A single domain antibody and its use in the preparation of a medicament for treating nasopharyngeal carcinoma

Abstract

The application 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, wherein the amino acid sequence of the single-domain antibody is shown as SEQ ID NO. 1; the application further provides a chimeric antigen receptor and a chimeric antigen receptor cell comprising the single-domain antibody; the single-domain antibody has high affinity and specificity, and can specifically recognize and combine with nasopharyngeal carcinoma cells; according to the embodiment, compared with a control group, the chimeric antigen receptor cell provided by the application can significantly kill tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a single-domain antibody and its application in the preparation of drugs for treating nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the nasopharyngeal mucosal epithelium. It exhibits significant geographical clustering, and 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. Simultaneously, it creates an immunosuppressive microenvironment by upregulating immune checkpoint molecules such as PD-L1 / B7-H3. Clinical data show that the 5-year survival rate for early-stage NPC patients treated with radiotherapy alone can reach over 80%. However, even with concurrent chemoradiotherapy, the 5-year progression-free survival rate for locally advanced patients is still less than 50%, while the median overall survival for 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, as a revolutionary breakthrough in cancer immunotherapy, has demonstrated significant efficacy in the field of tumor treatment. However, traditional CAR-T technology still faces multiple challenges: First, tumor-associated antigens (TAAs) in solid tumors are low in abundance and highly heterogeneous, and existing CAR structures are not sensitive enough to low-abundance antigens, resulting in limited therapeutic effects; Second, traditional antibody fragments (such as scFv) have problems such as poor tissue penetration and high immunogenicity, which affect the durability of efficacy.

[0004] In recent years, single-domain antibodies (Nanobody) 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 for targeted cancer therapy. For example, PET imaging probes based on single-domain antibodies have been successfully used for dynamic monitoring of PD-L1 expression, but their application in CAR-T cell construction remains in the exploratory stage. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a single-domain antibody and its application in the preparation of a drug for treating nasopharyngeal carcinoma; the single-domain antibody has high affinity and specificity. The chimeric antigen receptor constructed based on this single-domain antibody has shown excellent tumor-killing ability in in vitro and animal experiments.

[0006] This invention provides a single-domain antibody, the amino acid sequence of which 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 which is shown in SEQ ID NO.2;

[0010] SEQ ID NO.2:

[0011] GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGCAGCCTGGCGGATCTCTGCGGCTGAGCTGTGCCGCTTCTGGCTTTACATTCTCCGACAACTTCATGAGATGGGTGCGGCAGGCCCCGGCAAGGGCCTGGAATGGGTCGCTACCATCAACACCAACGGCGGATCTACC TTCTACCTGGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGATAATGCCAAGAACACACTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCCAGAAGGGCGTGCTGAAGACCGGCGCTAAAGGCCAGGGCACCCAAGTGACCGTGTCCAGC.

[0012] This invention provides the application of the single-domain antibody in the preparation of chimeric antigen receptors.

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

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

[0015] SEQ ID NO.3:

[0016] MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSDNFMRWVRQAPGKGLEWVATINTNGGSTFYLDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCQKGVLKTGAKGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC DIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

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

[0018] This invention provides the use of the single-domain antibody, the chimeric antigen receptor, or the chimeric antigen receptor cell in the preparation of a medicament for treating nasopharyngeal carcinoma.

[0019] This invention provides the use of the single-domain antibody, the chimeric antigen receptor, or the chimeric antigen receptor cell in the preparation of a drug that kills nasopharyngeal carcinoma cells.

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

[0021] Preferably, the ratio 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. By targeting CD276, it can specifically recognize and bind to nasopharyngeal carcinoma cells and exert a specific anti-tumor effect through an antigen-dependent manner; According to the embodiments, compared with the control group, the chimeric antigen receptor cells provided by the present invention can significantly kill tumor cells. Attached Figure Description

[0023] Figure 1 To detect the positivity rate of CAR-T cells by flow cytometry;

[0024] Figure 2 To detect the killing effect of CAR-T cells on tumor cells by flow cytometry. Detailed Implementation

[0025] This invention provides a single-domain antibody, the amino acid sequence of which 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 which is shown in SEQ ID NO.2;

[0029] SEQ ID NO.2:

[0030] GAAGTGCAACTAGTGGAAAGTGGTGGTGGTCTCGTGCAGCCTGGCGGATCTCTGCGGCTGAGCTGTGCCGCTTCTGGCTTTACATTCTCCGACAACTTCATGAGATGGGTGCGGCAGGCCCCGGCAAGGGCCTGGAATGGGTCGCTACCATCAACACCAACGGCGGATCTACC TTCTACCTGGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGATAATGCCAAGAACACACTGTACCTGCAGATGAACTCCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCCAGAAGGGCGTGCTGAAGACCGGCGCTAAAGGCCAGGGCACCCAAGTGACCGTGTCCAGC.

