Single-domain antibody and application thereof in preparation of medicine for treating nasopharynx cancer

Abstract

The invention provides a single-domain antibody and application thereof in preparation of a medicine for treating nasopharyngeal carcinoma, and belongs to the technical field of biological medicine, and the amino acid sequence of the single-domain antibody is shown as SEQ ID NO.1; the invention also provides a chimeric antigen receptor containing the single-domain antibody and a chimeric antigen receptor cell, and the single-domain antibody has high affinity and specificity, and can specifically recognize and bind nasopharyngeal carcinoma cells; according to the record of the embodiment, compared with a control group, the chimeric antigen recipient cell provided by the invention can obviously kill tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a single-domain antibody and its application in preparing a drug for treating nasopharyngeal carcinoma. Background Art

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the nasopharyngeal mucosal epithelium with significant geographical clustering. Its occurrence is closely related to Epstein-Barr virus (EBV) infection, genetic susceptibility, and environmental factors. EBV continuously activates the NF-κB pathway through latent membrane proteins (such as LMP1), inducing cell proliferation and inhibiting apoptosis. At the same time, it forms an immunosuppressive microenvironment by upregulating immune checkpoint molecules such as PD-L1 / B7-H3. Clinical data show that the 5-year survival rate of early-stage NPC patients can reach over 80% with radiotherapy alone, but the 5-year progression-free survival rate of locally advanced patients is still less than 50% even with concurrent chemoradiotherapy, and the median overall survival of recurrent / metastatic cases is only 12-18 months. Therefore, there is no effective treatment option for advanced or recurrent cases.

[0003] Chimeric antigen receptor T cell (CAR-T) therapy, a revolutionary breakthrough in cancer immunotherapy, has demonstrated significant efficacy in tumor treatment. However, traditional CAR-T technology still faces multiple challenges: First, tumor-associated antigens (TAAs) in solid tumors are of low abundance and high heterogeneity, and existing CAR structures are insufficiently sensitive to low-abundance antigens, resulting in limited therapeutic effects; second, traditional antibody fragments (such as scFv) have problems such as poor tissue permeability and high immunogenicity, which affect the durability of therapeutic effects.

[0004] In recent years, single-domain antibodies (Nanobodies) have become a research hotspot due to their unique advantages. With a molecular weight of only 15 kDa, approximately one-tenth that of traditional antibodies, single-domain antibodies possess high affinity, high stability, and excellent tissue penetration. Their low immunogenicity and ease of genetic engineering make them highly promising candidates for targeted tumor therapy. For example, single-domain antibody-based PET imaging probes have been successfully used to dynamically monitor PD-L1 expression, but their application in the development of CAR-T cells remains exploratory. Summary of the Invention

[0005] In light of this, the present invention aims to provide a single-domain antibody and its use in the preparation of a medicament for treating nasopharyngeal carcinoma; the single-domain antibody has high affinity and specificity. A chimeric antigen receptor constructed based on the single-domain antibody has demonstrated excellent tumor-killing ability in in vitro and animal experiments.

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

[0007] SEQ ID NO.1:

[0008] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLE WVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC QKGVLKTGAKGQGTQVTVSS.

[0009] The present invention provides a DNA molecule encoding the single domain antibody, the nucleotide sequence of the DNA molecule is shown as SEQ ID NO.2;

[0010] SEQ ID NO.2:

[0011] GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGCAGCCTGGCGGATCTCTGCGGCTGAGCTGTGCCGCTTCTGGCTTTACATTCTCCGACAACTTCATGAGATGGGTGCGGCAGGCCCCGGCAAGGGCCTGGAATGGGTCGCTACCATCAACACCAACGGCGGATCTACC TTCTACCTGGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGATAATGCCAAGAACACACTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCCAGAAGGGCGTGCTGAAGACCGGCGCTAAAGGCCAGGGCACCCAAGTGACCGTGTCCAGC.

[0012] The present invention provides the use of the single-domain antibody in preparing a chimeric antigen receptor.

[0013] The present invention provides a chimeric antigen receptor comprising the single-domain antibody.

[0014] Preferably, the amino acid sequence is shown in SEQ ID NO.3;

[0015] SEQ ID NO.3:

[0016] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLEWVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCQKGVLKTGAKGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0017] The present invention provides a chimeric antigen receptor cell, which expresses the chimeric antigen receptor.

[0018] The present invention provides use of the single-domain antibody, the chimeric antigen receptor or the chimeric antigen receptor cell in preparing a drug for treating nasopharyngeal carcinoma.

