A chimeric antigen receptor targeting TGFβRII and its use

Abstract

The present invention provides a chimeric antigen receptor, which is a chimeric antigen receptor capable of recognizing and binding to TGFβRII. The aforementioned chimeric antigen receptor is combined with a chimeric antigen receptor capable of recognizing and binding to tumor-specific antigens or tumor-associated antigens to construct a dual-target chimeric antigen receptor-modified immune cell, which exhibits excellent anti-tumor effects and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment, and specifically relates to a chimeric antigen receptor targeting TGFβRII and a use thereof. Background Art

[0002] Under normal circumstances, the immune system can identify and eliminate tumor cells in the tumor microenvironment, but in order to survive and grow, tumor cells can adopt different strategies to suppress the human immune system and prevent it from killing tumor cells normally, thereby surviving at all stages of the anti-tumor immune response. Tumor immunotherapy is a treatment method that controls and eliminates tumors by restoring the body's normal anti-tumor immune response. Tumor immunotherapy includes monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, etc. In recent years, tumor immunotherapy was rated as the most important scientific breakthrough of the year by Science magazine in 2013 due to its excellent efficacy and innovation. Among them, chimeric antigen receptor T cells (CAR-T) therapy and CTLA-4, PD-1 / PD-L1 antibody therapy are considered to be the three major advances in tumor immunotherapy.

[0003] CAR-T therapy uses antigen-antibody fragments, combined with T cell intracellular activation and proliferation signals, to construct CAR-T, which enables T cells to directly obtain the specific recognition ability of antibodies and become effector T cells that are not dependent on HLA (human leukocyte antigen) restrictions. Designed CAR-T cells can be cultured and grown in the laboratory, reaching billions. The amplified CAR-T cells are injected into the patient's body. After injection, the T cells will also proliferate in the patient's body and kill tumor cells with corresponding specific antigens, survive for a long time and form immune memory. There are also studies that express chimeric antigen receptors in immune cells other than T cells, such as monocytes, natural killer cells, neutrophils, etc., so that these immune cells can also obtain specific recognition of tumor cells and play the role of immunotherapy.

[0004] The core of the therapy of CAR-modified immune cells to achieve immunotherapy for tumors lies in the design of chimeric antigen receptors (CAR). CAR is mainly composed of three functional domains, namely the extracellular domain, the transmembrane domain and the intracellular domain. Generally speaking, the extracellular domain is a single-chain variable fragment (scFv) of a monoclonal antibody responsible for recognizing and binding to antigens, or scFv and a hinge region (Hinge) that acts as a connector. The intracellular domain includes a co-stimulatory domain and a signal transduction domain. The transmembrane domain connects the extracellular domain of CAR to the intracellular domain and anchors the receptor to the T cell membrane. Among them, scFv is the key to antigen recognition and targeting, and is an important factor affecting the therapeutic effect.

[0005] So far, 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. It exists during embryonic development and is related to the development of the central nervous system, heart, lung and bone tissue. Postnatally, except for a small amount of expression in adipose tissue, pancreas, lung and developing B cells (pre-B cells, immature B cells), it is not expressed in most normal tissues. However, it is expressed in many blood and solid malignancies. Current studies have shown that ROR1 is highly expressed to varying degrees in malignant tumors such as chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, melanoma, etc. And in ovarian cancer and triple-negative breast cancer, the degree of ROR1 expression is associated with poor prognosis. ROR1 tends to be expressed in poorly differentiated tumors. Cancer cells expressing ROR1 show stronger invasion, metastasis and recurrence capabilities, and express marker genes related to epithelial-mesenchymal transition (EMT). Therefore, ROR1 CAR-T developed based on the ROR1 target has been widely studied and has shown effective targeted killing of tumor cells.

[0006] Unfortunately, the clinical application of chimeric antigen receptors and immune cells expressing chimeric antigen receptors in solid tumors has been slow so far, with only limited therapeutic responses. One of the main reasons is that there are a large number of cytokines that inhibit immune cells in the tumor microenvironment, which prevent immune cells expressing chimeric antigen receptors from reaching and maintaining a high number level.

[0007] Among the many cytokines that inhibit immune cells, transforming growth factor β (TGF-β) is produced by tumor cells, regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), and other cells. It is expressed at high levels in most solid tumor lesions such as ovarian cancer, lung cancer, and colorectal cancer. TGF-β directly inhibits T cell activity by binding to TGF-β receptors TGFβRI and TGFβRII. After binding to dimerized TGFβRII, TGF-β recruits TGFβRI to form a heterotetramer, which causes the phosphorylation of intracellular SMAD2 and SMAD3. Phosphorylated SMADs induce inhibitory transcriptional programs, resulting in reduced cytokine production, reduced cell killing toxicity, and inhibition of T cell expansion when antigen binds. In addition, TGF-β can promote T cells to differentiate into Tregs, which in turn can produce TGF-β and further promote immunosuppression and tumor tolerance. Therefore, the immunosuppression of TGF-β is considered to be one of the main reasons for the failure of anti-tumor activity.

[0008] Therefore, based on the existing tumor-targeted CAR-T, in order to further improve its efficacy, how to use reasonable means to effectively avoid the influence of cytokines such as TGF-β that inhibit immune cells needs further exploration. Summary of the invention

[0009] The object of the present invention is to provide a chimeric antigen receptor targeting TGFβRII, and a dual-target chimeric antigen receptor formed by combining the chimeric antigen receptor with a traditional tumor-targeting chimeric antigen receptor.

