% alcohol cotton, and placing a pipette, a pipette gun, a gun tip box, a 15ml centrifuge tube, a centrifuge tube rack, and 10cm ² The new cell culture dish is placed in the biosafety cabinet, the cabinet door is closed, the ultraviolet switch of the biosafety cabinet is opened, and the cell culture dish is irradiated for half an hour to sterilize. DMEM and pancreatin containing 10% foetal calf serum and 100U/ml penicillin streptomycin were preheated in a 37℃water bath. Opening the biosafety cabinet, opening the ventilation switch, and separating 293T cell culture dish which has grown to 80% -90% from 37 ℃ and 5% CO ₂ Is taken out from the incubator and placed in a biosafety cabinet. Both hands, medium bottle mouth, pipette nozzle, etc. are sterilized with 75% alcohol. The culture medium in the dish was removed by a sterile pipette and discarded in a waste liquid jar. 2ml of pancreatin was added dropwise to the dishes, and the cells were observed under a microscope until the cells were rounded off and isolated, and pancreatin was removed by aspiration. 6-8ml of fresh complete medium was added to the dish and the cells gently blown down. The cell suspension was separated into other dishes and medium was added to reach 10ml per dish. Shaking the culture dish for several times, shaking the cells uniformly, observing under a mirror, and placing the cells in a 37 ℃ incubator. After 24 hours, observing the cell state, and carrying out the next subculture when the cell grows to 80% -90%.

2. Obtaining a lentivirus stock solution:

day 1, floor. 293T cells in good 90% density status were digested, passaged 1:3, approximately 4X10 ⁶ cells/10ml/10cm dish, 5% CO ₂ Culturing overnight at 37deg.C. The cell density is about 70-90% (not more than 90%) for 24h.

Day 2: and (5) transfection. All reagents were equilibrated to room temperature prior to transfection.

The transfection procedure was as follows:

a. the following DNA mixtures (per 10cm plate) were prepared in 10ml BD tubes, 3. Mu.g of psPAX2 (packaging plasmid); pMD.2G (evelope plasma) 1.5 μg; pWPXLd (lentivirus vector, prepared from example 1) 6. Mu.g.

b. The DMEM medium was added to a volume of 980. Mu.l and mixed well.

20 μl of Highgene transfection reagent (Ebolac) was slowly added to the DNA solution, and gently swirled until well mixed.

d. Standing at room temperature for 15min.

e. 1ml of the transfection mixture was dropped into a plate, gently shaken and mixed in a "cross" manner (10 times each) and returned to 5% CO ₂ Incubator at 37 ℃. After 8-12h, the medium was aspirated, and the medium was changed with pre-warmed DMEM complete medium and returned to 5% CO ₂ Incubator at 37 ℃ to 48h.

Day 4: 48h after transfection. Cell supernatants were harvested by addition of 10ml of pre-warmed DMEM complete medium, 5% CO ₂ Culturing at 37 ℃; the virus supernatant was filtered through a 0.45 μm filter and stored at 4℃for up to 1 week.

Day 5: after 72h of transfection, the virus supernatant was collected, filtered through a 0.45 μm filter and stored at 4 ℃.

3. Concentration of lentiviruses:

instrument: the ultra-high speed centrifugal instrument, the matched rotor and sleeve, the ultra-high speed centrifugal tube and the balance for balancing. And (5) sterilizing the sleeve and the balance under the biosafety cabinet ultraviolet instrument. After ensuring that there are no drops in each sleeve, the appropriate centrifuge tube is placed into the sleeve. The virus suspension filtered with a 0.45 μm filter was added to the centrifuge tube. Each centrifuge tube containing the virus suspension was tightly trimmed using a balance with an accuracy of 0.001g or more, and after the sleeve cap was closed, the balance was again used to verify complete trimming. The trimmed sleeves are loaded into the centrifuge rotor and ready for centrifugation. And (3) centrifuging: the centrifugation conditions were: 20 ℃,70000g and 2h. After the centrifuge speed increased to 70000g, it was removed. After centrifugation, the medium was decanted and the centrifuge tube was inverted and the remaining medium was blotted on sterile filter paper. Viral pellet was resuspended using PBS. The resuspended virus was aliquoted into 1.5ml EP tubes and stored in a refrigerator at-80℃until use.

