A chimeric antigen receptor against CD117 and its application

Abstract

The present invention relates to an anti-CD117 chimeric antigen receptor and its application, wherein the chimeric antigen receptor comprises an anti-CD117 single-chain antibody, a hinge region, a transmembrane region, a co-stimulatory domain and an intracellular signal transduction domain; the amino acid sequence of the heavy chain variable region of the anti-CD117 single-chain antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 2. The chimeric antigen receptor can specifically recognize and bind to CD117, thereby accurately locating and killing cells expressing CD117 in vivo.

Description

Technical Field

[0001] The present invention relates to the technical field of chimeric antigen receptors, and in particular to an anti-CD117 chimeric antigen receptor and applications thereof. Background Art

[0002] Hematopoietic stem cell transplantation (HSC) has always played a vital role in the treatment of hematological malignancies. However, in order to ensure the success of the transplant, patients must undergo a pretreatment phase known as myeloablative conditioning. This process aims to completely eliminate leukemia stem cells and tumor cells in the patient's body to make room for newly transplanted healthy hematopoietic stem cells. Although traditional myeloablative methods, such as radiotherapy and chemotherapy, can achieve the desired effects to a certain extent, the side effects they bring are daunting, especially for pediatric patients. The use of chemotherapy drugs such as alkylating agents may cause irreversible reproductive toxicity, bringing long-term pain and burden to patients and their families.

[0003] In recent years, scientists have made significant progress in the study of the surface protein CD117. CD117 is expressed on a variety of cell types, including normal hematopoietic stem cells and leukemia stem cells. Based on this discovery, researchers began to explore targeted therapeutic strategies for CD117 in order to effectively eliminate leukemia cells without damaging normal cells. The use of anti-CD117 antibodies in HSC transplantation, especially in myeloablative conditioning, provides new hope for reducing the side effects of traditional chemotherapy and radiotherapy. The anti-CD117 antibody JSP191 is a monoclonal antibody against the hematopoietic stem cell (HSC) surface marker CD117. CD117, also known as c-kit, is a receptor tyrosine kinase expressed on the surface of hematopoietic stem cells. The mechanism of action of JSP191 is to specifically eliminate abnormal hematopoietic stem cells in the body by targeting CD117, thereby creating conditions for the transplantation of healthy hematopoietic stem cells. In HSC transplantation, the use of JSP191 can reduce the risk of graft-versus-host disease (GVHD) and improve the success rate of transplantation. Currently, JSP191 is in the clinical trial stage, and researchers are evaluating its safety and effectiveness in treating blood system diseases. As research progresses, JSP191 is expected to become an important tool to improve the results of HSC transplantation.

[0004] Therefore, how to prepare an anti-CD117 chimeric antigen receptor has become a problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a chimeric antigen receptor against CD117. The chimeric antigen receptor can specifically recognize and bind to CD117, thereby accurately locating and killing cells expressing CD117 in vivo.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an anti-CD117 chimeric antigen receptor, wherein the chimeric antigen receptor comprises an anti-CD117 single-chain antibody, a hinge region, a transmembrane region, a co-stimulatory domain, and an intracellular signal transduction domain;

[0008] The amino acid sequence of the heavy chain variable region of the anti-CD117 single-chain antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2.

[0009] SEQ ID No.1:

[0010] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEW MGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARE RDTRFGNWGQGTLVTVSS.

[0011] SEQ ID No.2:

[0012] DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPK LLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPYTFG GGTKVEIK.

[0013] In this invention, an innovative anti-CD117 chimeric antigen receptor (CAR) is proposed, which can specifically recognize and bind to CD117, thereby accurately locating and killing cells expressing CD117 in vivo. By using this CAR, effective myeloablation can be performed before hematopoietic stem cell transplantation to maximize the removal of leukemia stem cells and tumor cells in the patient's body, while avoiding the serious side effects of traditional chemotherapy and radiotherapy.

[0014] Preferably, the amino acid sequence of the anti-CD117 single-chain antibody is as shown in SEQ ID No.3.

[0015] SEQ ID No.3:

[0016] DIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPYTFGGGTKVEIKGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARERDTRFGNWGQGTLVTVSS.

[0017] The hinge region includes any one of CD8α, CD28, human IgG1, IgG2, IgG4 or IgA, or a combination of at least two of them.

[0018] Preferably, the amino acid sequence of the hinge region includes that shown in SEQ ID No.4.

[0019] SEQ ID No.4:

[0020] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0021] Preferably, the transmembrane region includes any one or a combination of at least two of CD8α, CD4, CD28, CD3D, CD3E or CD3G.