[0010] The present invention provides a chimeric antigen receptor, which is a chimeric antigen receptor capable of recognizing and binding to TGFβRII.

[0011] Furthermore, 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 of TGFβRII.

[0012] Furthermore, the amino acid sequence of the single-chain antibody of TGFβRII is shown in SEQ ID NO.19.

[0013] Furthermore, the transmembrane domain is any one or more of CD28 transmembrane segment, CD8, CD3ζ, CD134, CD137, ICOS, DAP10, and CD27;

[0014] The intracellular domain includes a co-stimulatory signal domain, and the co-stimulatory signal domain is any one or more of the intracellular segment of CD28, 4-1BB, ICOS, CD27, OX40, MyD88, and CD40.

[0015] Furthermore, the transmembrane domain is CD8, and the amino acid sequence is shown in SEQ ID NO.21; the intracellular domain is 4-1BB, and the amino acid sequence is shown in SEQ ID NO.23.

[0016] Furthermore, the chimeric antigen receptor mentioned above further comprises a signal peptide 2. Preferably, the amino acid sequence of the signal peptide 2 is as shown in SEQ ID NO.15.

[0017] Furthermore, the chimeric antigen receptor mentioned above also includes a protein tag sequence, preferably a MYC tag sequence, and the amino acid sequence is shown in SEQ ID NO.17.

[0018] Furthermore, the chimeric antigen receptor comprises the following fragments connected in sequence: signal peptide 2, protein tag sequence, single-chain antibody of TGFβRII, CD8 transmembrane domain, 4-1BB co-stimulatory signal domain;

[0019] Preferably, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO.30.

[0020] The present invention also provides a dual-target chimeric antigen receptor, comprising a chimeric antigen receptor 1 and a chimeric antigen receptor 2;

[0021] Among them, chimeric antigen receptor 1 is a chimeric antigen receptor that can recognize and bind to tumor-specific antigens or tumor-associated antigens; chimeric antigen receptor 2 is the chimeric antigen receptor described in any one of claims 1 to 7.

[0022] Furthermore, the dual-target chimeric antigen receptor is a combination of chimeric antigen receptor 1 and chimeric antigen receptor 2, or a combination of chimeric antigen receptor 1 and chimeric antigen receptor 2.

[0023] Furthermore, the above-mentioned dual-target chimeric antigen receptor is formed by connecting chimeric antigen receptor 1 and chimeric antigen receptor 2 through a polypeptide fragment; preferably, the polypeptide fragment is a self-cleaving peptide fragment, more preferably a self-cleaving peptide P2A fragment, and the amino acid sequence is shown in SEQ ID NO.13.

[0024] Furthermore, the chimeric antigen receptor 1 comprises 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;

[0025] 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, and IGFR.

[0026] Furthermore, the above-mentioned single-chain antibody is a single-chain antibody of ROR1; preferably, the amino acid sequence of the single-chain antibody of ROR1 is shown in SEQ ID NO.3.

[0027] Furthermore, the transmembrane domain is any one or more of CD28 transmembrane segment, CD8, CD3ζ, CD134, CD137, ICOS, DAP10, and CD27;

[0028] The intracellular domain includes a co-stimulatory signal domain and a signal transduction domain, wherein the co-stimulatory signal domain is any one or more of the intracellular segment of CD28, 4-1BB, ICOS, CD27, OX40, MyD88, and CD40; and the signal transduction domain is CD3ζ or FcεRI.

[0029] Furthermore, the above-mentioned transmembrane domain is the CD28 transmembrane segment, and the amino acid sequence is shown in SEQ ID NO.7; the co-stimulatory signal domain is the CD28 intracellular segment, and the amino acid sequence is shown in SEQ ID NO.9; the signal transduction domain is CD3ζ, and the amino acid sequence is shown in SEQ ID NO.11.

[0030] Furthermore, 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 as shown in SEQ ID NO.1, and the amino acid sequence of the hinge region is as shown in SEQ ID NO.5.

[0031] Furthermore, the chimeric antigen receptor 1 comprises the following fragments connected in sequence: signal peptide 1, single-chain antibody of ROR1, hinge region, CD28 transmembrane domain, CD28 intracellular segment co-stimulatory signal domain, CD3ζ signal transduction domain;

[0032] Preferably, the amino acid sequence of the chimeric antigen receptor 1 is as shown in SEQ ID NO.29.

[0033] Furthermore, the amino acid sequence of the dual-target chimeric antigen receptor is shown in SEQ ID NO.26.

[0034] The present invention also provides a gene, which is a gene encoding the above-mentioned chimeric antigen receptor or the above-mentioned dual-target chimeric antigen receptor; preferably, the nucleotide sequence of the gene encoding the above-mentioned chimeric antigen receptor is shown in SEQ ID NO.27, and the nucleotide sequence of the gene encoding the above-mentioned dual-target chimeric antigen receptor is shown in SEQ ID NO.25.

[0035] The present invention also provides a vector, which is a vector containing the above gene; the vector is a plasmid or a virus.

[0036] The present invention also provides a host cell, which is a host cell expressing the above-mentioned chimeric antigen receptor or the above-mentioned dual-target chimeric antigen receptor, preferably a host cell containing the above-mentioned vector.

[0037] Furthermore, 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.

[0038] The present invention also provides the use of the chimeric antigen receptor, dual-target chimeric antigen receptor, gene, vector or host cell in preparing a drug for preventing and / or treating tumors.