4. Isolation of human peripheral blood T lymphocytes:

peripheral blood was collected using heparin sodium anticoagulation tube. The ratio of lymphocyte separation liquid volume to blood volume is 1:1, peripheral blood is slowly added to lymphocyte separation liquid, and the mixture is centrifuged. The conditions were 1000g,30min,18℃and a 1 acceleration/deceleration. After centrifugation, the blood was seen to be divided into 4 layers, with the PBMC layer being the middle white cloud flocculent layer. The gun tip slowly suctions the middle white cloud-like layer. The aspirated cells were added to 10ml of X VIVO medium and centrifuged at 300g for 10min. Discarding the supernatant, performing red cracking with 10ml of red cracking liquid, mixing uniformly, and standing for 5min. Centrifuge 300g,10min. The supernatant was discarded, resuspended in 10ml of X VIVO medium and counted. Centrifuge 300g,10min. The supernatant was discarded, lymphocytes were resuspended in 5% human serum, 100IU/ml IL-2X VIVO medium, and corresponding numbers of CD3/CD 28-stimulated beads were added depending on the number of cells. 5% CO ₂ Culturing at 37 ℃.

5. Virus infection of T cells

The day before infection of T cells with virus, twelve well plates (50. Mu.g/ml) were coated with a dilution of Retronectin (recombinant human fibronectin), 1ml per well. The well plate was sealed and placed at 4℃overnight for further use. On the day of infection, retronectin dilutions were blotted off and blocked for 30min with 2% BSA (bovine serum albumin) solution. The BSA liquid was aspirated and rinsed once with PBS. 1X 10 wells ⁶ The individual cells, as well as the virus concentrate, were placed in a centrifuge for centrifugation at 32℃for 1000g for 2h. Taking out the pore plate after centrifugation, and returning to 37 ℃ and 5% CO ₂ And (5) culturing the cells in a cell culture box. After 24h, the complete medium was changed.

6. Detecting T cell surface CAR molecule expression:

ROR1 CAR virus and ROR 1-tgfbetarii double CAR virus were infected with T cells for 72h and flow cytometry was performed to detect CAR expression (figure 2).

As shown in fig. 2, ROR1 CAR-T expressed the ROR1 CAR gene, while ROR 1-tgfbetarii CAR-T expressed the ROR1 CAR gene and scFv against tgfbetarii, indicating that the dual CAR construction was successful.

Comparative example 1 preparation of ROR1 CAR-T

The nucleotide sequence encoding chimeric antigen receptor 1 (signal peptide 1-Anti-ROR1 scFv-range-CD 28 transmembrane segment-CD 28 intracellular segment-CD 3 ζ, see FIG. 1A) was prepared according to the method of example 1, and then CAR-T, ROR1 CAR-T, was prepared only for chimeric antigen receptor 1 according to the method of example 2.

The following experiments prove the beneficial effects of the invention.

Experimental example 1, detection of tumor cell surface antigen expression

The expression levels of the different tumor cell lines ROR1 and TGF-beta RII were examined using a flow cytometer.

The method comprises the following steps: ovarian cancer cells SKOV3, breast cancer cells MDA-MB-231, and lung cancer cells a549 and ovarian cancer cells a2780 were incubated with PE-labeled antibodies specifically recognizing human ROR1 and APC-labeled antibodies specifically recognizing human tgfbetarii.

Results: and finally, detecting by a flow cytometer, and finding that the expression levels of the ROR1 and the TGF beta RII of different tumor cells are different. Wherein the MDA-MB-231 cells have the highest ROR1 and TGF-beta RII expression level; SKOV3 moderately expresses ROR1 and TGF beta RII; a549 moderately expresses ROR1, hardly expresses tgfbetarii; a2780 cells were negative for ROR1 expression and moderately expressed TGF-beta RII (see FIG. 3).

Experimental example 2 functional detection of double-Targeted CAR-T

1. Cytokine release detection after in vitro killing of tumor cells by CAR-T cells

The method comprises the following steps: t cells (ROR 1 CAR-T (comparative example 1), ROR1 TGF-beta RII CAR-T (example 2), control T cell) and 1X 10 ⁴ Target cells positive for ROR1 expression: ovarian cancer cell line SKOV3, breast cancer cell line MDA-MB-231, lung cancer cell line A549, or ROR1 expression negative target cells: ovarian cancer cell line A2780 was co-cultured with a ratio of effector cells to target cells (effective target ratio) of 1:1 or 2:1, and cultured for 24h in the absence of TGF- β or in the presence of 5ng/ml TGF- β. Detection of IFN-gamma (FIG. 4), IL-2 (FIG. 5) and TNF-alpha (FIG. 6) levels in cell culture supernatants using ELISA。

Results: compared with a single ROR1 CAR-T group, after the ROR1-TGF beta RII CAR-T group is co-cultured with tumor cells positive to ROR1 expression, more IFN-gamma, IL-2 and TNF-alpha cytokines can be released, and the killing capacity is stronger. And after co-culture with tumor cells negative for ROR1 expression, the double-targeted CAR-T also hardly releases cytokines, and has no off-target toxicity.

