Human papillomavirus antigen peptide and its application

Abstract

The present invention relates to a human papillomavirus antigen peptide and its application, belonging to the technical field of immunomedicine. The antigen peptide comprises the amino acid sequence shown in SEQ ID NO.1, or a small peptide having more than 90% similarity to the amino acid sequence shown in SEQ ID NO.1. The above-mentioned human papillomavirus antigen peptide is a new target located in the E7 gene of the HPV16 genome. This target is not expressed in normal human tissues during viral genome expression and can be used as an antigen target for applications in reagents, drugs, or vaccines for the diagnosis, treatment, and prognosis determination of clinical HPV16 / 18 infections or HPV16 / 18-positive tumors such as cervical cancer, esophageal cancer, head and neck cancer, oropharyngeal cancer, anogenital cancer, etc. For example, through adoptive T cell immunotherapy, it may bring new treatment possibilities to patients with advanced recurrent cervical cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and particularly to a human papillomavirus antigen peptide and its application. Background Art

[0002] Human Papilloma Virus (HPV) is an epitheliotropic virus with high specificity. Currently, it is known that HPV infection is associated with various cancers, including cervical cancer, esophageal cancer, head and neck cancer, oropharyngeal cancer, anogenital cancer, etc., especially cervical cancer. Cervical cancer is the third most common malignancy among women globally. It has been clearly established that persistent infection with high-risk HPV is a necessary factor for the occurrence of cervical cancer and precancerous lesions. The detection of HPV in cervical tissue specimens shows that the detection rate of HPV16 / 18 exceeds 70%. The genome of HPV integrates into the human cell genome and continuously expresses the E6 and E7 genes, which play a key role in the development of cervical cancer. The E6 and E7 proteins are viral proteins expressed by the viral genome and are not expressed in normal human tissues. Therefore, they are very ideal therapeutic targets for cervical cancer.

[0003] In recent years, targeted and immunotherapy have become new models for treating tumors, especially for patients with poor surgical, radiotherapy, and chemotherapy effects, postoperative metastasis, and late recurrence. Among them, the TCR-T cell therapy can reconstruct the patient's immune function against this virus, inhibit virus proliferation, kill infected cells, and establish long-term immune memory in the patient's body by in vitro expanding TCR-T cells that specifically recognize the virus and then transfusing them into the patient's body.

[0004] However, the discovery of individualized TCRs is difficult and costly. For example, when sequencing human peripheral blood T cells according to conventional techniques, although the cost is relatively low, it is difficult to determine the correctly paired and functional α-β pairs after obtaining sequence information of hundreds of thousands or millions of α and β chains; while using more advanced single-cell sequencing technology, the correctly paired α-β pairs can be obtained, but the synthesis cost of α and β peptide chains is high, the screening quantity is large, and the time consumption is long, resulting in numerous challenges in developing immunotherapy for specific targets.

[0005] In virus-infected cells, short peptides of viral protein antigens bind to the major histocompatibility complex (MHC) to form p-MHC complexes. After being presented on the cell membrane surface by antigen-presenting cells (APCs), the p-MHC complexes are specifically recognized and bound by T cell receptors (TCRs), activating T cells to specifically kill the infected cells. Human MHC, namely human leukocyte antigen (HLA), has a high degree of diversity in the population. Currently, the only universal TCR for treating HPV infection on the market or in clinical trials is specific to type A*02:01, and patients expressing other HLA types cannot be covered. Summary of the Invention

[0006] Based on this, in view of the problems of few immune therapy targets and high development difficulty related to HPV16 / 18 and HPV16 / 18-positive tumors, it is necessary to provide a human papillomavirus antigen peptide. This antigen peptide is a short peptide derived from the E7 gene of the HPV16 genome and also exists in the HPV18 E7 gene. It can be used as a new target for targeting HPV16 / 18 and is applicable to the diagnosis, treatment, and prognosis determination of HPV16 / 18 infections or HPV16 / 18-positive tumors such as cervical cancer, esophageal cancer, head and neck cancer, oropharyngeal cancer, and ano-genital cancer, as well as the development of HPV-positive tumor vaccines.

[0007] A human papillomavirus antigen peptide, which comprises the amino acid sequence shown in SEQ ID NO.1 or a peptide having more than 90% similarity to the amino acid sequence shown in SEQ ID NO.1.