[0039] Beneficial effects of the present invention: The present invention provides a chimeric antigen receptor targeting TGFβRII. On the one hand, it can bind to TGFβRII of tumor cells to enhance the recognition and targeting effect of immune cells on tumors. On the other hand, it can also target TGFβRII of immune cells themselves to achieve the blocking of TGFβRII of immune cells themselves, hindering the binding of TGFβ to TGFβ receptor of immune cells, thereby reducing the inhibitory effect of TGFβ on immune cells. At the same time, the chimeric antigen receptor sequence targeting TGF-βRII does not contain CD3ζ activation signal, and will not activate T cells to perform cytotoxic effects.

[0040] Furthermore, the chimeric antigen receptor targeting TGFβRII is used in combination with the traditional tumor-targeted chimeric antigen receptor to construct a dual-target chimeric antigen receptor-modified immune cell, which can not only achieve tumor-specific targeting and kill the tumor through the tumor-targeted chimeric antigen receptor, but also reduce the inhibitory effect of the inhibitory cytokine TGFβ on immune cells through the action of the chimeric antigen receptor targeting TGFβRII, while enhancing the tumor recognition and targeting effect, thereby achieving a synergistic anti-tumor effect.

[0041] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0042] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The figure is a structural composition diagram of the chimeric antigen receptor functional unit in the viral vector of the present invention, wherein: A is a chimeric antigen receptor that single-targets ROR1, and B is a chimeric antigen receptor that double-targets.

[0044] Figure 2 This is the expression of the CAR of the present invention on T cells.

[0045] Figure 3 The expression of antigens on the surface of tumor cells.

[0046] Figure 4 is the level of IFN-γ released after co-culture of CAR-T and tumor cells.

[0047] Figure 5 is the level of IL-2 released after co-culture of CAR-T and tumor cells.

[0048] Figure 6 is the level of TNF-α released after co-culture of CAR-T and tumor cells.

[0049] Figure 7 This is the detection of cells that secrete IFN-γ after co-culture of CAR-T and tumor cells.

[0050] Figure 8 This is a test to detect the release of T cell granzyme B after CAR-T cells kill tumor cells in vitro.

[0051] Fig. 9 This is the proliferation of T cells after co-culture of CAR-T cells and tumor cells.

[0052] Fig.10 To detect the intracellular pSMAD2 / 3 signaling pathway of T cells after CAR-T cells kill tumor cells in vitro.

[0053] Fig.11 The results show that CAR-T cells have an in vivo growth inhibitory effect on the SKOV3 ovarian cancer peritoneal dissemination model. DETAILED DESCRIPTION

[0054] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0055] Example 1: Synthesis of the full-length gene of the dual-target chimeric antigen receptor and completion of the construction of the recombinant plasmid vector

[0056] First, prepare the expression framework of the dual-target chimeric antigen receptor prepared in this example. The expression framework is as follows from the 5 end to the 3 end: signal peptide 1-Anti-ROR1 scFv-hinge-CD28 transmembrane segment-CD28 intracellular segment-CD3ζ-P2A-signal peptide 2-MYC protein tag-Anti-TGF-βRII scFv-CD8 transmembrane domain-4-1BB intracellular segment.

[0057] The sequence of each segment is:

[0058] Amino acid sequence of signal peptide 1 of chimeric antigen receptor 1 (SEQ ID NO.1):

[0059] MLLLVTSLLLCELPHPAFLLIP

[0060] Encoding nucleotide sequence (SEQ ID NO.2):

[0061] ATGCTGCTCCTGGTGACCTCCCTCCTGCTGTGCGAGCTGCCCCACCCCGCCTTCCTGCTGATTCCC

[0062] The amino acid sequence of the single-chain antibody of ROR1 (Anti-ROR1 scFv) (SEQ ID NO.3):

[0063] QEQLVESGGRLVTPGGSLTLSCKASGFDFSAYYMSWVRQAPGKGLEWIATIYPSSGKTYYATWVNGRFTISSDNAQNTVDLQMNSLTAADRATYFCARDSYADDGALFNIWGPGTLVTISSGGG GSGGGGSGGGGSELVLTQSPSVSAALGSPAKITCTLSSAHKTDTIDWYQQLQGEAPRYLMQVQSDGSYTKRPGVPDRFSGSSSGADRYLIIPSVQADDEADYYCGADYIGGYVFGGGTQLTVTG

[0064] Encoding nucleotide sequence (SEQ ID NO.4):

[0065] CAAGAGCAGCTGGTGGAGAGCGGGGGCAGACTGGTGACCCCCGGCGGCAGCCTGACCCTGAGCTGTAAGGCTAGCGGCTTCGACTTCAGCGCCTACTACATGAGCTGGGTGAGACAAGCCCCTGGGAAGGGCCTGGAATGGATCGCCACCATCTACCCTAGCAGCGGCAAGACCTACTACGCTACCTGGGTGAACGGCAGATTCACCATCTCCTCCGACAACGCTCAGAACACCGTGGACCTGCAGATGAACAGCCTGACCGCCGCCGACCGGGCCACCTACTTCTGCGCTAGAGACAGCTACGCCGACGACGGCGCCCTGTTCAACATCTGGGGGCCCGGCACCCTCGTGACAATTAGCAGCGGCGGGGGCGGCAGCGGCGGGGGCGGCAGCGGGGGGGGGGGCTCCGAGCTGGTCCTGACACAGAGCCCTAGCGTGAGCGCCGCTCTGGGCAGCCCCGCCAAGATCACCTGCACCCTGAGCAGCGCCCACAAGACCGACACCATCGACTGGTATCAGCAGCTGCAAGGCGAGGCCCCTAGATATCTGATGCAAGTGCAGAGCGACGGCAGCTACACCAAGAGACCCGGCGTGCCCGACCGGTTCAGCGGCTCCTCCTCCGGCGCCGACAGATACCTCATCATCCCTAGCGTGCAAGCCGACGACGAGGCCGACTACTACTGCGGCGCCGACTACATCGGCGGCTACGTGTTTGGCGGGGGCACACAGCTGACCGTGACCGGC

