Bispecific chimeric antigen receptor targeting CD117 and CD7 and its application

Abstract

The present invention relates to a bispecific chimeric antigen receptor targeting CD117 and CD7 and its application, wherein the bispecific chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and a signal transduction domain, wherein the antigen binding domain comprises a CD117 binding domain and a CD7 binding domain. The bispecific chimeric antigen receptor provided by the present invention combines targeted antibodies for CD117 and CD7 together, recognizes and binds to two different antigens at the same time, enhances the targeted elimination ability of malignant cells, and by acting on hematopoietic stem cells and immune cells at the same time, can more effectively eliminate tumor cells in the receptor, while retaining or reconstructing normal hematopoietic and immune functions.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a bispecific chimeric antigen receptor targeting CD117 and CD7 and applications thereof. Background Art

[0002] In the field of modern medicine, hematopoietic stem cell transplantation (HSCT) has become an important means of treating a variety of blood system diseases and certain solid tumors. In order to ensure the success of transplantation, myeloablative therapy is an indispensable step, which aims to completely eliminate malignant cells in the recipient and provide a tumor-free environment for newly transplanted healthy hematopoietic stem cells. However, traditional myeloablative methods, such as chemotherapy and radiotherapy, although they can achieve the purpose of myeloablative therapy to a certain extent, are often accompanied by a high relapse rate and treatment-related toxicity. These side effects not only affect the quality of life of patients, but may also endanger the safety of patients' lives.

[0003] With the continuous advancement of biotechnology, the emergence of bispecific chimeric antigen receptor (BiCAR) technology has provided new hope for improving the success rate of HSC transplantation. Compared with single-target CAR, BiCAR technology has shown obvious advantages in reducing treatment-related toxicity and relapse rate, greatly improving the success rate of HSC transplantation. In addition, BiCAR technology also has the potential advantage of personalized treatment, and can adjust the CAR structure according to the specific situation of the patient to achieve the best therapeutic effect. With the deepening of research and the improvement of technology, BiCAR technology is expected to play an important role in the field of hematopoietic stem cell transplantation in the future, bringing patients safer and more effective treatment options.

[0004] CD117, also known as stem cell factor receptor (c-Kit), is expressed on a variety of hematopoietic stem cells and early progenitor cells and is a key factor in maintaining hematopoietic function. It plays a vital role in the self-renewal and differentiation of hematopoietic stem cells. CD7 is mainly expressed on the surface of T cells and NK cells and is closely related to the development and function of immune cells. In recent years, single-target chimeric antigen receptor (CAR) T cell technology targeting CD117 and CD7 has shown certain efficacy in preclinical studies, but it has also exposed some limitations. For example, although CAR-T technology targeting CD117 can effectively target hematopoietic stem cells, it may cause damage to normal hematopoietic function; while CAR-T technology targeting CD7 shows potential in clearing tumor cells in T cells and NK cells, but it may affect normal immune function. These limitations have prompted researchers to seek new strategies to maximize the protection of normal hematopoiesis and immune function while clearing tumor cells.

[0005] In summary, providing a bispecific chimeric antigen receptor targeting CD117 and CD7, which can enhance the targeted clearance of malignant cells while retaining or reconstructing normal hematopoietic and immune functions, has become one of the urgent problems to be solved in this field. Summary of the invention

[0006] To solve the above technical problems, the present invention provides a bispecific chimeric antigen receptor targeting CD117 and CD7 and its application, which combines targeting antibodies against CD117 and CD7 together to simultaneously recognize and bind to two different antigens.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a bispecific chimeric antigen receptor targeting CD117 and CD7, wherein the bispecific chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and a signaling domain, wherein the antigen binding domain comprises a CD117 binding domain and a CD7 binding domain.

[0009] The bispecific chimeric antigen receptor provided by the present invention combines targeted antibodies against CD117 and CD7, and recognizes and binds to two different antigens at the same time. It not only enhances the targeted elimination ability of malignant cells, but also acts on hematopoietic stem cells and immune cells at the same time, which can more effectively eliminate tumor cells in the receptor while retaining or reconstructing normal hematopoietic and immune functions.

[0010] Preferably, the antigen binding domain comprises, from N-terminus to C-terminus, an anti-CD117 single-chain antibody heavy chain variable region, a first connecting peptide, an anti-CD7 single-chain antibody heavy chain variable region, a second connecting peptide, an anti-CD7 single-chain antibody light chain variable region, a third connecting peptide and an anti-CD117 single-chain antibody light chain variable region.