[0008] The above-mentioned human papillomavirus antigen peptide is a new target derived from the E7 gene of the HPV16 genome and also exists in the HPV18 E7 gene. This target is expressed by the viral genome and not expressed in normal human tissues. It can be used as an antigen target for the diagnosis, treatment, and prognosis determination of clinical HPV16 / 18 infections or HPV16 / 18-positive tumors such as cervical cancer, esophageal cancer, head and neck cancer, oropharyngeal cancer, and ano-genital cancer.

[0009] In one embodiment, the anchor points for the antigen peptide to bind to HLA are the second amino acid residue histidine and the last amino acid residue leucine in the antigen peptide sequence.

[0010] The present invention also discloses a p-MHC complex, which is characterized in that the p-MHC complex comprises the above-mentioned human papillomavirus antigen peptide and MHC.

[0011] In one embodiment, the MHC is selected from at least one of the following HLA-B subtypes: B*38:01, B*38:02, and B*38:15. The p-MHC complexes are peptide-B*38:01, peptide-B*38:02, and peptide-B*38:15.

[0012] The present invention also discloses a TCR that has antigen specificity for the above-mentioned human papillomavirus antigenic peptide.

[0013] It can be understood that TCRs function by recognizing specific types of p-HLA complexes. However, HLA exhibits high diversity in the population, resulting in low universality of conventional TCRs and difficulties and high costs in developing personalized immunotherapies for a specific target.

[0014] The above-mentioned TCR can cover multiple HLA subtypes such as B*38:01, B*38:02, and B*38:15, improving the applicability of the TCR.

[0015] In one embodiment, the TCR is selected from TCR-362 and / or TCR-2495.

[0016] The α and β chains of the TCR-362 contain the following CDR (complementary determining region):

[0017] CDR3α of TCR-362: AVRDRNPGGNKLV (SEQ ID NO.2);

[0018] CDR3β of TCR-362: ASSLDWGGWDYGYT (SEQ ID NO.3);

[0019] The α and β chains of the TCR-2495 contain the following CDR (complementary determining region):

[0020] CDR3α of TCR-2495: GGVSGAGSYQLT (SEQ ID NO.4);

[0021] CDR3β of TCR-2495: GGVSGAGSYQLT (SEQ ID NO.5).

[0022] In one embodiment, the α and β chains of the TCR-362 contain the following CDR (complementary determining region):

[0023] CDR1α of TCR-362: VSGNPY (SEQ ID NO.6)

[0024] CDR2α of TCR-362: YITGDNLV (SEQ ID NO.7)

[0025] CDR1β of TCR-362: SGHNT (SEQ ID NO.8)

[0026] CDR2β of TCR-362: YYREEE (SEQ ID NO.9)

[0027] The α-chain and β-chain of the said TCR-2495 comprise the following CDR (Complementary Determining Region):

[0028] CDR1α of TCR-2495: KTLYG (SEQ ID NO.10)

[0029] CDR2α of TCR-2495: LQKGGEE (SEQ ID NO.11)

[0030] CDR1β of TCR-2495: MDHEN (SEQ ID NO.12)

[0031] CDR2β of TCR-2495: SYDVKM (SEQ ID NO.13)

[0032] In one embodiment, the amino acid sequence of the said TCR-362 is as shown in SEQ ID NO.14, and the amino acid sequence of the said TCR-2495 is as shown in SEQ ID NO.15.

[0033] The present invention also discloses a nucleic acid molecule, comprising a nucleotide sequence encoding the above-mentioned human papillomavirus antigen peptide or the above-mentioned TCR.

[0034] The present invention also discloses a T cell, expressing the above-mentioned TCR.

[0035] The present invention also discloses the applications of the above-mentioned human papillomavirus antigen peptide, the above-mentioned p-MHC complex, and the above-mentioned TCR in the preparation of reagents, drugs or vaccines for the diagnosis, treatment and prognosis determination of anti-HPV infection or HPV-positive tumors.

[0036] It can be understood that the above-mentioned HPV-positive tumors refer to tumor types related to positive HPV infection, which can be obtained by those skilled in the art according to the correlation between the two.