[0066] Amino acid sequence of the hinge region (SEQ ID NO.5): ESKYGPPCPPCP

[0067] Coding nucleotide sequence (SEQ ID NO.6):

[0068] GAGAGCAAATACGGCCCCCCCTGCCCCCCCTGTCCT

[0069] Amino acid sequence of the CD28 transmembrane segment (SEQ ID NO.7):

[0070] MFWVLVVVGGVLACYSLLVTVAFIIFWV

[0071] Encoding nucleotide sequence (SEQ ID NO.8):

[0072] ATGTTCTGGGTGCTGGTGGTCGTGGGCGGCGTGCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTTATCATCTTCTGGGTG

[0073] The amino acid sequence of the intracellular segment of CD28 (SEQ ID NO.9): RSKRSRGCHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRS

[0074] Encoding nucleotide sequence (SEQ ID NO.10):

[0075] AGAAGCAAGAGAAGCAGAGGCTGCCACAGCGACTACATGAACATGACCCCTAGAAGACCCGGCCCCACAAGAAAGCACTATCAGCCCTACGCCCCCCCTAGAGACTTCGCCGCCTACAGAAGC

[0076] The amino acid sequence of CD3ζ (SEQ ID NO.11):

[0077] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0078] Encoding nucleotide sequence (SEQ ID NO.12):

[0079] AGAGTGAAGTTCAGCAAGCGCCGACGCCCCCGCCTATCAGCAAGGGCAGAATCAGCTGTACAATGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCTAGAAGAAAGAACCCCCAAGAG GGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGAGACGGAGAGGCAAGGGCCACGACGGCCTGTACCAAGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAAGCCCTGCCCCTAGA

[0080] Amino acid sequence of P2A (SEQ ID NO. 13): GSGATNFSLLKQAGDVEENPGP

[0081] Encoding nucleotide sequence (SEQ ID NO.14):

[0082] GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAAGCCGGGGATGTGGAAGAAAACCCCGGCCCC

[0083] The amino acid sequence of CD8 signal peptide (signal peptide 2) (SEQ ID NO.15):

[0084] MALPVTALLLPLALLLHAARP

[0085] Encoding nucleotide sequence (SEQ ID NO.16):

[0086] ATGGCCCTGCCCGTGACCGCCCTGCTCCTGCCCCTGGCCCTGCTCCTGCATGCTGCTAGACCC

[0087] Amino acid sequence of MYC tag (SEQ ID NO.17): EQKLISEEDL

[0088] Encoding nucleotide sequence (SEQ ID NO.18):

[0089] GAGCAGAAGCTGATCAGCGAGGAGGACCTG

[0090] The amino acid sequence of Anti-TGFβRII-scFv (SEQ ID NO.19):

[0091] QLQVQESGPGLVKPSETLSLTCTVSGGSISNSYFSWGWIRQPPGKGLEWIGSFYYGEKTYYNPSLKSRATISIDTSKSQFSLKLSSVTAADTAVYYCPRGPTMIRGVIDSWGQGTLVTVSS GGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDEAVYYCQQRSNWPPTFGQGTKVEIK

[0092] Encoding nucleotide sequence (SEQ ID NO.20):

[0093] CAGCTCCAAGTGCAAGAGAGCGGCCCTGGGCTGGTGAAGCCTAGCGAGACCCTGAGCCTCACATGCACCGTGAGCGGCGGCAGCATCAGCAACAGCTACTTCAGCTGGGGCTGGATCAGACAGCCCCCCGGCAAGGGCCTGGAGTGGATTGGCAGCTTCTACTACGGCGAGAAAACCTACTACAACCCTAGCCTGAAGAGCAGAGCCACCATCAGCATCGACACAAGCAAGAGCCAATTCAGCCTGAAGCTGAGCAGCGTCACAGCCGCCGACACCGCCGTGTATTACTGCCCTAGAGGCCCCACCATGATCAGAGGCGTGATCGATAGCTGGGGGCAAGGCACACTGGTGACCGTCAGCAGCGGCGGCGGGGGGAGCGGGGGGGGCGGCAGCGGCGGGGGCGGCTCCGAAATTGTCCTGACACAGAGCCCTGCCACACTGTCCCTCAGCCCCGGCGAGAGAGCCACCCTGAGCTGCAGAGCTAGCCAAAGCGTGAGAAGCTACCTGGCCTGGTACCAACAAAAGCCCGGCCAAGCCCCTAGACTGCTGATCTACGACGCTAGCAACAGAGCCACCGGCATCCCCGCTAGATTCTCCGGCAGCGGGAGCGGCACCGACTTCACCCTCACCATCAGCAGCCTCGAGCCCGAGGACGAGGCCGTGTACTACTGTCAGCAGAGAAGCAACTGGCCCCCCACCTTCGGCCAAGGCACCAAGGTGGAGATTAAG