[0011] Preferably, CDR1, CDR2 and CDR3 of the light chain variable region of the anti-CD117 single-chain antibody include the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0012] Preferably, the light chain variable region of the anti-CD117 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO.4.

[0013] Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region of the anti-CD117 single-chain antibody include the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively.

[0014] Preferably, the heavy chain variable region of the anti-CD117 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO.8.

[0015] SEQ ID NO. 1 (CD117-VL-CDR1): RASESVDIYGNSFMH.

[0016] SEQ ID NO. 2 (CD117-VL-CDR2): LASNLES.

[0017] SEQ ID NO. 3 (CD117-VL-CDR3): QQNNEDPYT.

[0018] SEQ ID NO. 4 (CD117-VL): DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMH WYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPY TFGGGTKVEIK.

[0019] SEQ ID NO. 5 (CD117-VH-CDR1): SYNMH.

[0020] SEQ ID NO. 6 (CD117-VH-CDR2): VIYSGNGDTSYNQKFKG.

[0021] SEQ ID NO. 7 (CD117-VH-CDR3): ERDTRFGN.

[0022] SEQ ID NO. 8 (CD117-VH): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHW VRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSSTAYMELSSLRSEDTAVYYC ARERDTRFGNWGQGTLVTVSS.

[0023] Preferably, CDR1, CDR2 and CDR3 of the light chain variable region of the anti-CD7 single-chain antibody include the amino acid sequences shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, respectively.

[0024] Preferably, the anti-CD7 single-chain antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO.12.

[0025] Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region of the anti-CD7 single-chain antibody include the amino acid sequences shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively.

[0026] Preferably, the heavy chain variable region of the anti-CD7 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO.16.

[0027] SEQ ID NO. 9 (CD7-VL-CDR1): SASQGISNYLN.

[0028] SEQ ID NO. 10 (CD7-VL-CDR2): YTSSLHS.

[0029] SEQ ID NO. 11 (CD7-VL-CDR3): QQYSKLPYT.

[0030] SEQ ID NO. 12 (CD7-VL): DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQK PDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGT KLEIKR.

[0031] SEQ ID NO. 13 (CD7-VH-CDR1): SYAMS.

[0032] SEQ ID NO. 14 (CD7-VH-CDR2): SISSGGFTYYPDSVKG.

[0033] SEQ ID NO. 15 (CD7-VH-CDR3): DEVRGYLDV.

[0034] SEQ ID NO. 16 (CD7-VH): EVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVR QTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEV RGYLDVWGAGTTVTVSS.

[0035] Preferably, the first connecting peptide comprises the amino acid sequence shown in SEQ ID NO.17.

[0036] Preferably, the second connecting peptide comprises the amino acid sequence shown in SEQ ID NO.18.

[0037] Preferably, the third connecting peptide comprises the amino acid sequence shown in SEQ ID NO.19.

[0038] SEQ ID NO. 17: GGGGSGGGGSGGGGS.

[0039] SEQ ID NO. 18: GGGGSGGGGSGGGGSGGGGS.

[0040] SEQ ID NO. 19: GGGGSGGGGSGGGGS.

[0041] Preferably, the signal peptide comprises a CD8α signal peptide, and the CD8α signal peptide comprises the amino acid sequence shown in SEQ ID NO.20.

[0042] Preferably, the hinge region comprises a CD8α hinge region, and the CD8α hinge region comprises the amino acid sequence shown in SEQ ID NO.21.

[0043] Preferably, the transmembrane domain includes a CD8α transmembrane domain, and the CD8α transmembrane domain includes the amino acid sequence shown in SEQ ID NO.22.

[0044] Preferably, the signaling domains are a 4-1BB co-stimulatory domain and a CD3ζ signaling domain, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO.23, and the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO.24.

[0045] SEQ ID NO.20 (CD8α signal peptide): MALPVTALLLPLALLLHAARP.

[0046] SEQ ID NO.21 (CD8α hinge region): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0047] SEQ ID NO.22 (CD8α transmembrane domain): IYIWAPLAGTCGVLLLSLVITLYC.

[0048] SEQ ID NO.23 (4-1BB co-stimulatory domain): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0049] SEQ ID NO. 24 (CD3ζ signaling domain): RVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR.