[0037] In one embodiment, the said HPV is HPV16 or HPV18; the said HPV-positive tumors are selected from: cervical cancer, esophageal cancer, head and neck cancer, oropharyngeal cancer, ano-genital cancer.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] A human papillomavirus antigen peptide of the present invention is a new target derived from the E7 gene of the HPV16 genome and simultaneously present in the HPV18 E7 gene. This target is expressed by the viral genome and not expressed in normal human tissues, and can be used as an antigen target for the diagnosis, treatment, and prognosis determination of clinical anti-HPV16 / 18 infection or HPV16 / 18-positive tumors such as cervical cancer, esophageal cancer, head and neck cancer, oropharyngeal cancer, and anogenital cancer. For example, adoptive T cell immunotherapy can bring new treatment possibilities to patients with advanced recurrent tumors.

[0040] A TCR of the present invention has antigen specificity for the above-mentioned human papillomavirus antigen peptide, and this TCR can cover HLA typing B*38:01, B*38:02, and B*38:15, improving the applicability of the TCR and facilitating the development of individualized immunotherapy regimens. Brief Description of the Drawings

[0041] Figure 1 It is a graph of the CD137 signal of 362 TCR-T cells;

[0042] Figure 2 It is a graph of the CD137 signal of 2495 TCR-T cells;

[0043] Figure 3 It is a graph of the CD107a and IFNγ signals of 362 TCR-T cells;

[0044] Figure 4 It is a graph of the CD107a and IFNγ signals of 2495 TCR-T cells;

[0045] Figure 5 It is a graph of the flow cytometry detection signal results of experimental group 1 in the HLA typing identification experiment;

[0046] Figure 6 It is a graph of the flow cytometry detection signal results of experimental group 2 in the HLA typing identification experiment;

[0047] Figure 7 It is a graph of the flow cytometry detection signal results of experimental group 5 in the HLA typing identification experiment;

[0048] Figure 8 It is a graph of the flow cytometry detection signal results of experimental group 1 in the antigen target experiment;

[0049] Figure 9 It is a graph of the flow cytometry detection signal results of experimental group 2 in the antigen target experiment;

[0050] Figure 10 It is a graph of the flow cytometry detection signal results of experimental group 3 in the antigen target experiment;

[0051] Figure 11It is the flow cytometry detection signal result graph of experimental group 4 in the antigen target experiment;

[0052] Figure 12 It is the flow cytometry detection signal result graph of experimental group 5 in the antigen target experiment;

[0053] Figure 13 It is the flow cytometry detection signal result graph of experimental group 6 in the antigen target experiment;

[0054] Figure 14 It is the flow cytometry detection signal result graph of experimental group 7 in the antigen target experiment;

[0055] Figure 15 It is the flow cytometry detection signal result graph of experimental group 8 in the antigen target experiment;

[0056] Figure 16 It is the flow cytometry detection signal result graph of experimental group 9 in the antigen target experiment;

[0057] Figure 17 It is the flow cytometry detection signal result graph of experimental group 1 in the target sequence determination experiment;

[0058] Figure 18 It is the flow cytometry detection signal result graph of experimental group 2 in the target sequence determination experiment;

[0059] Figure 19 It is the flow cytometry detection signal result graph of experimental group 3 in the target sequence determination experiment;

[0060] Figure 20 It is the flow cytometry detection signal result graph of experimental group 4 in the target sequence determination experiment;

[0061] Figure 21 It is the flow cytometry detection signal result graph of experimental group 5 in the target sequence determination experiment;

[0062] Figure 22 It is the flow cytometry detection signal result graph of experimental group 6 in the target sequence determination experiment;

[0063] Figure 23 It is the flow cytometry detection signal result graph of experimental group 7 in the target sequence determination experiment;

[0064] Figure 24 It is the flow cytometry detection signal result graph of experimental group 8 in the target sequence determination experiment;

[0065] Figure 25 It is the flow cytometry detection signal result graph of experimental group 9 in the target sequence determination experiment;

[0066] Figure 26 It is the flow cytometry detection signal result graph of experimental group 10 in the anchor point experiment;

[0067] Figure 27 It is a flow cytometry detection signal result graph of experimental group 11 in the anchor point experiment.

[0068] Among them, Figure 5-27 in it, A is the CD137 signal result of 362 TCR-T cells, B is the CD107a + IFNγ signal result of 362 TCR-T cells, C is the CD137 signal result of 2495 TCR-T cells, and D is the CD107a + IFNγ signal result of 2495 TCR-T cells. Detailed implementation mode

[0069] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure content of the present invention more thorough and comprehensive.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0071] The reagents used in the following examples are commercially available unless otherwise specified; the methods used in the following examples are achievable by conventional methods unless otherwise specified.