2. Detection of intracellular INF-gamma release of T cells after in vitro killing of tumor cells by CAR-T cells

The method comprises the following steps: will be 1X 10 ⁶ Individual T cells (ROR 1 CAR-T group, ROR 1-tgfbetarii CAR-T group, control T cell group) were co-cultured with breast cancer cell line MDA-MB-231, ovarian cancer cell line SKOV3 at an effective target ratio of 3:1 for 24h. The level of intracellular IFN-gamma release by CD3 positive T cells was detected using flow cytometry (FIG. 7).

Results: compared with a single ROR1 CAR-T group, after the ROR1-TGF beta RII CAR-T group is co-cultured with tumor cells positive for ROR1 expression, the number of cells secreting intracellular IFN-gamma is more, and the killing capacity is stronger.

3. T cell granzyme B release detection after in vitro killing of tumor cells by CAR-T cells

The method comprises the following steps: will be 1X 10 ⁶ Individual T cells (ROR 1 CAR-T group, ROR 1-tgfbetarii CAR-T group, control T cell group) were co-cultured with lung cancer cell line a549 at an effective target ratio of 3:1 for 4h. CD8 positive T cells were tested for intracellular granzyme B release levels using flow cytometry (fig. 8).

Results: compared with a pure ROR1 CAR-T group, the ROR1-TGF beta RII CAR-T group can release granzyme B more quickly after being co-cultured with tumor cells positive for ROR1 expression, and has stronger cytotoxicity.

4. Proliferation of T cells after Co-culture of CAR-T cells and tumor cells

The method comprises the following steps: labeling T cells with FAR RED cell tracer fluorescent dye; the cell tracing fluorescent dye is attached to the cell membrane, if the cell divides, the membrane dye on one cell is distributed to the membranes of two new cells along with the division, so that the fluorescence on the cell is reduced by half; the weaker the fluorescence intensity, the less fluorescent dye on the cell membrane, the more cell division and proliferation.

Co-culturing two CAR-T cells and breast cancer cells MDA-MB-231, wherein the effective target ratio is 3 to 1; after 24h, the fluorescence of the T cells was detected.

Results: as shown in fig. 9, the horizontal axis represents FAR RED fluorescence signal intensity, the vertical axis represents cell number in a certain intensity interval, and the fluorescence signal is weaker as the peak formed by the curve is closer to the origin. From the figure, it can be seen that in the CD8 and CD4 positive cell populations, the ROR 1-TGF-beta RII CAR-T division degree is greater than that of the ROR1 CAR-T group, namely the group has stronger cell proliferation capacity.

4. Detection of intracellular pSMAD2/3 signal path of T cells after in vitro killing of tumor cells by CAR-T cells

The method comprises the following steps: will be 1X 10 ⁶ Individual T cells (ROR 1 CAR-T group, ROR 1-tgfbetarii CAR-T group, control T cell group) were co-cultured with ovarian cancer cell line SKOV3 at an effective target ratio of 3:1, cultured for 24h in the absence of TGF- β or in the presence of 5ng/ml, 10ng/ml, 20ng/ml TGF- β. The expression level of TGF- β downstream signaling pathway pSMAD2/3 in CAR-positive or CAR-negative T cells was detected using flow cytometry (fig. 10).

Results: after co-incubation of CAR-T with tumor cells in the presence of TGF- β, TGF- βrii dimerizes on the T cell surface and binds to TGF- β, forming heterotetramers with TGF- βri, thereby activating downstream signaling pathways, phosphorylating intracellular SMAD proteins. The dual-targeting ROR1-TGF beta RII CAR-T group can block the TGF beta RII on the surface of the human body because the human body expresses scfv of the anti-TGF beta RII, thereby preventing the TGF beta RII from combining with the TGF beta and blocking the phosphorylation of intracellular SMAD proteins, thus resisting the inhibition of the TGF beta on T cells. The experimental results show that the dual-targeting ROR 1-tgfbetarii CAR-T group, in the presence of TGF- β, the T cell intracellular pSMAD2/3 expression of the CAR positive fraction was lower than that of the ROR1 CAR-T group alone.