[0050] Preferably, the bispecific chimeric antigen receptor comprises, from N-terminus to C-terminus, a CD8α signal peptide, an anti-CD117 single-chain antibody heavy chain variable region, a first connecting peptide, an anti-CD7 single-chain antibody heavy chain variable region, a second connecting peptide, an anti-CD7 single-chain antibody light chain variable region, a third connecting peptide, an anti-CD117 single-chain antibody light chain variable region, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB co-stimulatory domain and a CD3ζ signaling domain.

[0051] Preferably, the bispecific chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO.25.

[0052] SEQ ID NO. 25 (BiCAR): MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGAS.

[0053] In a second aspect, the present invention provides a nucleic acid molecule comprising a gene encoding the bispecific chimeric antigen receptor described in the first aspect.

[0054] Preferably, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO.26.

[0055]

[0056] In a third aspect, the present invention provides an expression vector, which comprises the nucleic acid molecule described in the second aspect.

[0057] Preferably, the expression vector further comprises a ribosome binding sequence and a CD7-PEBL1 endoplasmic reticulum arrest signal coding sequence connected in series.

[0058] The CD7 surface antigen can also be detected on normal T lymphocytes and NK cells, as well as on the progenitors of thymocytes, lymphocytes, and myeloid cells. Therefore, in naturally selected CD7 CAR-T cells, the expression of CD7 will trigger the phenomenon of fratricide. At the same time, it affects its own proliferation function and cytotoxicity in vivo. Infusing CD7 CAR-T cells into patients may unexpectedly deplete T cells and NK cells, increasing the risk of opportunistic infections in patients. At present, CD7 CAR-T cells can be modified through gene editing technologies such as CRISPR / CAS9, protein blocking technology, nano-antibody technology, etc., to reduce the fratricide of CD7 CAR-T cells and improve the efficacy of CD7 CAR-T cells.

[0059] The present invention introduces the IRES2 sequence, allowing CAR-T cells to synchronously express a crucial endoplasmic reticulum blocking signal - CD7-PEBL1, which consists of a CD7 single-chain antibody sequence (including VL and VH regions), a Myc tag protein, and an efficient endoplasmic reticulum blocking short peptide KDEL, ensuring that the CD7 molecule is effectively locked in the endoplasmic reticulum and cannot reach the cell surface. The realization of this mechanism greatly reduces the fratricide between CAR-T cells caused by the surface expression of CD7 molecules, thereby improving the safety and effectiveness of CAR-T cell therapy. Through this innovative strategy, the present invention not only optimizes the performance of CAR-T cells, but also brings new hope and possibilities to the field of cancer treatment.

[0060] Preferably, the ribosome binding sequence comprises IRES2, and the IRES2 comprises the nucleic acid sequence shown in SEQ ID NO.27.

[0061] Preferably, the CD7-PEBL1 endoplasmic reticulum arrest signal coding sequence includes the nucleic acid sequence shown in SEQ ID NO.28.

[0062] Preferably, the CD7-PEBL1 endoplasmic reticulum arrest signal comprises the amino acid sequence shown in SEQ ID NO.29.

[0063] SEQ ID NO.27:CCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTG GAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACC。

[0064] SEQ ID NO.28:ATGGCCACAACCATGGATATGAGAGTGCCAGCCCAGCTCCTCGGA CTGCTGCTCCTGTGGCTGCGCGGAGCACGGTGTGCAGCTTACAAGGATATTCAGATGACACAAACCACCAGTAGCCTCTCCGCTAGTCTGGGAGATCGCGTTACCATCAGCTGCTCTGCCAGCCAAGGGATAAGCAATTATCTGAACTGGTATCAGCAGAAACCTGATGGGACAGTGAAGCTGCTGATATACTATACATCTAGCCTCCACAGTGGCGTGCCAAGCAGGTTCTCCGGGAGCGGATCTGGTACAGATTATTCTCTCACAATCAGCAACCTCGAACCCGAGGATATAGCTACCTACTACTGTCAACAGTACTCCAAACTGCCTTACACTTTCGGAGGAGGCACCAAGCTGGAGATCAAAAGAGGCGGAGGCGGATCAGGTGGTGGCGGCTCAGGTGGAGGAGGTTCCGGAGGCGGAGGCTCCGAAGTCCAGCTGGTGGAATCAGGCGGTGGTCTGGTCAAGCCTGGCGGATCTCTGAAGCTCAGCTGTGCTGCTAGTGGCCTGACTTTCAGTAGCTACGCCATGTCCTGGGTGAGGCAAACACCTGAGAAGCGGCTGGAATGGGTTGCCAGCATATCCTCAGGCGGGTTCACATACTATCCAGACTCTGTGAAAGGGAGGTTTACCATTAGCAGAGATAACGCCAGAAATATTCTCTACCTGCAGATGAGCAGCCTGAGATCCGAAGATACTGCTATGTACTACTGCGCCAGGGATGAAGTTCGCGGATACCTCGACGTGTGGGGGGCTGGGACCACCGTCACCGTCTCCAGCGAGCAGAAGCTCATCAGCGAGGAGGATCTGAAAGATGAGCTGTGA。