[0019] The present invention provides the use of the single-domain antibody, the chimeric antigen receptor or the chimeric antigen receptor cell in preparing a drug for killing nasopharyngeal cancer cells.

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

[0021] Preferably, the ratio of the number of the chimeric antigen receptor cells to the nasopharyngeal carcinoma cell line HK1-EBV is (1-3):1.

[0022] Compared with the prior art, the present invention has the following beneficial effects: the single-domain antibody provided by the present invention is an alpaca single-domain antibody obtained through screening, which can specifically recognize and bind to nasopharyngeal cancer cells by targeting CD276, and exert specific anti-tumor effects in an antigen-dependent manner; according to the examples, compared with the control group, the chimeric antigen receptor cells provided by the present invention can significantly kill tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The positive rate of CAR-T cells was detected by flow cytometry;

[0024] Figure 2 Flow cytometry was used to detect the killing of tumor cells by CAR-T cells. DETAILED DESCRIPTION

[0025] The present invention provides a single domain antibody, the amino acid sequence of the single domain antibody is shown in SEQ ID NO.1;

[0026] SEQ ID NO.1:

[0027] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLE WVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC QKGVLKTGAKGQGTQVTVSS.

[0028] The present invention provides a DNA molecule encoding the single domain antibody, the nucleotide sequence of the DNA molecule is shown as SEQ ID NO.2;

[0029] SEQ ID NO.2:

[0030] GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGCAGCCTGGCGGATCTCTGCGGCTGAGCTGTGCCGCTTCTGGCTTTACATTCTCCGACAACTTCATGAGATGGGTGCGGCAGGCCCCGGCAAGGGCCTGGAATGGGTCGCTACCATCAACACCAACGGCGGATCTACC TTCTACCTGGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGATAATGCCAAGAACACACTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCCAGAAGGGCGTGCTGAAGACCGGCGCTAAAGGCCAGGGCACCCAAGTGACCGTGTCCAGC.

[0031] The present invention provides the use of the single-domain antibody in preparing a chimeric antigen receptor. In the present invention, preferably, the single-domain antibody is connected to the CD8a signal peptide upstream, and the CD8a hinge region, CD8a transmembrane region, 41BB costimulatory region and CD3z signaling domain are connected downstream in sequence to obtain a chimeric antigen receptor.

[0032] The present invention also provides a chimeric antigen receptor comprising the single-domain antibody, the amino acid sequence of which is shown in SEQ ID NO.3;

[0033] SEQ ID NO.3:

[0034] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLEWVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCQKGVLKTGAKGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0035] The present invention provides a chimeric antigen receptor cell, which expresses a chimeric antigen receptor. The present invention does not particularly limit the method for preparing the chimeric antigen receptor cell; the chimeric antigen receptor cell can be prepared using conventional chimeric antigen receptor cell preparation methods in the art. In a specific embodiment of the present invention, a DNA molecule encoding the chimeric antigen receptor is preferably recombined into a lentiviral vector, which is then transfected into CD3 / CD28-stimulated PBMCs and cultured.

[0036] The present invention also provides the use of the single-domain antibody, the chimeric antigen receptor, or the chimeric antigen receptor cell in the preparation of a drug for treating nasopharyngeal carcinoma. The present invention does not particularly limit the dosage form of the drug, as long as it can ensure the specific recognition activity of the single-domain antibody.

[0037] The present invention also provides the use of the single-domain antibody, the chimeric antigen receptor or the chimeric antigen receptor cell in the preparation of a drug for killing nasopharyngeal cancer cells.

[0038] In the present invention, the nasopharyngeal carcinoma cell line is preferably the nasopharyngeal carcinoma cell line HK1-EBV. The present invention does not particularly limit the source or preparation method of the nasopharyngeal carcinoma cell line HK1-EBV. The nasopharyngeal carcinoma cell line HK1-EBV is a nasopharyngeal carcinoma cell line infected with the Epstein-Barr virus (EBV). The primary cell line HK1 is derived from tumor tissue of a nasopharyngeal carcinoma (NPC) patient and becomes an immortalized cell model after being transformed by EBV. This cell line retains the complete EBV genome and continuously expresses viral proteins (such as EBNA1 and LMP1), simulating the latent EBV infection state in vivo.

[0039] In the present invention, the ratio of the chimeric antigen receptor cells to the nasopharyngeal carcinoma cell line HK1-EBV is (1-3):1, preferably (1.5-2.5):1, and more preferably 2:1.

