A single domain antibody targeting B7-H3, a chimeric antigen receptor molecule, a chimeric antigen receptor cell and use thereof in preparing a drug for treating nasopharyngeal carcinoma

Abstract

The present invention provides a single domain antibody targeting B7-H3, a chimeric antigen receptor molecule, a chimeric antigen receptor cell and an application thereof in preparing a drug for treating nasopharyngeal carcinoma, belonging to the technical field of antibody drugs; the amino acid sequence of the single domain antibody targeting B7-H3 provided by the present invention is shown in SEQ ID NO.1, and the amino acid sequence of the chimeric antigen receptor molecule is shown in SEQ ID NO.2; the chimeric antigen receptor cell provided by the present invention can specifically bind to tumor cells by targeting B7-H3, and has a specific anti-tumor effect in an antigen-dependent manner, and is one of the potential effective methods for treating recurrent and metastatic NPC; according to the description in the embodiments, the chimeric antigen receptor cell provided by the present invention can significantly kill nasopharyngeal carcinoma cells after being co-cultured with the nasopharyngeal carcinoma cell line HK1-EBV.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody drugs, and in particular relates to a single domain antibody targeting B7-H3, a chimeric antigen receptor molecule, a chimeric antigen receptor cell and applications thereof in the preparation of drugs for treating nasopharyngeal carcinoma. Background Art

[0002] With the development of tumor immunotherapy and clinical biotechnology, chimeric antigen receptor T cell (CAR-T) immunotherapy is currently one of the most promising tumor immunotherapy methods. Chimeric antigen receptor (CAR) contains three domains, namely, extracellular antigen binding domain, transmembrane domain and intracellular signal transduction domain. The extracellular antigen binding domain contains a single-chain variable region fragment (scFv) targeting tumor antigens. CAR is expressed on the surface of T cells through gene transfection technology. After co-culture with tumor cells, CAR molecules bind to tumor antigens, CAR activates T cells, and performs specific anti-tumor effects in an antigen-dependent manner, and is not restricted by the major histocompatibility complex (MHC).

[0003] Single-domain antibodies (sdAbs) are a type of antibody that lacks the light chain of an antibody and only has the variable region of the heavy chain. Because of their small molecular weight, they are also called nanobodies. Camelids produce sdAbs with only heavy chains. The antigen-binding fragment of heavy-chain antibodies has a variable domain of heavy chain of heavy-chain antibody (VHH). VHH can maintain high affinity for antigens in the absence of light chains.

[0004] Nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor that is prone to occur in the nasopharynx. It is often accompanied by Epstein-Barr virus infection and has a regional clustering pattern. It is one of the most common malignant tumors. Clinically, NPC may present with hematuria, tinnitus, nasal congestion, headache, decreased vision, and cranial nerve damage. The etiology of NPC has not yet been fully elucidated, but it is considered to be closely related to Epstein-Barr virus infection. Most NPC patients are sensitive to radiotherapy, and early-stage NPC is mainly treated with radiotherapy alone, while locally advanced cases are treated with comprehensive treatments such as radiotherapy and chemotherapy. However, there is currently a lack of effective means for advanced cases and recurrences after treatment. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a single domain antibody, chimeric antigen receptor molecule, chimeric antigen receptor cell targeting B7-H3 and their use in the preparation of drugs for treating nasopharyngeal carcinoma; Epstein-Barr virus infection promotes NPC cells to upregulate the expression of B7-H3, and B7-H3 is lowly expressed in normal tissues. The single domain antibody, chimeric antigen receptor molecule or chimeric antigen receptor cell targeting B7-H3 provided by the present invention can specifically bind to tumor cells by targeting B7-H3, and has a specific anti-tumor effect in an antigen-dependent manner, which is one of the potential effective methods for treating recurrent and metastatic NPC.

[0006] The present invention provides a single-domain antibody targeting B7-H3, and the amino acid sequence of the single-domain antibody is shown in SEQ ID NO.1.