[0065] SEQ ID NO.29:MATTMDMRVPAQLLGLLLLWLRGARCAAYKDIQMTQTTSSLSASLG DRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGG LVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSEQKLISEEDLKDEL.

[0066] In a fourth aspect, the present invention provides a recombinant lentivirus, which is prepared by mammalian cells transfected with the expression vector and helper plasmid described in the third aspect.

[0067] In a fifth aspect, the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the bispecific chimeric antigen receptor described in the first aspect.

[0068] Preferably, the starting cells of the chimeric antigen receptor immune cells include any one or a combination of at least two of T cells, NK cells, NKT cells, iNKT cells, γδT cells, B cells, macrophages or iPSC-induced immune cells.

[0069] In a sixth aspect, the present invention provides a pharmaceutical composition, comprising the chimeric antigen receptor immune cell described in the fifth aspect.

[0070] Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

[0071] In the seventh aspect, the present invention provides the use of any one or a combination of at least two of the bispecific chimeric antigen receptor described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant lentivirus described in the fourth aspect, the chimeric antigen receptor immune cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of tumor therapeutic drugs.

[0072] Preferably, the tumor comprises liver cancer.

[0073] Compared with the prior art, the present invention has the following beneficial effects:

[0074] The present invention provides a bispecific chimeric antigen receptor targeting CD117 and CD7, and prepares CD117-CD7 bispecific CAR-T cells, which can simultaneously recognize CD117 and CD7 antigens. Compared with single-target CAR-T, bispecific CAR-T has stronger killing ability and has great clinical transformation value in tumor treatment, especially in the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 Schematic diagram of the structures of BiCAR, CD117-specific CAR and CD7-specific CAR targeting CD117 and CD7.

[0076] Figure 2 Figure 2 is a diagram of the lentiviral expression vector for CD117 and CD7 bispecific CAR.

[0077] Figure 3 This is the result of CAR-T expression positive rate detection. DETAILED DESCRIPTION

[0078] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0079] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0080] Example 1

[0081] In this example, a BiCAR targeting CD117 and CD7 was constructed. The schematic diagrams of the structures of the BiCAR targeting CD117 and CD7, the CD117 monospecific CAR, and the CD7 monospecific CAR are shown in FIG. Figure 1 As shown. The BiCAR includes a signal peptide sequence (SP) of CD8α, an anti-CD117 single-chain antibody heavy chain variable region (CD117 VH), a first connecting peptide, an anti-CD7 single-chain antibody heavy chain variable region (CD7 VH), a second connecting peptide, an anti-CD7 single-chain antibody light chain variable region (CD7 VL), a third connecting peptide, an anti-CD117 single-chain antibody light chain variable region (CD117 VL), a CD8α hinge region, a CD8α transmembrane domain (the hinge region and the transmembrane domain are represented as H+TM in the figure), a 4-1BB costimulatory domain, and a CD3ζ signaling domain.

[0082] Example 2

[0083] This example constructs a chimeric antigen receptor expression vector, the vector structure is as follows Figure 2 As shown, the following steps are included:

[0084] (1) Based on the theoretical protein sequence of the CAR gene, the CAR gene was optimized so that it could be efficiently expressed in human cells. The CAR gene was prepared by codon optimization and whole gene synthesis, and whole gene synthesis was performed at Guangzhou Aigene Biotechnology Co., Ltd.

[0085] (2) The fully synthesized CAR gene and the empty vector pCDH-EF1-MCS (lentiviral vector) were double-digested with EcoRI and BamHI. After digestion in a 37°C water bath for 30 min, DNA was electrophoresed using 1.5% agarose gel, and then purified and recovered using Tiangen's agarose gel kit.