5. The method for inhibiting the growth of the CAR-T cells on the SKOV3 ovarian cancer peritoneal dissemination model in vivo comprises the following steps: and (3) using NSG immunodeficiency mice, using SKOV3 (SKOV 3-luc) cells stably expressing luciferase to establish a human ovarian cancer peritoneal cavity spreading model, and performing peritoneal injection on reinfusion T cells to evaluate the growth inhibition effect of the T cells on tumors.

(1) NSG mice of 5 weeks of age were purchased and kept in the animal house for 1 week to adapt to the environment, and 5X 10 mice were kept ⁵ The individual SKOV3-luc cells were intraperitoneally injected to establish the model. One week later bioluminescence imaging was performed to detect tumor burden in vivo. Mice were divided into four groups on average, based on tumor burden, into ROR1 CAR-T, ROR 1-TGF-beta RII CAR-T, unmodified control T cells, and PBS, with 5 mice per group. The preparation of CAR-T cells was performed as in example 2.

(2) CAR-T groups were infused back 2X 10 per group by intraperitoneal injection ⁶ The number of CAR positive T cells, unmodified control T cells, was the same as the total amount of CAR-T groups, and cells were resuspended in 100 μl PBS and injected intraperitoneally back into mice.

(3) Tumor burden was assessed by intraperitoneal injection of fluorescein per peripheral mouse and imaging using a bioluminescence imaging system.

Results: both CAR-T inhibited SKOV3 tumor cell growth and ROR 1-tgfbrii CAR-T had a stronger tumor inhibitory effect in vivo compared to PBS, unmodified control T cell group (fig. 11).

In conclusion, compared with the unmodified ROR1 CAR-T, the double-target CAR-T has stronger killing activity on tumor cells, is characterized in that more cytokines can be released, and has stronger release of granzyme B; meanwhile, the preparation has stronger proliferation activity and is beneficial to the long-term survival of the CAR-T. Also exhibits stronger anti-tumor effect in the ovarian cancer peritoneal metastasis model. This shows that the double-target CAR-T designed in the invention can significantly increase the curative effect of T cells on solid tumors when TGF-beta is inhibited.

The principle of the invention is that a chimeric antigen receptor targeting TGF-beta RII is designed, on one hand, the chimeric antigen receptor targeting TGF-beta RII can be combined with TGF beta RII of tumor cells to enhance the recognition targeting effect of immune cells on tumors, on the other hand, the chimeric antigen receptor targeting TGF-beta RII can also be used for targeting TGF beta RII of immune cells to realize the blocking of the TGF beta RII of the immune cells and prevent the TGF beta from combining with the TGF beta receptor of the immune cells so as to lighten the inhibition effect of the TGF beta on the immune cells, and meanwhile, the chimeric antigen receptor sequence targeting the TGF-beta RII does not contain CD3 zeta activation signals and does not activate T cells to execute cytotoxicity.

Based on this principle, the chimeric antigen receptor of the target TGF- βrii of the present invention may be combined with the tumor-targeted chimeric antigen receptor (e.g., the chimeric antigen receptor of ROR 1) by means of self-cleaving peptide, and modified immune cells (e.g., T cells) to prepare CAR-T, but at the same time, according to the basic common sense in the art, the combined forms thereof may be reasonably changed, for example, plasmids expressing the chimeric antigen receptor of the target TGF- βrii and plasmids expressing the tumor-targeted chimeric antigen receptor (e.g., the chimeric antigen receptor of ROR 1) are transferred into immune cells (e.g., T cells), so that the immune cell surface is modified with two chimeric antigen receptors simultaneously, and the two chimeric antigen receptors are not connected, and may also form a combined form, thereby achieving the same synergistic antitumor effect as the embodiment of the present invention.

Claims

1. A chimeric antigen receptor, characterized in that it is a chimeric antigen receptor capable of recognizing and binding tgfbetarii.

2. The chimeric antigen receptor of claim 1, comprising an antigen recognition domain, a transmembrane domain, an intracellular domain; the antigen recognition domain is a single chain antibody to tgfbetarii.

3. A chimeric antigen receptor according to claim 2, wherein the single chain antibody amino acid sequence of tgfbetarii is shown in SEQ ID No. 19.

4. The chimeric antigen receptor of claim 2, wherein the transmembrane domain is any one or more of CD28 transmembrane segment, CD8, cd3ζ, CD134, CD137, ICOS, DAP10, CD 27;

5. The chimeric antigen receptor of claim 4, wherein the transmembrane domain is CD8 and has an amino acid sequence shown in SEQ ID No. 21; the intracellular domain is 4-1BB, and the amino acid sequence is shown as SEQ ID NO. 23.