[0094] Amino acid sequence of the CD8 transmembrane domain (SEQ ID NO.21):

[0095] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC

[0096] Coding nucleotide sequence (SEQ ID NO.22):

[0097] ACCACAACCCCTGCCCCTAGACCCCCTACACCTGCCCCCACCATTGCTAGCCAACCCCTCAGCCTCAGACCCGAAGCCTGTAGACCTGCTGCCGGCGGCGCTGTGCACACAAGAGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTCCTGAGCCTGGTGATCACCCTGTACTGC

[0098] Amino acid sequence of the intracellular segment of 4-1BB (SEQ ID NO.23):

[0099] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0100] Encoding nucleotide sequence (SEQ ID NO.24):

[0101] AAGAGAGGCAGAAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACCACCCAAGAGGAGGACGGCTGCAGCTGTAGATTCCCCGAGGAAGAGGAGGGCGGCTGCGAGCTG

[0102] The complete nucleotide sequence encoding the dual-target chimeric antigen receptor (SEQ ID NO.25):

[0103]

[0104]

[0105] The italicized portion is the nucleotide sequence encoding chimeric antigen receptor 1 (SEQ ID NO.26), the underlined portion is the nucleotide sequence encoding the polypeptide connecting chimeric antigen receptor 1 and chimeric antigen receptor 2 (SEQ ID NO.14), and the remaining portion is the nucleotide sequence encoding chimeric antigen receptor 2 (SEQ ID NO.27)

[0106] The complete amino acid sequence of the dual-target chimeric antigen receptor (SEQ ID NO.28):

[0107]

[0108]

[0109] Among them, 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 connecting 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).

[0110] The nucleotide sequence encoding the complete dual-target chimeric antigen receptor (SEQ ID NO. 25) was synthesized and inserted into the EcoRI-BamH1 site of the lentiviral pWPXLd vector, which was preceded by the EF-1α promoter, and transformed into Escherichia coli competent cells. After correct sequencing, the plasmid was extracted and purified using the plasmid purification kit of Qiagen. The purification steps were referred to the instructions of the kit to obtain high-quality plasmids of the recombinant expression vector. The structure of the inserted target fragment was referred to Figure 1 B.

[0111] Example 2: Transformation of cells with recombinant vectors

[0112] 1. Cultivation and passaging of 293T cells:

[0113] Open the biosafety cabinet, wipe the table with 75% alcohol cotton, and put the pipette, pipette gun, tip box, 15ml centrifuge tube, centrifuge tube rack, 10cm 2 Place the new cell culture dish in the biosafety cabinet, close the door, turn on the UV switch of the biosafety cabinet, and irradiate for half an hour to sterilize. Preheat DMEM containing 10% fetal bovine serum and 100U / ml penicillin-streptomycin and trypsin in a 37℃ water bath. Open the biosafety cabinet, turn on the ventilation switch, and transfer the 293T cell culture dish that has grown to 80%-90% from 37℃ and 5% CO to the water bath. 2 Take it out of the incubator and place it in a biosafety cabinet. Disinfect your hands, the mouth of the culture medium bottle, the mouth of the pipette, etc. with 75% alcohol. Use a sterile pipette to suck out the culture medium in the culture dish and discard it in the waste tank. Drop 2 ml of trypsin into the culture dish and observe the cells under a microscope until the cells become round and separate, then remove the trypsin. Add 6-8 ml of fresh complete culture medium to the culture dish and gently blow down the cells. Divide the cell suspension into other culture dishes and add culture medium to reach 10 ml per dish. Shake the culture dish in a cross shape several times to shake the cells evenly, observe under a microscope and place it in a 37°C incubator. Observe the cell status after 24 hours, and carry out the next subculture when the cells grow to 80%-90%.

[0114] 2. Obtaining lentiviral stock solution:

[0115] Day 1: Plate. 293T cells with good 90% density were digested and passaged at 1:3, about 4x10 6cells / 10ml / 10cm dish, 5% CO 2 , incubate at 37°C overnight. After 24 hours, the cell density is about 70-90% (not more than 90%).

[0116] Day 2: Transfection. All reagents were equilibrated to room temperature before transfection.

[0117] The transfection steps are as follows:

[0118] a. Prepare the following DNA mixture in a 10 ml BD tube (per 10 cm dish): psPAX2 (packaging plasmid) 3 μg; pMD.2G (envelope plasmid) 1.5 μg; pWPXLd (lentivirus vector, prepared in Example 1) 6 μg.

[0119] b. Add DMEM medium to make up to 980 μl and mix well.

[0120] c. Slowly drip 20 μl of Highgene transfection reagent (Aibotec) into the DNA solution and gently pipette until mixed.

[0121] d. Let stand at room temperature for 15 minutes.

[0122] e. Drop 1 ml of the transfection mixture into the plate, shake gently in a "cross" pattern (10 times each) to mix well, and then return to 5% CO 2 , 37℃ incubator. After 8-12h, remove the culture medium and replace it with pre-warmed DMEM complete medium. Return to 5% CO 2 , incubator at 37°C for 48h.

[0123] Day 4: 48 hours after transfection. Collect cell supernatant and add 10 ml of pre-warmed DMEM complete medium, 5% CO 2 , cultured at 37°C; the viral supernatant was filtered through a 0.45 μm filter and stored at 4°C (up to 1 week).

[0124] Day 5: 72 hours after transfection, collect the viral supernatant, filter with a 0.45 μm filter, and store at 4°C.