[0072] Example 1

[0073] I. Screening TCR reactive to HPV16 E7

[0074] Obtain the sequence information of HPV16 E7 from NCBI, GeneID: 1489079. Referring to the methods disclosed in WO2020036875A1 and CN111875698B, two TCRs targeting HPV16 E7 are screened from the tumor tissues of cervical cancer patients, namely TCR-362 and TCR-2495. The amino acid sequences of TCR-362 and TCR-2495 are shown in SEQ ID NO.14 and SEQ ID NO.15 respectively, and the nucleotide sequences of TCR-362 and TCR-2495 are shown in SEQ ID NO.16 and SEQ ID NO.17 respectively.

[0075] The key variable regions in the α-chain and β-chain of the above TCR and their CDR (complementarity-determining regions) structures are shown in the following table.

[0076] Table 1. TCR targeting HPV16 E7

[0077]

[0078] II. TCR function verification at the cellular level

[0079] 1. Method

[0080] 1.1 Preparation of CD8+ TCR-T cells expressing TCR-362 and TCR-2495

[0081] CD8+ T cells were sorted from commercial PBMC (Miaotong (Shanghai) Biotechnology Co., Ltd.), frozen after stimulation and amplification, and after 48 h of resuscitation, they were resuspended in Buffer R supplied with the Neon cell electroporator (Thermo Fisher, MPK5000) to a density of 2×10 7 cells / ml. 300 μl of cells were taken, 60 μg of mRNA of TCR-362 / TCR-2495 (i.e., the mRNA sequences corresponding to SEQ ID NO.16 and SEQ ID NO.17 above) was added, and after mixing, it was transferred to an electroporation cuvette for electroporation.

[0082] After the electroporation, it was left standing for 30 - 60 s, and the mixture was added to a 12-well plate containing 2.5 ml / well of pre-warmed ICXF medium (Stemcell, catalog number 10981), and 30 IU / ml of IL-2 was added to each well. Two wells were cultured in parallel for the detection of T cell activation markers CD137 and CD107a+IFN-γ. Then it was cultured in a 37 °C CO2 incubator for 4 h.

[0083] 1.2 Preparation of K562 cells expressing HLA and HPV16 E7

[0084] K562 cells (from ATCC) were resuspended in Buffer R supplied with the Neon cell electroporator to a density of 1×10 6cells / ml, take 400 μl of cells, add 40 μg of mRNA of HLA-I6mix (6.7 μg each of HLA-A*24:02, HLA-A*33:03, HLA-B*38:15, HLA-B*55:02, HLA-C*03:02, HLA-C*12:03) and 40 μg of mRNA of E7 protein (HPV16 E7 sequence information obtained from NCBI, GeneID: 1489079). After mixing, transfer to an electroporation cuvette for electroporation. The DNA sequences of the above HLA are referred to the public information at https: / / www.ebi.ac.uk / ipd / imgt / hla.

[0085] After the electroporation, let it stand for 30 - 60 s, and add the mixture to 1 ml of pre-warmed DMEM medium containing 10% FBS (Gibco, catalog number 11995065). At 0.1×10 6 cells / well, seed in a 96-well plate and culture in a 37 °C CO2 incubator for 4 h. Centrifuge at 250×g for 4 min to discard the supernatant. Add 200 μl of 1×PBS to each well and wash twice, then add 100 μl of pre-warmed DMEM medium containing 10% FBS to resuspend each well.

[0086] 1.3 Co-culture

[0087] (1) Co-culture the prepared CD8+ TCR-T cells expressing TCR-362 and TCR-2495 with K562 cells. The specific method is as follows:

[0088] Take the prepared K562 cells expressing HLA and HPV16 E7, add 100 μl of the above-prepared TCR-T culture (about 0.2×10 6 cells / well) to each well, and set up four groups of experiments:

[0089] Experimental group 1: TCR-T cells only;

[0090] Experimental group 2: TCR-T cells + 1 wt‰ PMA (phorbol ester, PKC activator) + 1 wt‰ Ionomycin (calcium ionophore);

[0091] Experimental group 3: TCR-T cells + wild-type K562 cells;

[0092] Experimental group 4: TCR-T cells + K562 cells (HLAI6mix + HPV16 E7).

[0093] The above cells were cultured overnight at 37°C and stained with DAPI, CD3-FITC, mTCRβ-PE, and CD137-PECy5 antibodies, and the signal of CD137 was detected by flow cytometry.