[0007] The present invention provides a chimeric antigen receptor molecule, comprising a CD8a signal peptide, the single domain antibody, a CD8a hinge region, a transmembrane region of CD8a, a 41BB costimulatory region and a CD3z signal transduction domain which are sequentially connected.

[0008] Preferably, the amino acid sequence of the chimeric antigen receptor molecule is as shown in SEQ ID NO.2.

[0009] The present invention provides a lentiviral vector targeting B7-H3, and the construction method comprises the following steps:

[0010] 1) cloning the DNA sequence encoding the chimeric antigen receptor molecule into the pHIV-EGFP vector to obtain the recombinant expression plasmid pHIV-B7-H3-EGFP;

[0011] 2) The recombinant expression plasmid pHIV-B7-H3-EGFP, packaging plasmids pAdVntage, pBabTR and pCMV-dR8.91 obtained in step 1) are transfected into cells at a mass ratio of (4.5-5.5): (2.5-3.5): 1: (0.4-0.6) for culture;

[0012] 3) Collect the culture supernatant to obtain the lentiviral vector targeting B7-H3.

[0013] Preferably, after step 3), the method further comprises: mixing the supernatant with PEG 8000, centrifuging, collecting the precipitate, and resuspending the precipitate to obtain a concentrated lentiviral vector targeting B7-H3.

[0014] Preferably, the volume ratio of the supernatant to PEG 8000 is 1:(4-6).

[0015] Preferably, the resuspension is performed using a PBS solution, and the volume ratio of the precipitate to the PBS solution is 1:(80-120).

[0016] The present invention provides a chimeric antigen receptor cell targeting B7-H3, wherein the chimeric antigen receptor cell expresses the chimeric antigen receptor molecule.

[0017] The present invention provides a method for preparing the chimeric antigen receptor cell targeting B7-H3, comprising the following steps: transfecting the lentiviral vector into PBMCs cells to obtain the cell.

[0018] The present invention provides the use of the single domain antibody targeting B7-H3, the chimeric antigen receptor molecule, the chimeric antigen receptor cell targeting B7-H3 or the lentiviral vector in the preparation of a drug for treating nasopharyngeal carcinoma.

[0019] The present invention also provides the use of the single domain antibody targeting B7-H3, the chimeric antigen receptor molecule, the chimeric antigen receptor cell targeting B7-H3 or the lentiviral vector in the preparation of a reagent for killing nasopharyngeal cancer cells.

[0020] Preferably, the nasopharyngeal carcinoma cells are HK1-EBV cell lines.

[0021] Compared with the prior art, the present invention has the following beneficial effects: the single-domain antibody, chimeric antigen receptor molecule or chimeric antigen receptor cell targeting B7-H3 provided by the present invention can specifically bind to tumor cells by targeting B7-H3, and exert specific anti-tumor effects in an antigen-dependent manner, and is one of the potentially effective methods for treating recurrent and metastatic NPC; according to the description in the examples, the chimeric antigen receptor cell provided by the present invention can significantly kill nasopharyngeal cancer cells after co-culture with the nasopharyngeal cancer cell line HK1-EBV. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the molecular structure of the chimeric antigen receptor targeting B7-H3;

[0023] Figure 2 This is the structure map of pHIV-B7-H3-EGFP expression plasmid;

[0024] Figure 3 The structure map of pBabTR plasmid

[0025] Figure 4 is the positive rate of chimeric antigen receptor cells targeting B7-H3;

[0026] Figure 5 The results of chimeric antigen receptor cells targeting B7-H3 killing nasopharyngeal carcinoma cells in vitro, where the left picture is the control group and the right picture is the B7-H3 CAR-T treatment group. DETAILED DESCRIPTION

[0027] The present invention provides a single domain antibody targeting B7-H3, the amino acid sequence of the single domain antibody is shown in SEQ ID NO.1, and is specifically as follows:

[0028] EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYAMSWYRQAPGKERE LVANIGGPSGYSTYYKEAVKGRFTISRDNAKTTVYLHMNSLKPDDTAVYF CKAVARIGVPQEYWGQGTQVTVSS.