[0125] 3. Concentration of Lentivirus:

[0126] Instruments: Ultracentrifuge, matching rotor and sleeve, ultracentrifuge tube, balance. Disinfect the sleeve and balance under the UV instrument of the biosafety cabinet. After ensuring that there are no droplets in each sleeve, put the appropriate centrifuge tube into the sleeve. Add the virus suspension filtered with a 0.45μm filter to the centrifuge tube. Each centrifuge tube containing the virus suspension is strictly balanced, using a balance with an accuracy of 0.001g or above. After covering the sleeve cover, use the balance again to verify whether it is completely balanced. Install each balanced sleeve into the centrifuge rotor and prepare for centrifugation. Centrifugation: Centrifugation conditions are: 20℃, 70000g, 2h. Wait until the centrifuge speed rises to 70000g before leaving. After centrifugation, pour out the culture medium and invert the centrifuge tube on the sterilized filter paper to absorb the remaining culture medium. Resuspend the virus precipitate with PBS. Divide the resuspended virus into 1.5ml EP tubes and store in a -80℃ refrigerator for use.

[0127] 4. Isolation of human peripheral blood T lymphocytes:

[0128] Peripheral blood was collected using a sodium heparin anticoagulation tube. The ratio of lymphocyte separation solution volume to blood volume was 1:1. The peripheral blood was slowly added to the lymphocyte separation solution, and the mixture was centrifuged. The conditions were 1000g, 30min, 18℃, and acceleration / deceleration of 1. After centrifugation, the blood was divided into 4 layers, and the layer where PBMC was located was the middle white cloudy layer. The tip of the gun slowly aspirated the middle white cloudy layer. The aspirated cells were added to 10ml X VIVO medium and centrifuged at 300g for 10min. The supernatant was discarded, and 10ml of lysate was used for lysing. After mixing, the cells were allowed to stand for 5min. Centrifuged at 300g for 10min. The supernatant was discarded, and the cells were resuspended in 10ml of X VIVO medium and counted. Centrifuged at 300g for 10min. Discard the supernatant, resuspend the lymphocytes in X VIVO medium containing 5% human serum and 100 IU / ml IL-2, and add the corresponding number of CD3 / CD28 stimulating magnetic beads according to the number of cells. 5% CO 2 , cultured at 37℃.

[0129] 5. Virus-infected T cells

[0130] One day before the virus infected T cells, coat the 12-well plate with Retronectin (recombinant human fibronectin) dilution (concentration 50μg / ml), 1ml per well. After sealing the plate, place it at 4℃ overnight for use. On the day of infection, remove the Retronectin dilution and use 2% BSA (bovine serum albumin) solution to block for 30 minutes. Remove the BSA liquid and rinse once with PBS. Add 1×10 6 The cells and virus concentrate were placed in a centrifuge for centrifugal infection at 32°C, 1000g, and 2h. After centrifugation, the well plate was removed and placed back at 37°C and 5% CO 2Culture in a cell culture incubator. Replace the medium with complete culture medium after 24 hours.

[0131] 6. Detection of CAR molecule expression on T cell surface:

[0132] 72h after T cells were infected with ROR1 CAR virus and ROR1-TGFβRII dual CAR virus, flow cytometry was used to detect the expression of CAR ( Figure 2 ).

[0133] like Figure 2 As shown, ROR1 CAR-T expressed the ROR1 CAR gene, while ROR1-TGFβRII CAR-T expressed the ROR1 CAR gene as well as anti-TGFβRII scFv, indicating that the dual CAR was successfully constructed.

[0134] Comparative Example 1: Preparation of ROR1 CAR-T

[0135] Prepare the chimeric antigen receptor 1 (signal peptide 1-Anti-ROR1scFv-hinge-CD28 transmembrane segment-CD28 intracellular segment-CD3ζ, see Figure 1 A) was prepared by the method of Example 2 to obtain a CAR-T cell with only chimeric antigen receptor 1, namely, ROR1 CAR-T cell.

[0136] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0137] Experimental Example 1: Detection of tumor cell surface antigen expression

[0138] Flow cytometry was used to detect the expression levels of ROR1 and TGFβRII in different tumor cell lines.

[0139] Methods: Ovarian cancer cells SKOV3, breast cancer cells MDA-MB-231, lung cancer cells A549 and ovarian cancer cells A2780 were incubated with PE-labeled antibodies that specifically recognize human ROR1 and APC-labeled antibodies that specifically recognize human TGFβRII.

[0140] Results: Flow cytometry showed that different tumor cells had different expression levels of ROR1 and TGFβRII. MDA-MB-231 cells had the highest expression levels of ROR1 and TGFβRII; SKOV3 cells had moderate expression of ROR1 and TGFβRII; A549 cells had moderate expression of ROR1 and almost no expression of TGFβRII; A2780 cells had negative expression of ROR1 and moderate expression of TGFβRII (see Figure 3 ).

[0141] Experimental Example 2: Functional detection of dual-targeting CAR-T

[0142] 1. Detection of cytokine release after CAR-T cells kill tumor cells in vitro

[0143] Methods: T cells (ROR1 CAR-T (Comparative Example 1) group, ROR1-TGFβRII CAR-T (Example 2) group, control T cell group) were mixed with 1×10 4 ROR1-positive target cells: ovarian cancer cell line SKOV3, breast cancer cell line MDA-MB-231, lung cancer cell line A549, or ROR1-negative target cells: ovarian cancer cell line A2780, were co-cultured at a ratio of effector cells to target cells (effector-target ratio) of 1:1 or 2:1, and cultured for 24 hours in the absence of TGF-β or in the presence of 5 ng / ml TGF-β. IFN-γ ( Figure 4 )、IL-2( Figure 5 ) and TNF-α( Figure 6 ) level.