[0094] Results Figure 1-2 As shown, among them, Figure 1 are the CD137 signal results of 362TCR-T (T cells expressing TCR-362) cells, where Figures A-D are the results of experimental groups 1-4 respectively. Figure 2 are the CD137 signal results of 2495TCR-T (T cells expressing TCR-2495) cells, where Figures A-D are the results of experimental groups 1-4 respectively.

[0095] The results showed that compared with experimental group 1 (blank control group) and experimental group 3 (negative control group), both experimental group 2 (positive control group) and experimental group 4 had obvious CD137 response signals.

[0096] (2) The above-prepared CD8+ TCR-T cells expressing TCR-362 and TCR-2495 were co-cultured with K562 cells. The specific method was as follows:

[0097] Collect the above-prepared TCR-T cells, centrifuge at 300×g for 5 min to discard the supernatant, resuspend with 500 μl of pre-warmed ICXF medium containing 60 U / mL IL-2, and add it to a 96-well plate with the above-resuspended K562 cells expressing HLA and HPV16E7 at 50 μl / well (about 0.2×10 6 cells / well), and set up four groups of experiments:

[0098] Experimental group 1: TCR-T cells only;

[0099] Experimental group 2: TCR-T cells + 1 wt‰ PMA (phorbol ester, PKC activator) + 1 wt‰ Ionomycin (calcium ionophore);

[0100] Experimental group 3: TCR-T cells + wild-type K562 cells;

[0101] Experimental group 4: TCR-T cells + K562 cells (HLA I6mix + HPV16 E7).

[0102] 5 μl of CD107a-PE was added to each co-culture well. For culture wells with a volume less than 200 μl, the volume was made up to 200 μl / well with ICXF medium, and cultured at 37°C for 4 h.

[0103] Stain with Zombie NIR, CD3-FITC, mTCRβ-APC, CD107a-PE, and IFNγ-BV421 antibodies, and detect the signals of CD107a and IFNγ by flow cytometry.

[0104] Results Figure 3-4 As shown, among them, Figure 3 are the results of CD107a and IFNγ signals of 362TCR-T (T cells expressing TCR-362) cells, where Figures A-D are the results of experimental groups 1-4 respectively. Figure 4 are the results of CD107a and IFNγ signals of 2495TCR-T (T cells expressing TCR-2495) cells, where Figures A-D are the results of experimental groups 1-4 respectively. The results show that compared with experimental group 1 (blank control group) and experimental group 3 (negative control group), both experimental group 2 (positive control group) and experimental group 4 have obvious CD107a+IFNγ response signals.

[0105] It can be seen from the above results that TCR-362 and TCR-2495 are HPV16 E7-reactive TCRs.

[0106] III. HLA Typing Determination

[0107] Refer to the above TCR function verification experimental method to verify the HLA typing recognized by TCR-362 and TCR-2495. The difference is that when preparing K562 cells expressing HLA and HPV16 E7, in addition to HLA-I6mix, K562 cells expressing one of the following subtypes of HLA and HPV16 E7 are also prepared: HLA-A*24:02, HLA-A*33:03, HLA-B*38:15, HLA-B*55:02, HLA-C*03:02, HLA-C*12:03 (the expression method refers to item 1.2, only need to replace HLA-I6mix with the above HLAs).

[0108] The experimental groups are:

[0109] Experimental group 1: Only K562 cells;

[0110] Experimental group 2: TCR-T cells + K562 cells (HLA-I6mix + HPV16 E7);

[0111] Experimental group 3: TCR-T cells + K562 cells (HLA-A*24:02 + HPV16 E7);

[0112] Experimental group 4: TCR-T cells + K562 cells (HLA-A*33:03 + HPV16 E7);

[0113] Experimental group 5: TCR-T cells + K562 cells (HLA-B*38:15 + HPV16 E7);

[0114] Experimental group 6: TCR-T cells + K562 cells (HLA-B*55:02 + HPV16 E7);

[0115] Experimental group 7: TCR-T cells + K562 cells (HLA-C*03:02 + HPV16 E7);

[0116] Experimental group 8: TCR-T cells + K562 cells (HLA-C*12:03 + HPV16 E7).

[0117] Stain with DAPI, CD3-FITC, mTCRβ-APC, CD137-PECy5 antibodies, and detect the signal of CD137 by flow cytometry. Stain with Zombie NIR, CD3-FITC, mTCRβ-APC, CD107a-PEcy7, IFNγ-PE antibodies, and detect the signals of CD107a and IFNγ by flow cytometry.