[0086] (3) The pCDH-EF1-MCS vector was connected to the CAR gene fragment at 22°C for 1 hour. The ligation product was directly transformed into Stbl3 E. coli competent cells. 200 μL of the transformation product was coated on an ampicillin-resistant LB plate, and the LB plate was incubated in an incubator at 37°C overnight. The next day, three single clones were randomly selected for colony PCR identification, and the positive clones were sent for sequencing. The ligation system is shown in Table 1.

[0087] Table 1

[0088]

[0089]

[0090] Example 3

[0091] In this example, the lentiviral expression vector provided in Example 2 is packaged with lentivirus using a four-plasmid system. The specific steps are as follows:

[0092] (1) The four-plasmid system respectively expresses gag / pol, Rev, VSV-G and the CAR expression vector provided by the present invention required for lentiviral vector packaging: the four plasmids are transiently transfected into 293T cells, and the DNA content is 2 μg / mL.

[0093] (2) The plasmids were mixed with PEI transfection reagent, added to a certain volume of serum-free DMEM, mixed and allowed to stand for 15 min, and the mixture was added to a T75 culture flask containing 293T cells, mixed gently, and cultured in a cell culture incubator at 37°C and 5% CO2 for 6 h.

[0094] (3) After 6 hours, the culture medium was replaced with fresh medium, and the culture was continued. A 10 mM sodium butyrate solution was added. After 72 hours, the culture supernatant of the lentivirus was collected for purification and detection.

[0095] Example 4

[0096] In this example, the lentivirus prepared in Example 3 was used to infect T cells, including the following steps: after isolating PBMC, activating them with X-VIVO containing 50 ng / mL OKT3 and 300 IU / mL IL-2, and replacing the culture medium with 300 IU / mL X-VIVO for expansion culture after 2 days; using RetroNectin to improve the infection efficiency of lentivirus on T cells, 30 μg RetroNectin was coated in a 6-well plate, and placed in a 37°C cell culture incubator for 2 hours; absorbing RetroNectin, blocking the coated 6-well plate with Hank's solution containing 2.5% BSA, and placing it in a 37°C cell culture incubator for 0.5 hours; absorbing the blocking solution, washing the 6-well plate with Hank's solution containing 2% Hepes, adding X-VIVO culture medium, adding an appropriate amount of lentivirus solution, and centrifuging at 2000×g for 2 hours; discarding the supernatant, adding 1×10 6 The activated PBMC cells were centrifuged at 1000×g for 10 min and cultured in a cell culture incubator at 37°C, 5% CO2 and a certain humidity. The cells were counted and replaced with 300 IU / mL X-VIVO every two days, and the cell concentration was maintained at 0.5×10 6 -1×10 6 / mL, and cultured continuously for 8 days. The expansion of CAR-T cells was evaluated using the American Countstar IC1000 automatic cell counter. The results showed that each CAR-T group expanded well and there was no significant difference.

[0097] Example 5

[0098] In this example, the function of the CAR molecule was evaluated. The expression of CAR molecules on the surface of CAR-T cells and their binding ability with the corresponding antigen protein were detected by flow cytometry. The T cell population was labeled with APC-anti-CD3 antibody, and then the CD117 protein and CD7 protein (ACRO Biosystems) were used to detect the CAR expression positivity rate.

[0099] like Figure 3 As shown, the CAR expression rate of CD117 CAR-T cells was 19.34%, and the CAR expression rate of CD7 CAR-T cells was 10.35%. The ability of CD117-CD7 bispecific CAR-T (BiCAR-T) to recognize CD117 and CD7 antigen proteins was further tested, and the positive rates were 45.98% and 43.15%, respectively, indicating that bispecific CAR-T can effectively recognize two antigens.

[0100] In summary, the present invention provides a bispecific chimeric antigen receptor targeting CD117 and CD7, and prepares CD117-CD7 bispecific CAR-T cells, which can simultaneously recognize CD117 and CD7 antigens. Compared with single-target CAR-T, bispecific CAR-T has stronger killing ability and has great clinical transformation value in tumor treatment, especially in the treatment of liver cancer.

[0101] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A bispecific chimeric antigen receptor targeting CD117 and CD7, characterized in that The bispecific chimeric antigen receptor comprises a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain and a signaling domain, wherein the antigen binding domain comprises a CD117 binding domain and a CD7 binding domain.