[0031] This invention provides the application of the aforementioned single-domain antibody in the preparation of chimeric antigen receptors. In this invention, preferably, a chimeric antigen receptor is obtained by linking a CD8a signal peptide upstream of the single-domain antibody, and sequentially linking the CD8a hinge region, the transmembrane region of CD8a, the 41BB co-stimulatory region, and the CD3z signal transduction domain downstream.

[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] This invention provides a chimeric antigen receptor cell that expresses the chimeric antigen receptor. The invention does not specify a particular method for preparing the chimeric antigen receptor cell; conventional methods for preparing chimeric antigen receptor cells in the art can be used. In a preferred embodiment of this invention, the DNA molecule encoding the chimeric antigen receptor is recombined into a lentiviral vector, and then the lentiviral vector is transfected into CD3 / CD28-stimulated PBMCs for culture.

[0036] This 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 medicament for treating nasopharyngeal carcinoma. This invention does not impose any particular limitation on the dosage form of the medicament, as long as the specific recognition activity of the single-domain antibody is guaranteed.

[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 carcinoma cells.

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

[0039] In this invention, the ratio of chimeric antigen receptor cells to 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 will be 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 chimeric antigen receptor molecules targeting CD276:

[0043] Alpaca single-domain antibody against CD276 (SEQ ID No. 1) was screened out, and a chimeric antigen receptor (SEQ ID No. 3) was obtained by linking the CD8a signal peptide upstream and the CD8a hinge region, CD8a transmembrane region, 41BB co-stimulatory region and CD3z signal transduction domain downstream.

[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 (construction was outsourced to a biotechnology company).

[0049] Empty vector plasmids or successfully constructed target expression plasmids were transfected into 293T cells (purchased from the American Type Culture Collection Center, ATCC) at a ratio of 5:3:1:0.5 with packaging plasmids pAdVntage (purchased from Shanghai Hewu Biotechnology Co., Ltd., catalog number: P2356), pBabTR (Beijing Ruiboxingke Biotechnology Co., Ltd.), and pCMV-dR8.91 (Shanghai Hewu Biotechnology Co., Ltd., catalog number: P3110). After 48 h and 72 h, the culture supernatant was collected, 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 4000g for 45 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in PBS at a ratio of 1:100 and frozen at -80 °C for later use. PBMCs derived from healthy individuals stimulated with CD3 / CD28 were infected with concentrated virus; the volume ratio of concentrated virus to PBMCs was 50 μl:500 μl, and the concentration of PBMCs was 10. 6 PBMCs were cultured at a concentration of [number] cells / ml, and 500 IU / ml IL-2 was added in vitro to maintain the growth of infected PBMCs. After 20 days of culture, 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 (from Sun Yat-sen University Cancer Center) at a ratio of CAR-T cells:nasopharyngeal carcinoma cells = 2:1 for 6 hours. Tumor cells were then labeled with 7-AAD antibody by flow cytometry, and apoptosis was detected by flow cytometry. A control group was set up, in which T cells transfected with an empty vector were co-cultured with the nasopharyngeal carcinoma cell line HK1-EBV, with other conditions identical to the experimental group. Compared with the control group, the chimeric antigen receptor cells provided by this invention significantly killed tumor cells, such as... Figure 2 As shown, the positivity rate of 7-AAD reached 30.44%.

[0051] As can be seen from the above embodiments, 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 description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered 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 of 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. The use of the single-domain antibody according to claim 1 in the preparation of chimeric antigen receptors.

4. 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 cells express the chimeric antigen receptor as described in claim 4 or 5.

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

8. The use of the single-domain antibody of claim 1, the chimeric antigen receptor of claim 4 or 5, or the chimeric antigen receptor cell of claim 6 in the preparation of a drug for killing nasopharyngeal carcinoma cells.

9. The application according to claim 8, characterized in that, The nasopharyngeal carcinoma cells are the nasopharyngeal carcinoma cell line HK1-EBV.

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

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