[0118] Results Figure 5-7 As shown Figure 5-7 The signal results of experimental group 1 (negative control group), experimental group 2 (positive control group), and experimental group 5 are shown respectively. In each figure, A and B are the CD137 and CD107a+IFNγ signal results of 362 TCR-T cells, and C and D are the CD137 and CD107a+IFNγ signal results of 2495 TCR-T cells.

[0119] The results showed that no signals of CD137 and CD107a+IFNγ were detected in experimental group 1 (negative control group), while signals of CD137 and CD107a+IFNγ were detected in both experimental group 2 (positive control group) and experimental group 5.

[0120] The results of experimental groups 3, 4, 6, 7, and 8 were similar to those of experimental group 1 (negative control group), and no signals of CD137 and CD107a+IFNγ were detected.

[0121] From the above results, it can be determined that the HLA genotypes recognized by TCR-362 and TCR-2495 are both HLA-B*38:15.

[0122] IV. Determination of antigen targets

[0123] 1. Preliminary screening of antigen targets

[0124] Referring to the above TCR function verification experimental method, verify the antigen targets recognized by TCR-362 and TCR-2495. The difference is that when preparing K562 cells expressing HLA and HPV16 E7, the HLA typing is B*38:15, and HPV16 E7 is decomposed into 7 overlapping 39bp fragments with a length of 75bp each, covering the entire gene length.

[0125] Set up the experimental groups as follows:

[0126] Experimental group 1: TCR-T cells + K562 cells (HLA-B*38:15);

[0127] Experimental group 2: TCR-T cells + K562 cells (HLA-B*38:15 + E7);

[0128] Experimental group 3: TCR-T cells + K562 cells (HLA-B*38:15 + E7(1-75));

[0129] Experimental group 4: TCR-T cells + K562 cells (HLA-B*38:15 + E7(37-111));

[0130] Experimental group 5: TCR-T cells + K562 cells (HLA-B*38:15 + E7(73-147));

[0131] Experimental group 6: TCR-T cells + K562 cells (HLA-B*38:15 + E7(109-183));

[0132] Experimental group 7: TCR-T cells + K562 cells (HLA-B*38:15 + E7(145-249));

[0133] Experimental group 8: TCR-T cells + K562 cells (HLA-B*38:15 + E7(181-255));

[0134] Experimental group 9: TCR-T cells + K562 cells (HLA-B*38:15 + E7(217-294)).

[0135] Use a flow cytometer to detect the signals of CD137 and CD107a+IFNγ.

[0136] Results Figure 8-16 as shown Figure 8-16Signal results of experimental group 1 (negative control), experimental group 2 (positive control), and experimental groups 3 - 9 respectively. In each figure, A and B are the CD137 and CD107a + IFNγ signal results of 362 TCR-T cells, and C and D are the CD137 and CD107a + IFNγ signal results of 2495 TCR-T cells.

[0137] The results showed that no obvious CD137 and CD107a + IFNγ signals were detected in experimental group 1 (negative control group) and experimental groups 3 - 7, while obvious CD137 and CD107a + IFNγ signals were detected in experimental group 2 (positive control group), experimental group 8, and experimental group 9.

[0138] It can be seen from the above results that both the E7(181 - 255) and E7(217 - 294) fragments contain antigen targets. Therefore, it can be further inferred that the CDS 217 - 255 of the HPV16 E7 gene (peptide 72 - 84, that is, a short peptide containing amino acids 72 - 84 of the E7 protein amino acid sequence, with the sequence THVDIRTLEDLLM (SEQ ID NO.18), hereinafter referred to as E7(p72 - 84)) contains antigen targets.

[0139] 2. Determination of the target sequence (the peptide sequence with the highest affinity)

[0140] The above screening results were used to predict and verify the interaction between HLA-B*38:15 and antigen peptides using a website tool (https: / / openvax.github.io / mhcflurry-motifs / HLA-B-38-15.html). It was speculated that the length of the corresponding antigen peptides was mainly 8 - 12 amino acid residues, and the anchor points were very likely the second amino acid residue (H) and the last amino acid residue (L). This prediction result was consistent with the previous experimental results, and the inventor further designed experiments for testing and verification.