[0022] Preferably, the amino acid sequence of the transmembrane region includes that shown in SEQ ID No.5.

[0023] SEQ ID No.5:

[0024] IYIWAPLAGTCGVLLLSLVITLYC.

[0025] Preferably, the co-stimulatory domain comprises any one or a combination of at least two of 4-1BB, CD28, OX40, CD27 or ICOS.

[0026] Preferably, the amino acid sequence of the co-stimulatory domain includes that shown in SEQ ID No.6.

[0027] SEQ ID No.6:

[0028] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0029] Preferably, the intracellular signal transduction domain includes any one of CD3ζ, CD3D, CD3E or CD3G, or a combination of at least two of them.

[0030] Preferably, the amino acid sequence of the intracellular signal transduction domain includes that shown in SEQ ID No.7.

[0031] SEQ ID No.7:

[0032] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR.

[0033] Preferably, the anti-CD117 chimeric antigen receptor further comprises a signal peptide.

[0034] Preferably, the signal peptide comprises any one of CD8α, IL-2, CD33, IgG or Insulin, or a combination of at least two of them.

[0035] Preferably, the amino acid sequence of the signal peptide includes that shown in SEQ ID No.8.

[0036] SEQ ID No.8:

[0037] MALPVTALLLPLALLLHAARP.

[0038] Preferably, the amino acid sequence of the anti-CD117 chimeric antigen receptor includes that shown in SEQ ID No.9.

[0039] SEQ ID No.9:

[0040] MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPYT FGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCA RERDTRFGNWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0041] In a second aspect, the present invention provides a nucleic acid molecule encoding the anti-CD117 chimeric antigen receptor described in the first aspect.

[0042] Preferably, the nucleic acid molecule comprises that shown in SEQ ID No.10.

[0043] SEQ ID No.10:

[0044]

[0045] In a third aspect, the present invention provides an expression vector, comprising the nucleic acid molecule described in the second aspect; and after transfection, transduction or transformation of a host cell, the expression vector enables the host cell to express the anti-CD117 chimeric antigen receptor described in the first aspect.

[0046] Preferably, the expression vector further comprises a promoter;

[0047] Preferably, the promoter comprises any one or a combination of at least two of EF1α, CMV, CAG, RPBSA, hPGK or Ubiquitin.

[0048] Preferably, the sequence of the promoter is shown as SEQ ID No.11.

[0049] SEQ ID No.11:

[0050] GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC TTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACG CGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACC GGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATC.

[0051] In a fourth aspect, the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the anti-CD117 chimeric antigen receptor described in the first aspect.

[0052] Preferably, the host cell comprises 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.

[0053] In a fifth aspect, the present invention provides an application of the anti-CD117 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, or the chimeric antigen receptor immune cell described in the fourth aspect in the preparation of a drug for treating diseases associated with high expression of CD117.

[0054] Compared with the prior art, the present invention has at least the following beneficial effects:

[0055] 1. The present invention proposes an innovative anti-CD117 chimeric antigen receptor (CAR), which can specifically recognize and bind to CD117, thereby accurately locating and killing cells expressing CD117 in the body. By using this CAR, effective myeloablation can be performed before hematopoietic stem cell transplantation to maximize the removal of leukemia stem cells and tumor cells in the patient's body, while avoiding the serious side effects of traditional radiotherapy and chemotherapy. The advantage of this technology is its high degree of targeting and low risk of side effects, especially in pediatric patients, which can significantly reduce the potential damage of chemotherapy drugs such as alkylating agents to the reproductive system.

[0056] 2. In terms of social significance, the emergence of this invention has far-reaching impact. It not only provides a new way to improve the safety and effectiveness of hematopoietic stem cell transplantation, but also is expected to improve the long-term survival rate and quality of life of patients. With the continuous deepening of CD117 research, anti-CD117 CAR technology is expected to become an important part of the treatment of hematological malignancies in the future, bringing hope and good news to countless patients. In addition, the promotion and application of this technology will also promote the innovation and development of the entire medical industry in the field of cell therapy, and open up new paths for medical research and clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the structure of anti-CD117 CAR.

[0058] Figure 2 This is the map of the lentiviral expression vector of pCDH-CD117-CAR.

[0059] Figure 3 This is the flow cytometry detection chart of CAR-T expression positivity rate.