[0029] The present invention provides a chimeric antigen receptor molecule, comprising a CD8a signal peptide, the single domain antibody, a CD8a hinge region, a transmembrane region of CD8a, a 41BB costimulatory region and a CD3z signal transduction domain which are sequentially connected.

[0030] In the present invention, the amino acid sequence of the chimeric antigen receptor molecule is shown in SEQ ID NO.2, which is as follows:

[0031] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASG FPFSNYAMSWYRQAPGKERELVANIGGPSGYSTYYKEAVKGRFTISRDNAKTTVYLHMNSLKPDDTAVYFCKAVARIGVPQEYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVI TLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0032] The present invention provides a chimeric antigen receptor cell targeting B7-H3, wherein the chimeric antigen receptor cell expresses the chimeric antigen receptor molecule. In the present invention, the chimeric antigen receptor cell targeting B7-H3 is preferably obtained by transfecting a lentiviral vector targeting B7-H3 into PBMCs stimulated with CD3 / CD28 and culturing them.

[0033] The present invention provides a lentiviral vector targeting B7-H3, and the construction method comprises the following steps:

[0034] 1) cloning the DNA sequence encoding the chimeric antigen receptor molecule into the pHIV-EGFP vector to obtain the recombinant expression plasmid pHIV-B7-H3-EGFP;

[0035] 2) The recombinant expression plasmid pHIV-B7-H3-EGFP, packaging plasmids pAdVntage, pBabTR and pCMV-dR8.91 obtained in step 1) are transfected into cells at a ratio of (4.5-5.5): (2.5-3.5): 1: (0.4-0.6) for culture;

[0036] 3) Collect the culture supernatant to obtain the lentiviral vector targeting B7-H3.

[0037] In the present invention, the DNA sequence encoding the chimeric antigen receptor molecule is cloned into the pHIV-EGFP vector to obtain the recombinant expression plasmid pHIV-B7-H3-EGFP; in the present invention, the pHIV-EGFP vector is preferably purchased from Shanghai Hewu Biotechnology Co., Ltd., item number P25226; the DNA sequence encoding the chimeric antigen receptor molecule is synthesized by a biological company and then cloned into the pHIV-EGFP vector; the above operation is preferably performed by a biological technology company.

[0038] After obtaining the recombinant expression plasmid pHIV-B7-H3-EGFP, the present invention transfects the recombinant expression plasmid pHIV-B7-H3-EGFP, the packaging plasmid pAdVntage, pBabTR and pCMV-dR8.91 into cells for culture in a mass ratio of (4.5-5.5):(2.5-3.5):1:(0.4-0.6); in the present invention, the mass ratio of the recombinant expression plasmid pHIV-B7-H3-EGFP, the packaging plasmid pAdVntage, pBabTR and pCMV-dR8.91 is preferably (4.8-5.2):(2.8-3.2):1:(0.45-0.55), and more preferably 5:3:1:0.5. In the present invention, the packaging plasmids pAdVntage and pCMV-dR8.91 are preferably purchased from Shanghai Hewu Biotechnology Co., Ltd.; the pBabTR plasmid is synthesized by Beijing Ruibo Xingke Biotechnology Co., Ltd. The structure map of the plasmid is as shown in Figure 3As shown. In the present invention, the cells are preferably 293T cells, and the 293T cells are preferably purchased from the American Type Culture Collection (ATCC). In the present invention, the culture time is preferably 48 to 72 hours. The present invention preferably collects the supernatant at 48 hours and 72 hours of culture, respectively, and the collection is preferably performed by filtering with a filter membrane, and the pore size of the filter membrane is preferably 0.45 μm. After collecting the supernatant, the present invention preferably further includes: mixing the supernatant with PEG 8000, centrifuging, collecting the precipitate, and resuspending to obtain a concentrated lentiviral vector targeting B7-H3. In the present invention, the volume ratio of the supernatant to PEG 8000 is preferably 1: (4-6), more preferably 1: 5, and the PEG 8000 is preferably purchased from Sigma-Aldrich, with a catalog number of 89510; in the present invention, the centrifugal temperature is preferably 3-5°C, more preferably 4°C; the centrifugal force of the centrifugation is preferably 3500-4500g, more preferably 3800-4200g, and more preferably 4000g; the centrifugal time is preferably 40-50min, more preferably 42-48min, and most preferably 45min. After the centrifugation, the precipitate is collected and resuspended with a PBS solution. The ratio of the precipitate to the PBS solution is preferably 1: (80-120), more preferably 1: (90-110), and more preferably 1: 100. In the present invention, the concentrated B7-H3-targeting lentiviral vector is preferably stored at low temperature, and more preferably stored at -80°C.