[0141] The experiment was conducted with reference to the above TCR function verification experimental method, with the difference that when preparing K562 cells expressing HLA and viral antigens, the HLA typing was B*38:15, and the antigen was sequentially reduced by 1 amino acid from the right and left sides of E7(p72 - 84), with an addition amount of 0.01 μg / ml.

[0142] The following experimental groups were set up:

[0143] Experimental group 1: TCR-T cells + K562 cells (HLA-B*38:15);

[0144] Experimental group 2: TCR-T cells + K562 cells (HLA-B*38:15 + E7);

[0145] Experimental group 3: TCR-T cells + K562 cells (HLA-B*38:15 + E7 (p72-84));

[0146] Experimental group 4: TCR-T cells + K562 cells (HLA-B*38:15 + E7 (p72-83));

[0147] Experimental group 5: TCR-T cells + K562 cells (HLA-B*38:15 + E7 (p72-82));

[0148] Experimental group 6: TCR-T cells + K562 cells (HLA-B*38:15 + E7 (p72-81));

[0149] Experimental group 7: TCR-T cells + K562 cells (HLA-B*38:15 + E7 (p72-80));

[0150] Experimental group 8: TCR-T cells + K562 cells (HLA-B*38:15 + E7 (p73-83));

[0151] Experimental group 9: TCR-T cells + K562 cells (HLA-B*38:15 + E7 (p74-83)).

[0152] Detect the signals of CD137 and CD107a+IFNγ by flow cytometry.

[0153] Results Figure 17-25 As shown Figure 17-25 They are the signal results corresponding to experimental group 1 (negative control), experimental group 2 (positive control), and experimental groups 3-9 respectively. In each figure, A and B are the CD137 and CD107a+IFNγ signal results of 362 TCR-T cells, and C and D are the CD137 and CD107a+IFNγ signal results of 2495 TCR-T cells.

[0154] The results showed that no obvious CD137 and CD107a+IFNγ signals were detected in experimental group 1 (negative control group), experimental groups 5-7, and experimental group 9, while obvious CD137 and CD107a+IFNγ signals were detected in experimental group 2 (positive control group), experimental groups 3-4, and experimental group 8, and the signal of experimental group 4 was stronger than that of experimental group 8.

[0155] It can be seen from the above results that the antigen target is located at p72-83, and the sequence is: THVDIRTLEDLL (SEQ ID NO.1).

[0156] 3. Determine the anchor points of the antigenic peptide E7 (p72-83)

[0157] In the above experiment, two groups of TCR-T cell + K562 cell (HLA-B*38:15 + E7(p72-83)) experiments were conducted simultaneously. In the E7(p72-83), parallel experiments with H73A substitution (Experimental Group 10: TCR-T cell + HLA-B*38:15 + E7(p72-83, H73A substitution)) and L83A substitution (Experimental Group 11: TCR-T cell + HLA-B*38:15 + E7(p72-83, L83A substitution)) were carried out to verify its specificity in binding to TCR.

[0158] Flow cytometry was used to detect the signals of CD137 and CD107a + IFNγ.

[0159] The results are as Figure 26-27 shown, Figure 26-27 which are the signal results of Experimental Groups 10 - 11 respectively. Among them, A and B are the CD137 and CD107a + IFNγ signal results of 362 TCR-T cells, and C and D are the CD137 and CD107a + IFNγ signal results of 2495 TCR-T cells.

[0160] The results showed that compared with Experimental Group 4, the CD137 and CD107a + IFNγ signals in Experimental Groups 10 and 11 were significantly decreased, approaching the results of Experimental Group 1 (negative control group).

[0161] From the above results, it can be seen that the anchor points of the antigenic peptide E7(p72-83) are H73 and L83.

[0162] Example 2

[0163] Verification experiment on the applicability of TCR to HLA-B*38:01 and HLA-B*38:02.

[0164] According to the Chinese common and confirmed HLA allele table (CWD table) version 2.2 released by the China Marrow Donor Program, the population coverage rate of B*38:15 is relatively low, with a frequency of 0.00161%. The inventor's research found that the sequence of B*38:15 only differs from the sequence of B*38:02:01 by one amino acid. Moreover, B*38:01 is more common in East Asia and Southeast Asian countries such as China and Japan. If the two TCRs of the present invention can recognize B*38:01 and B*38:02, its applicability can be improved.