[0144] Results: Compared with the simple ROR1 CAR-T group, the ROR1-TGFβRII CAR-T group released more IFN-γ, IL-2 and TNF-α cytokines after co-culture with ROR1-positive tumor cells, and had stronger killing ability. After co-culture with ROR1-negative tumor cells, the dual-targeted CAR-T almost did not release cytokines and had no off-target toxicity.

[0145] 2. Detection of intracellular INF-γ release of T cells after CAR-T cells kill tumor cells in vitro

[0146] Method: 1×10 6 T cells (ROR1 CAR-T group, ROR1-TGFβRII CAR-T group, control T cell group) were co-cultured with breast cancer cell line MDA-MB-231 and ovarian cancer cell line SKOV3, with an effector-target ratio of 3:1, and cultured for 24 hours. Flow cytometry was used to detect the level of intracellular IFN-γ released by CD3-positive T cells ( Figure 7 ).

[0147] Results: Compared with the simple ROR1 CAR-T group, the ROR1-TGFβRII CAR-T group had more cells secreting intracellular IFN-γ and stronger killing ability after co-culture with ROR1-positive tumor cells.

[0148] 3. Detection of T cell granzyme B release after CAR-T cells kill tumor cells in vitro

[0149] Method: 1×10 6 T cells (ROR1 CAR-T group, ROR1-TGFβRII CAR-T group, control T cell group) were co-cultured with lung cancer cell line A549, with an effector-target ratio of 3:1, and cultured for 4 hours. Flow cytometry was used to detect the level of intracellular granzyme B released by CD8-positive T cells ( Figure 8 ).

[0150] Results: Compared with the simple ROR1 CAR-T group, the ROR1-TGFβRII CAR-T group released granzyme B faster and had stronger cytotoxicity after co-culture with ROR1-positive tumor cells.

[0151] 4. T cell proliferation after co-culture of CAR-T cells and tumor cells

[0152] Method: T cells were labeled with FAR RED cell-tracing fluorescent dye. The cell-tracing fluorescent dye is attached to the cell membrane. If the cell divides, the membrane dye on one cell will be distributed to the membranes of two new cells as the cell divides, so that the fluorescence on the cell will be weakened by half. Therefore, the weaker the fluorescence intensity, the less fluorescent dye there is on the cell membrane, and the more the cell divides and proliferates.

[0153] The two CAR-T cells were co-cultured with breast cancer cells MDA-MB-231, with an effector-target ratio of 3 to 1; after 24 hours, the fluorescence of T cells was detected.

[0154] Results: Fig. 9 As shown in the figure, the horizontal axis represents the intensity of FAR RED fluorescence signal, and the vertical axis represents the number of cells in a certain intensity range. The closer the peak of the curve is to the origin, the weaker the fluorescence signal. As can be seen from the figure, in the CD8 and CD4 positive cell populations, the degree of division of ROR1-TGFβRII CAR-T is greater than that of the ROR1 CAR-T group, that is, the cell proliferation ability of this group is stronger.

[0155] 4. Detection of pSMAD2 / 3 signaling pathway in T cells after CAR-T cells kill tumor cells in vitro

[0156] Method: 1×10 6 T cells (ROR1 CAR-T group, ROR1-TGFβRII CAR-T group, control T cell group) were co-cultured with ovarian cancer cell line SKOV3, with an effector-target ratio of 3:1, and cultured for 24 hours in the absence of TGF-β or in the presence of 5ng / ml, 10ng / ml, and 20ng / ml TGF-β. Flow cytometry was used to detect the expression level of pSMAD2 / 3, a downstream signaling pathway of TGF-β, in CAR-positive or CAR-negative T cells ( Fig.10 ).

[0157] Results: In the presence of TGF-β, after CAR-T cells were co-incubated with tumor cells, TGF-βRII on the surface of T cells dimerized and bound to TGF-β, and then formed a heterotetramer with TGF-βRI, thereby activating downstream signaling pathways and phosphorylating intracellular SMAD proteins. In the dual-targeted ROR1-TGFβRII CAR-T group, because it expressed anti-TGFβRII scfv, it could block TGFβRII on its own surface, thereby preventing TGFβRII from binding to TGF-β, blocking intracellular SMAD protein phosphorylation, and thus resisting the inhibitory effect of TGF-β on T cells. The experimental results showed that in the dual-targeted ROR1-TGFβRII CAR-T group, in the presence of TGF-β, the expression of intracellular pSMAD2 / 3 in the CAR-positive part of T cells was lower than that in the simple ROR1 CAR-T group.

[0158] 5. In vivo growth inhibitory effect of CAR-T cells on the SKOV3 ovarian cancer peritoneal dissemination model Methods: Using NSG immunodeficient mice, a human ovarian cancer peritoneal dissemination model was established using SKOV3 (SKOV3-luc) cells that stably expressed luciferase. T cells were intraperitoneally injected and infused to evaluate the inhibitory effect of T cells on tumor growth.