[0039] The present invention provides a chimeric antigen receptor cell targeting B7-H3, wherein the chimeric antigen receptor cell expresses the chimeric antigen receptor molecule.

[0040] The present invention also provides a method for preparing the chimeric antigen receptor cell targeting B7-H3, comprising the following steps: transfecting the lentiviral vector into PBMCs cells to obtain the cell. The present invention has no special limitation on the specific operation of the transfection, and conventional transfection operations in the art can be used.

[0041] The present invention also provides the use of the single domain antibody targeting B7-H3, the chimeric antigen receptor molecule, the chimeric antigen receptor cell targeting B7-H3 or the lentiviral vector in the preparation of a drug for treating nasopharyngeal carcinoma. In the present invention, the chimeric antigen receptor cell targeting B7-H3 can significantly kill the nasopharyngeal carcinoma cell line HK1-EBV, and can be used as a drug for inhibiting the nasopharyngeal carcinoma cell line HK1-EBV or inhibiting nasopharyngeal carcinoma.

[0042] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] Design of chimeric antigen receptor molecules targeting B7-H3

[0045] The alpaca single domain antibody (shown in SEQ ID No. 1) screened out against B7-H3 was connected to the CD8a signal peptide, hinge region and transmembrane region, 41BB co-stimulatory and CD3z signaling domains, respectively. The structure of the chimeric antigen receptor molecule targeting B7-H3 is as follows: Figure 1 The amino acid sequence is shown in SEQ ID NO.2.

[0046] Construction of chimeric antigen receptor CAR-T cells targeting B7-H3

[0047] The DNA sequence encoding the above-mentioned B7-H3 chimeric antigen receptor molecule was constructed into the pHIV-EGFP vector to form the pHIV-B7-H3-EGFP expression plasmid (commissioned to a biological company). The plasmid map is shown in Figure 2 shown.

[0048] The empty plasmid or the successfully constructed target expression plasmid was respectively combined with the packaging plasmid pAdVntage (purchased from Shanghai Hewu Biotechnology Co., Ltd., catalog number: P2356), pBabTR (Beijing Ruibo Xingke Biotechnology Co., Ltd., plasmid map as shown in Figure 3 ) and pCMV-dR8.91 (Shanghai Hewu Biotechnology Co., Ltd., catalog number: P3110) were transfected into 293T cells (purchased from the American Type Culture Collection Center ATCC) at a ratio of 5:3:1:0.5. The culture supernatant was collected after 48h and 72h, filtered through a 0.45μm filter, and PEG 8000 (purchased from Sigma-Aldrich, catalog number 89510) was added. After thorough mixing, centrifugation was performed at 4000g for 45min at 4℃, the supernatant was discarded, and the precipitate was resuspended in PBS at 1:100 and frozen at -80 degrees for later use. The concentrated virus was used to infect PBMCs from healthy subjects after CD3 / CD28 stimulation; the volume ratio of concentrated virus to PBMCs was 50μl:500μl, and the concentration of PBMCs was 10 6 500 IU / ml IL-2 was added in vitro to maintain the growth of infected PBMCs. After culturing for 20 days, the expression of CAR molecules was detected by VHH flow cytometry antibody labeling. The results are as follows: Figure 4 As shown, the positivity rate is approximately 77%.