[0060] Figure 4 This is the IFN-γ factor secretion detection diagram of the blank T cell group and the CAR-T cell group. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0062] Example 1

[0063] This example designs a chimeric antigen receptor

[0064] In this example, an anti-CD117 chimeric antigen receptor (anti-CD117 CAR) was constructed, and the schematic diagram of the structure is shown in Figure 1 The chimeric antigen receptor includes a CD8α signal peptide sequence (Leader), an anti-CD117 single-chain antibody sequence (anti-CD117scFv), a CD8α hinge region (Hinge) and a transmembrane region sequence (Transmembrane), a 4-1BB co-stimulatory domain sequence and a CD3ζ signaling domain sequence.

[0065] Example 2

[0066] The chimeric antigen receptor expression vector constructed in this example

[0067] (1) CAR gene optimization and sequence synthesis

[0068] According to 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.

[0069] Among them, the signal peptide sequence is shown in SEQ ID No.8, the single-chain antibody sequence against CD117 is shown in SEQ ID No.3, the hinge region of CD8α is shown in SEQ ID No.4, the transmembrane region sequence is shown in SEQ ID No.5, the 4-1BB co-stimulatory domain sequence is shown in SEQ ID No.6, and the CD3ζ signaling domain sequence is shown in SEQ ID No.7.

[0070] (2) Enzyme digestion and identification

[0071] The fully synthesized CAR gene and the empty vector pCDH-MCS were double-digested with EcoRI and BamHI, and digested in a 37°C water bath for 30 minutes. DNA electrophoresis was performed using 1.5% agarose gel, and then purified and recovered using Tiangen's agarose gel kit. The empty vector pCDH-MCS includes the promoter EF1α, and the sequence is shown in SEQ ID No.11.

[0072] (3) Ligation of pCDH-MCS vector and CAR gene fragment

[0073] The pCDH-MCS vector and the CAR gene fragment were connected using the connection system described in Table 1 and incubated at 22°C for 1 hour. The connection product was directly transformed into Stbl3 Escherichia coli competent cells, and 200 μl of the transformation product was coated on an ampicillin-resistant LB plate. The LB plate was inverted and cultured overnight in a 37°C incubator. The next morning, three single clones were randomly selected for colony PCR identification, and the positive clones were sent for sequencing. The sequencing results were correct, that is, the pCDH-MCS vector and the CAR gene fragment were successfully connected. The expression vector map after connection is shown in the figure below. Figure 2 As shown, it was named pCDH-CD117-CAR.

[0074] Example 3

[0075] This example performs lentiviral packaging

[0076] In this embodiment, a four-plasmid system is used to package the lentiviral expression vector, and the specific steps are as follows. The four-plasmid system respectively expresses gag / pol, Rev, VSV-G required for lentiviral vector packaging and the CAR expression vector constructed in Example 2: The four plasmids are transiently transfected into 293T cells, wherein the DNA content is 2 μg / mL. The above plasmids are mixed with PEI transfection reagent, added to a certain volume of serum-free DMEM, mixed and placed for 15 minutes, and the above mixture is added to a T75 culture flask with 293T cells, gently mixed, and cultured in a 37°C, 5% CO2 cell culture incubator for 6 hours. After 6 hours, fresh culture medium is replaced, culture is continued, and 10mM sodium butyrate solution is added. After 72 hours, the culture supernatant of the lentivirus is collected for purification and detection.

[0077] Example 4

[0078] In this example, lentivirus was used to infect T cells

[0079] After PBMCs were isolated, they were activated with X-VIVO containing 50 ng / mL OKT3 and 300 IU / ml IL-2. After 2 days, the culture medium was replaced with X-VIVO containing 300 IU / mL for expanded culture. RetroNectin was used to improve the infection efficiency of lentivirus on T cells. 30 μg of RetroNectin was coated in a 6-well plate and placed in a 37°C cell culture incubator for 2 hours. RetroNectin was aspirated and the coated 6-well plate was blocked with Hank's solution containing 2.5% BSA and placed in a 37°C cell culture incubator for 0.5 hours. The blocking solution was aspirated and the 6-well plate was washed with Hank's solution containing 2% Hepes, X-VIVO culture medium was added, an appropriate amount of lentivirus solution was added, and centrifuged at 2000×g for 2 hours. The supernatant was discarded and 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.

[0080] Example 5

[0081] In this example, CAR expression

[0082] Flow cytometry was used to detect the expression of CAR molecules on the surface of CAR-T cells, APC-anti-CD3 antibody was used to mark the T cell population, and then CD117 protein (ACRO Biosystems) was used to detect the positive rate of CAR expression. Figure 3 The results showed that the average CAR positivity rate of pCDH-CD117-CAR transduction was 71.7% on day 7 of culture. The results showed that CD117 CAR-T can effectively recognize CD117 antigen protein.