[0159] ① Purchase 5-week-old NSG mice, keep them in the animal room for 1 week to adapt to the environment, and give them 5×10 5 SKOV3-luc cells were injected intraperitoneally to establish the model. Bioluminescence imaging was performed one week later to detect the tumor burden in vivo. The mice were randomly divided into four groups according to the tumor burden, namely, ROR1 CAR-T group, ROR1-TGFβRII CAR-T group, unmodified control T cells and PBS group, with 5 experimental mice in each group. The preparation process of CAR-T cells was carried out according to Example 2.

[0160] ②In each CAR-T group, 2×10 6 The number of CAR-positive T cells and unmodified control T cells infused was the same as the total amount of the CAR-T group. The cells were resuspended in 100 μl PBS and injected intraperitoneally back into the mice.

[0161] ③ Tumor burden was assessed by intraperitoneal injection of luciferin into mice every week and imaging using a bioluminescence imaging system.

[0162] Results: Compared with the PBS group and the unmodified control T cell group, both CAR-T cells could inhibit the growth of SKOV3 tumor cells, and ROR1-TGFβRII CAR-T had a stronger tumor inhibitory effect in vivo ( Fig.11 ).

[0163] In summary, compared with the unmodified ROR1 CAR-T, the dual-target CAR-T has stronger killing activity against tumor cells, which is reflected in the release of more cytokines and stronger release of granzyme B; at the same time, it has stronger proliferation activity, which is conducive to the long-term survival of CAR-T. It also showed a stronger anti-tumor effect in the ovarian cancer peritoneal metastasis model. This shows that the dual-target CAR-T designed in the present invention can significantly increase the efficacy of T cells against solid tumors when TGF-β is inhibited.

[0164] The principle of the present invention is to design a chimeric antigen receptor targeting TGF-βRII. On the one hand, it can bind to the TGFβRII of tumor cells to enhance the recognition and targeting effect of immune cells on tumors. On the other hand, it can also target the TGFβRII of immune cells themselves to achieve the blocking of TGFβRII of immune cells themselves, hindering the binding of TGFβ to the TGFβ receptor of immune cells, thereby reducing the inhibitory effect of TGFβ on immune cells. At the same time, the chimeric antigen receptor sequence targeting TGF-βRII does not contain CD3ζ activation signal, and will not activate T cells to perform cytotoxic effects.

[0165] Furthermore, the chimeric antigen receptor targeting TGFβRII is used in combination with the traditional tumor-targeted chimeric antigen receptor to construct a dual-target chimeric antigen receptor-modified immune cell, which can not only achieve tumor-specific targeting and kill the tumor through the tumor-targeted chimeric antigen receptor, but also reduce the inhibitory effect of the inhibitory cytokine TGFβ on immune cells through the action of the chimeric antigen receptor targeting TGFβRII, while enhancing the tumor recognition and targeting effect, thereby achieving a synergistic anti-tumor effect.

[0166] Based on this principle, the method provided in the embodiment of the present invention can be adopted, in which the chimeric antigen receptor targeting TGF-βRII of the present invention is connected to the chimeric antigen receptor targeting the tumor (such as the chimeric antigen receptor of ROR1) through a self-cleaving peptide, and the immune cells (such as T cells) are modified to make CAR-T. However, at the same time, according to the basic common sense in the field, the combination form can also be reasonably changed. For example, the plasmid expressing the chimeric antigen receptor targeting TGF-βRII and the plasmid expressing the chimeric antigen receptor targeting the tumor (such as the chimeric antigen receptor of ROR1) are respectively transferred into the immune cells (such as T cells), so that the surface of the immune cells is modified with two chimeric antigen receptors at the same time, and the two chimeric antigen receptors are not connected, which can also constitute a combination form to achieve the same synergistic anti-tumor effect as the embodiment of the present invention.

Claims

1. A dual-target chimeric antigen receptor, characterized in that: Includes chimeric antigen receptor 1 and chimeric antigen receptor 2; Wherein, chimeric antigen receptor 1 is a chimeric antigen receptor capable of recognizing and binding to a tumor-specific antigen or a tumor-associated antigen; chimeric antigen receptor 2 is a chimeric antigen receptor capable of recognizing and binding to TGFβ RII; The chimeric antigen receptor capable of recognizing and binding to TGFβRII comprises an antigen recognition domain, a transmembrane domain, and an intracellular domain; the antigen recognition domain is a single-chain antibody of TGFβRII; The amino acid sequence of the dual-target chimeric antigen receptor is shown in SEQ ID NO.

28.

2. A gene, characterized in that It is a gene encoding the dual-target chimeric antigen receptor described in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

25.

4. A carrier, characterized in that It is a vector containing the gene described in claim 2; the vector is a plasmid or a virus.

5. A host cell, characterized in that It is a host cell that expresses the dual-target chimeric antigen receptor described in claim 1.

6. A host cell, characterized in that This is a host cell containing the vector according to claim 4.

7. The host cell according to claim 5 or 6, characterized in that The host cell is an immune response cell.

8. The host cell according to claim 7, characterized in that The host cell is at least one of a T cell, a monocyte, a natural killer cell or a neutrophil.

9. The host cell according to claim 8, characterized in that The host cell is a T cell.

10. Use of the dual-target chimeric antigen receptor according to claim 1, the gene according to claim 2 or 3, the vector according to claim 4, or the host cell according to any one of claims 5 to 9 in the preparation of a drug for treating a tumor, wherein the tumor is selected from at least one of the following types: chronic lymphocytic leukemia, acute lymphocytic leukemia, non-Hodgkin's lymphoma, ovarian cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, and melanoma.

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