[0165] According to the method for TCR function verification in Example 1, prepare 362TCR-T cells, 2495TCR-T cells, and K562 cells (HLA-B*38:01+HPV16 E7(p72-83)), K562 cells (HLA-B*38:02+HPV16 E7(p72-83)). The addition amount of the antigen short peptide is 0.01 μg / ml, and the experimental groups are set as follows:

[0166] Experimental group 1: TCR-T cells + K562 cells (HLA-B*38:01);

[0167] Experimental group 2: TCR-T cells + K562 cells (HLA-B*38:01+E7(p72-83));

[0168] Experimental group 3: TCR-T cells + K562 cells (HLA-B*38:02);

[0169] Experimental group 4: TCR-T cells + K562 cells (HLA-B*38:02+E7(p72-83)).

[0170] Detect the signals of CD137 and CD107a+IFNγ by flow cytometry.

[0171] Results Figure 21-24 As shown Figure 21-24 are the signal results corresponding to Experimental groups 1, 2, 3, and 4 respectively, where A and B are the CD137 and CD107a+IFNγ signal results of 362TCR-T cells, and C and D are the CD137 and CD107a+IFNγ signal results of 2495TCR-T cells.

[0172] It can be seen from the above results that TCR-362 and TCR-2495 of the present invention can recognize HLA-B*38:01 and HLA-B*38:02, and this TCR expands the applicable range of conventional TCRs.

[0173] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0174] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A human papillomavirus antigenic peptide, characterized in that, The amino acid sequence of the antigenic peptide is shown in SEQ ID NO.

1.

2. A p-MHC complex, characterized in that, The p-MHC complex comprises the human papillomavirus antigenic peptide as claimed in claim 1 and MHC; the MHC is selected from at least one of the B*38:01, B*38:02 and B*38:15 subtypes.

3. A TCR, characterized in that, The TCR has antigenic specificity for the human papillomavirus antigenic peptide as claimed in claim 1; the TCR is selected from TCR-362 and / or TCR-2495. The α-chain and β-chain of the TCR-362 comprise the following CDR complementary determining regions: CDR1α of TCR-362: VSGNPY (SEQ ID NO.6); CDR2α of TCR-362: YITGDNLV (SEQ ID NO.7); CDR3α of TCR-362: AVRDRNPGGNKLV (SEQ ID NO.2); CDR1β of TCR-362: SGHNT (SEQ ID NO.8); CDR2β of TCR-362: YYREEE (SEQ ID NO.9); CDR3β of TCR-362: ASSLDWGGWDYGYT (SEQ ID NO.3); The α-chain and β-chain of the TCR-2495 comprise the following CDR complementary determining regions: CDR1α of TCR-2495: KTLYG (SEQ ID NO.10); CDR2α of TCR-2495: LQKGGEE (SEQ ID NO.11); CDR3α of TCR-2495: GGVSGAGSYQLT (SEQ ID NO.4); CDR1β of TCR-2495: MDHEN (SEQ ID NO.12); CDR2β of TCR-2495: SYDVKM (SEQ ID NO.13); CDR3β of TCR-2495: GGVSGAGSYQLT (SEQ ID NO.5).

4. The TCR according to claim 3, wherein The amino acid sequence of the TCR-362 is shown in SEQ ID NO.14, and the amino acid sequence of the TCR-2495 is shown in SEQ ID NO.

15.

5. A nucleic acid molecule, characterized in that, Comprising a nucleotide sequence encoding the human papillomavirus antigenic peptide as claimed in claim 1 or the nucleotide sequence of the TCR as claimed in claim 3.

6. A T cell, characterized in that: Expressing the TCR as claimed in any one of claims 3-4.

7. Use of the TCR as claimed in any one of claims 3-4 in the preparation of a reagent for the diagnosis, treatment and prognosis determination of anti-HPV infection or HPV-positive tumors, wherein the HPV is HPV16 or HPV18; the HPV-positive tumors are selected from: cervical cancer.

8. Use of the TCR as claimed in any one of claims 3-4 in the preparation of a drug for the diagnosis, treatment and prognosis determination of anti-HPV infection or HPV-positive tumors, wherein the HPV is HPV16 or HPV18; the HPV-positive tumors are selected from: cervical cancer.

9. Use of the TCR according to any one of claims 3-4 in the preparation of a vaccine for the diagnosis, treatment and prognosis determination of anti-HPV infection or HPV-positive tumors, wherein the HPV is HPV16 or HPV18; the HPV-positive tumors are selected from: cervical cancer.

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