2. The bispecific chimeric antigen receptor according to claim 1, characterized in that The antigen binding domain includes, from N-terminus to C-terminus, an anti-CD117 single-chain antibody heavy chain variable region, a first connecting peptide, an anti-CD7 single-chain antibody heavy chain variable region, a second connecting peptide, an anti-CD7 single-chain antibody light chain variable region, a third connecting peptide and an anti-CD117 single-chain antibody light chain variable region; Preferably, CDR1, CDR2 and CDR3 of the light chain variable region of the anti-CD117 single-chain antibody include the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively; Preferably, the anti-CD117 single-chain antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO.4; Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region of the anti-CD117 single-chain antibody include the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; Preferably, the heavy chain variable region of the anti-CD117 single-chain antibody comprises the amino acid sequence shown in SEQ ID NO.8; Preferably, CDR1, CDR2 and CDR3 of the light chain variable region of the anti-CD7 single-chain antibody include the amino acid sequences shown in SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11, respectively; Preferably, the anti-CD7 single-chain antibody light chain variable region comprises the amino acid sequence shown in SEQ ID NO.12; Preferably, CDR1, CDR2 and CDR3 of the heavy chain variable region of the anti-CD7 single-chain antibody include the amino acid sequences shown in SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15, respectively; Preferably, the anti-CD7 single-chain antibody heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.16; Preferably, the first connecting peptide comprises the amino acid sequence shown in SEQ ID NO.17; Preferably, the second connecting peptide comprises the amino acid sequence shown in SEQ ID NO.18; Preferably, the third connecting peptide comprises the amino acid sequence shown in SEQ ID NO.

19.

3. The bispecific chimeric antigen receptor according to claim 1 or 2, characterized in that The signal peptide includes a CD8α signal peptide, and the CD8α signal peptide includes an amino acid sequence shown in SEQ ID NO.20; Preferably, the hinge region comprises a CD8α hinge region, and the CD8α hinge region comprises the amino acid sequence shown in SEQ ID NO.21; Preferably, the transmembrane domain comprises a CD8α transmembrane domain, and the CD8α transmembrane domain comprises an amino acid sequence shown in SEQ ID NO.22; Preferably, the signaling domains are a 4-1BB co-stimulatory domain and a CD3ζ signaling domain, wherein the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO.23, and the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO.

24.

4. The bispecific chimeric antigen receptor according to any one of claims 1 to 3, characterized in that: The bispecific chimeric antigen receptor includes, from N-terminus to C-terminus, a CD8α signal peptide, an anti-CD117 single-chain antibody heavy chain variable region, a first connecting peptide, an anti-CD7 single-chain antibody heavy chain variable region, a second connecting peptide, an anti-CD7 single-chain antibody light chain variable region, a third connecting peptide, an anti-CD117 single-chain antibody light chain variable region, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ signaling domain; Preferably, the bispecific chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO.

25.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a gene encoding the bispecific chimeric antigen receptor according to any one of claims 1 to 4; Preferably, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO.

26.

6. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 5; Preferably, the expression vector further comprises a ribosome binding sequence and a CD7-PEBL1 endoplasmic reticulum arrest signal coding sequence connected in series; Preferably, the ribosome binding sequence comprises IRES2, and the IRES2 comprises the nucleic acid sequence shown in SEQ ID NO.27; Preferably, the CD7-PEBL1 endoplasmic reticulum arrest signal coding sequence includes the nucleic acid sequence shown in SEQ ID NO.28; Preferably, the CD7-PEBL1 endoplasmic reticulum arrest signal comprises the amino acid sequence shown in SEQ ID NO.

29.

7. A recombinant lentivirus, characterized in that: The recombinant lentivirus is prepared by mammalian cells transfected with the expression vector and helper plasmid according to claim 6.

8. A chimeric antigen receptor immune cell, characterized in that The chimeric antigen receptor immune cell expresses the bispecific chimeric antigen receptor according to any one of claims 1 to 4; Preferably, the starting cells of the chimeric antigen receptor immune cells include any one or a combination of at least two of T cells, NK cells, NKT cells, iNKT cells, γδT cells, B cells, macrophages or iPSC-induced immune cells.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimeric antigen receptor immune cell of claim 8; Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

10. Use of any one or a combination of at least two of the bispecific chimeric antigen receptor according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the recombinant lentivirus according to claim 7, the chimeric antigen receptor immune cell according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of a tumor therapeutic drug.

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