[0049] Example 2

[0050] The B7-H3 CAR-T cells prepared in Example 1 were co-cultured with the nasopharyngeal carcinoma cell line HK1-EBV (derived from the Cancer Prevention and Treatment Center of Sun Yat-sen University) at a ratio of CAR-T cells: nasopharyngeal carcinoma cells = 5:1 for 6 hours, and the tumor cells were labeled with flow cytometry antibodies 7-AAD and ANNEXIN-V, and the apoptosis of tumor cells was detected by flow cytometry. A control group was set up, in which T cells transfected with empty vectors were co-cultured with the nasopharyngeal carcinoma cell line HK1-EBV, and the other conditions were consistent with the experimental group. Compared with the control group, B7-H3 CAR-T cells significantly killed tumor cells, as shown in Figure 2. Figure 5 .

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A single domain antibody targeting B7-H3, characterized in that: The amino acid sequence of the single domain antibody is shown in SEQ ID NO.

1.

2. A chimeric antigen receptor molecule, characterized in that It comprises a CD8a signal peptide, the single domain antibody according to claim 1, a CD8a hinge region, a transmembrane region of CD8a, a 41BB costimulatory region and a CD3z signaling domain which are sequentially connected.

3. The chimeric antigen receptor molecule according to claim 2, characterized in that The amino acid sequence of the chimeric antigen receptor molecule is shown in SEQ ID NO.

2.

4. A lentiviral vector targeting B7-H3, characterized in that: The construction method includes the following steps: 1) cloning the DNA sequence encoding the chimeric antigen receptor molecule of claim 3 into the pHIV-EGFP vector to obtain the recombinant expression plasmid pHIV-B7-H3-EGFP; 2) The recombinant expression plasmid pHIV-B7-H3-EGFP, packaging plasmids pAdVntage, pBabTR and pCMV-dR8.91 obtained in step 1) are transfected into cells at a mass ratio of (4.5-5.5): (2.5-3.5): 1: (0.4-0.6) for culture; 3) Collect the culture supernatant to obtain the lentiviral vector targeting B7-H3.

5. The lentiviral vector according to claim 4, characterized in that Step 3) further includes: mixing the supernatant with PEG8000, centrifuging, collecting the precipitate, and resuspending the precipitate to obtain a concentrated lentiviral vector targeting B7-H3.

6. The lentiviral vector according to claim 5, characterized in that The volume ratio of the supernatant to PEG 8000 is 1:(4-6).

7. The lentiviral vector according to claim 5 or 6, characterized in that The resuspension is performed using a PBS solution, and the volume ratio of the precipitate to the PBS solution is 1:(80-120).

8. A chimeric antigen receptor cell targeting B7-H3, characterized in that: The chimeric antigen receptor cell expresses the chimeric antigen receptor molecule of claim 2 or 3.

9. The method for preparing a chimeric antigen receptor cell targeting B7-H3 according to claim 8, characterized in that: The following steps are involved: The method is obtained by transfecting PBMCs cells with the lentiviral vector according to any one of claims 4 to 7.

10. Use of the single domain antibody targeting B7-H3 according to claim 1, the chimeric antigen receptor molecule according to claim 2 or 3, the lentiviral vector according to any one of claims 4 to 7, and the chimeric antigen receptor cell targeting B7-H3 according to claim 8 in the preparation of a drug for treating nasopharyngeal carcinoma.

Images