[0083] Example 6

[0084] This example is used to identify the function of CAR-T cells

[0085] In this CAR-T killing experiment, flow cytometry was used to detect the binding ability of CAR molecules on the surface of CAR-T cells with the corresponding antigen proteins. HEL-luc was selected as the positive target cell (expressing CD117) and U937-luc as the negative target cell (not expressing CD117). The in vitro killing effect of CAR-T cells and the above target cells was observed at effector-target ratios of 1:1, 2:1, and 5:1.

[0086] The results are as follows Figure 4 It can be seen that the IFN-γ factor was secreted in large quantities in the CAR-T and HEL-luc cell co-incubation group, which was higher than that in the blank T cell group and the CAR-T and U937-luc co-incubation group, especially in the effector-target ratio 5:1 group, indicating that CD117CAR-T has a specific killing effect on HEL-luc.

[0087] The above results indicate that anti-CD117 CAR-T has a killing effect on CD117 positive target cells, but has no killing toxicity to negative cells.

[0088] In summary, anti-CD117 CAR-T can simultaneously recognize CD117 antigens and exhibit specific killing ability. The anti-CD117 CAR-T provided by the present invention has great clinical transformation value.

[0089] 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. An anti-CD117 chimeric antigen receptor, characterized in that: The chimeric antigen receptor comprises an anti-CD117 single-chain antibody, a hinge region, a transmembrane region, a co-stimulatory domain and an intracellular signal transduction domain; The amino acid sequence of the heavy chain variable region of the anti-CD117 single-chain antibody is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The anti-CD117 chimeric antigen receptor according to claim 1, characterized in that The amino acid sequence of the anti-CD117 single-chain antibody is shown in SEQ ID No.

3.

3. The anti-CD117 chimeric antigen receptor according to claim 1 or 2, characterized in that: The hinge region includes any one or a combination of at least two of CD8α, CD28, IgG1, IgG2, IgG4 or IgA; Preferably, the amino acid sequence of the hinge region includes the sequence shown in SEQ ID No.4; Preferably, the transmembrane region includes any one or a combination of at least two of CD8α, CD4, CD28, CD3D, CD3E or CD3G; Preferably, the amino acid sequence of the transmembrane region includes the sequence shown in SEQ ID No.

5.

4. The anti-CD117 chimeric antigen receptor according to any one of claims 1 to 3, characterized in that: The co-stimulatory domain includes any one or a combination of at least two of 4-1BB, CD28, OX40, CD27 or ICOS; Preferably, the amino acid sequence of the co-stimulatory domain includes the sequence shown in SEQ ID No.6; Preferably, the intracellular signal transduction domain comprises any one or a combination of at least two of CD3ζ, CD3D, CD3E or CD3G; Preferably, the amino acid sequence of the intracellular signal transduction domain includes the sequence shown in SEQ ID No.

7.

5. The anti-CD117 chimeric antigen receptor according to any one of claims 1 to 4, characterized in that: The anti-CD117 chimeric antigen receptor further comprises a signal peptide; Preferably, the signal peptide comprises any one or a combination of at least two of CD8α, IL-2, CD33, IgG or Insulin; Preferably, the amino acid sequence of the signal peptide includes the sequence shown in SEQ ID No.8; Preferably, the amino acid sequence of the anti-CD117 chimeric antigen receptor includes the sequence shown in SEQ ID No.

9.

6. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-CD117 chimeric antigen receptor according to any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, characterized in that The nucleic acid molecule includes the sequence shown in SEQ ID No.

10.

8. An expression vector, characterized in that The expression vector contains the nucleic acid molecule of claim 6 or 7; and after transfection, transduction or transformation of the host cell, the expression vector causes the host cell to express the anti-CD117 chimeric antigen receptor of any one of claims 1-5; Preferably, the expression vector further comprises a promoter; Preferably, the promoter comprises any one or a combination of at least two of EF1α, CMV, CAG, RPBSA, hPGK or Ubiquitin; Preferably, the sequence of the promoter is shown as SEQ ID No.

11.

9. A chimeric antigen receptor immune cell, characterized in that: The chimeric antigen receptor immune cell expresses the anti-CD117 chimeric antigen receptor according to any one of claims 1 to 5; 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.

10. Use of an anti-CD117 chimeric antigen receptor according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to claim 8 or the chimeric antigen receptor immune cell according to claim 9 in the preparation of a drug for treating a disease associated with high expression of CD117.

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