6. The chimeric antigen receptor according to any one of claims 1 to 5, further comprising a signal peptide 2, preferably wherein the amino acid sequence of the signal peptide 2 is shown in SEQ ID No. 15.

7. Chimeric antigen receptor according to claim 6, further comprising a protein tag sequence, preferably a MYC tag sequence, the amino acid sequence of which is shown in SEQ ID No. 17.

8. The chimeric antigen receptor of claim 7, wherein the chimeric antigen receptor comprises the following fragments, linked in sequence: signal peptide 2, protein tag sequence, single chain antibody of TGF beta RII, CD8 transmembrane domain, 4-1BB costimulatory signal domain;

9. A dual-target chimeric antigen receptor, comprising chimeric antigen receptor 1 and chimeric antigen receptor 2;

10. The dual-target chimeric antigen receptor according to claim 9, wherein the chimeric antigen receptor is a combination of chimeric antigen receptor 1 and chimeric antigen receptor 2, or wherein chimeric antigen receptor 1 is linked to chimeric antigen receptor 2.

11. The double-target chimeric antigen receptor according to claim 10, wherein the chimeric antigen receptor 1 and the chimeric antigen receptor 2 are linked by a polypeptide fragment; preferably, the polypeptide fragment is a self-cleaving peptide fragment, more preferably a self-cleaving peptide P2A fragment, having the amino acid sequence shown in SEQ ID NO. 13.

12. The dual-target chimeric antigen receptor of claim 9, wherein the chimeric antigen receptor 1 comprises an antigen recognition domain, a transmembrane domain, an intracellular domain; the antigen recognition domain is a single chain antibody capable of recognizing and binding to a tumor-specific antigen or a tumor-associated antigen;

13. The dual-target chimeric antigen receptor of claim 12, wherein the single chain antibody is a single chain antibody of ROR 1; preferably, the amino acid sequence of the single chain antibody of ROR1 is shown as SEQ ID NO. 3.

14. The dual-target chimeric antigen receptor of claim 12, wherein the transmembrane domain is any one or more of CD28 transmembrane segment, CD8, cd3ζ, CD134, CD137, ICOS, DAP10, CD 27;

15. The dual-target chimeric antigen receptor of claim 14, wherein the transmembrane domain is a CD28 transmembrane segment and has an amino acid sequence as set forth in SEQ ID No. 7; the co-stimulatory signal domain is a CD28 intracellular segment, and the amino acid sequence is shown as SEQ ID NO. 9; the signal transduction domain is CD3 zeta, and the amino acid sequence is shown as SEQ ID NO. 11.

16. The dual-target chimeric antigen receptor according to any one of claims 12 to 15, wherein the chimeric antigen receptor 1 further comprises a signal peptide 1, a hinge region; preferably, the amino acid sequence of the signal peptide 1 is shown as SEQ ID NO.1, and the amino acid sequence of the hinge region is shown as SEQ ID NO. 5.

17. The dual-target chimeric antigen receptor of claim 16, wherein the chimeric antigen receptor 1 comprises the following fragments linked in sequence: signal peptide 1, single chain antibody of ROR1, hinge region, CD28 transmembrane domain, CD28 intracellular segment costimulatory signal domain, cd3ζ signal transduction domain;

18. The dual-target chimeric antigen receptor according to any one of claims 9 to 17, wherein the amino acid sequence is as shown in SEQ ID No. 26.

19. A gene encoding the chimeric antigen receptor according to any one of claims 1 to 8 or encoding the dual-target chimeric antigen receptor according to any one of claims 9 to 18; preferably, the nucleotide sequence of the gene encoding the chimeric antigen receptor according to any one of claims 1 to 8 is shown as SEQ ID NO.27, and the nucleotide sequence of the gene encoding the double-target chimeric antigen receptor according to any one of claims 9 to 18 is shown as SEQ ID NO. 25.

20. A vector comprising the gene of claim 19; the vector is a plasmid or a virus.

21. A host cell, characterized in that it is a host cell expressing the chimeric antigen receptor according to any one of claims 1 to 8 or expressing the dual-target chimeric antigen receptor according to any one of claims 9 to 18, preferably a host cell comprising the vector according to claim 20.

22. The host cell of claim 21, wherein the host cell is an immune response cell, preferably at least one of a T cell, a monocyte, a natural killer cell or a neutrophil, more preferably a T cell.

23. Use of the chimeric antigen receptor of any one of claims 1 to 8, the dual-target chimeric antigen receptor of any one of claims 9 to 18, the gene of claim 19, the vector of claim 20 or the host cell of any one of claims 21 to 22 in the preparation of a medicament for the prevention and/or treatment of a tumor.