[0040] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] Design of chimeric antigen receptor molecules targeting CD276:

[0043] An alpaca single-domain antibody against CD276 (SEQ ID No. 1) was screened out, and the CD8a signal peptide was connected upstream, and the CD8a hinge region, CD8a transmembrane region, 41BB costimulatory region and CD3z signaling domain were connected downstream in sequence to obtain a chimeric antigen receptor (SEQ ID No. 3).

[0044] SEQ ID NO.1:

[0045] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLE WVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC QKGVLKTGAKGQGTQVTVSS.

[0046] SEQ ID NO.3:

[0047] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLEWVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCQKGVLKTGAKGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0048] The DNA sequence encoding the chimeric antigen receptor was constructed into the pHIV-EGFP vector to form the pHIV-CD276-EGFP expression plasmid (commissioned to a biological company).

[0049] The empty plasmid or the successfully constructed target expression plasmid was transfected into 293T cells (purchased from the American Type Culture Collection) with the packaging plasmids pAdVntage (purchased from Shanghai Hewu Biotechnology Co., Ltd., catalog number: P2356), pBabTR (Beijing Ruibo Xingke Biotechnology Co., Ltd.) and pCMV-dR8.91 (Shanghai Hewu Biotechnology Co., Ltd., catalog number: P3110) at a ratio of 5:3:1:0.5. The culture supernatant was collected after 48 h and 72 h, filtered through a 0.45 μm filter, and PEG8000 (purchased from Sigma-Aldrich, catalog number 89510) was added. After thorough mixing, the cells were centrifuged at 4000 g at 4°C for 45 min, the supernatant was discarded, and the precipitate was resuspended in PBS at a ratio of 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 50 μl:500 μl, 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 1 As shown, the positive rate was 87.59%.

[0050] The chimeric antigen receptor cells prepared above 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 = 2:1 for 6 hours. The tumor cells were labeled with flow cytometry antibody 7-AAD, and the apoptosis of the tumor cells was detected by flow cytometry. A control group was set up. The control group used T cells transfected with an empty vector to co-culture with the nasopharyngeal carcinoma cell line HK1-EBV. The other conditions were the same as those of the experimental group. Compared with the control group, the chimeric antigen receptor cells provided by the present invention significantly killed tumor cells, such as Figure 2 As shown, the 7-AAD positive rate reached 30.44%.

[0051] It can be seen from the above examples that the single-domain antibody provided by the present invention can specifically recognize and bind to nasopharyngeal carcinoma cells by targeting CD276. Compared with the control group, the chimeric antigen receptor cells provided by the present invention can significantly kill tumor cells.

[0052] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A single domain antibody, characterized in that The amino acid sequence of the single domain antibody is shown in SEQ ID NO.1; SEQ ID NO.1: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLE WVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYC QKGVLKTGAKGQGTQVTVSS.

2. A DNA molecule encoding the single domain antibody according to claim 1, characterized in that The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.2; SEQ ID NO.2: GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGCAGCCTGGCGGATCTCTGCGGCTGAGCTGTGCCGCTTCTGGCTTTACATTCTCCGACAACTTCATGAGATGGGTGCGGCAGGCCCCGGCAAGGGCCTGGAATGGGTCGCTACCATCAACACCAACGGCGGATCTACC TTCTACCTGGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGATAATGCCAAGAACACACTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCCAGAAGGGCGTGCTGAAGACCGGCGCTAAAGGCCAGGGCACCCAAGTGACCGTGTCCAGC.

3. Use of the single domain antibody according to claim 1 in the preparation of a chimeric antigen receptor. A chimeric antigen receptor comprising the single domain antibody of claim 1 .

5. The chimeric antigen receptor according to claim 4, characterized in that The amino acid sequence is shown in SEQ ID NO. 3; SEQ ID NO.3: MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLEWVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCQKGVLKTGAKGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

6. A chimeric antigen receptor cell, characterized in that The chimeric antigen receptor cell expresses the chimeric antigen receptor according to claim 4 or 5.

7. Use of the single domain antibody according to claim 1, the chimeric antigen receptor according to claim 4 or 5, or the chimeric antigen receptor cell according to claim 6 in the preparation of a medicament for treating nasopharyngeal carcinoma.

8. Use of the single domain antibody according to claim 1, the chimeric antigen receptor according to claim 4 or 5, or the chimeric antigen receptor cell according to claim 6 in the preparation of a drug for killing nasopharyngeal cancer cells.

9. The use according to claim 8, characterized in that The nasopharyngeal carcinoma cell line is HK1-EBV.

10. The use according to claim 9, characterized in that The number ratio of the chimeric antigen receptor cells to the nasopharyngeal carcinoma cell line HK1-EBV is (1-3):1.

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