CN113754756B - A TCR Recognizing HLA-A*02:01/E629-38 and Its Application - Google Patents

Abstract

The invention disclosesbase:Sub>A method for identifying HLA-A02_29‑38And uses thereof. CDR1, CDR2 and CDR3 of the alpha chain variable region of the TCR comprise amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 respectively, or comprise amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 respectively, or comprise amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 15 respectively. The TCRs of the invention have high affinity for pMHC (HLA-base:Sub>A × 02_DThe value can reach 4.2E-06M.

Description

A TCR for recognizing HLA-A*02:01/E629-38 and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a TCR for recognizing HLA-A*02:01/E6 _29-38 and its application.

Background technique

Human papillomavirus (HPV) is an epitheliophilic virus that easily infects human epidermis and mucosal squamous epithelium, and is associated with a variety of cancers (cervical, anal, penile, vaginal, vulvar, and head and neck cancer, etc.) (Lancet Oncol, 2010, 11, 781; Clin Cancer Res, 2019, 25, 1486), in the past ten years, the mortality rate of head and neck cancer related to HPV infection has continued to rise (CA Cancer J Clin, 2021, 71, 7). There are dozens of subtypes of HPV virus (Virology, 2015, 476, 341), and the above-mentioned tumors are mainly related to HPV16 infection (JNatl Cancer Inst, 2015, 107, djv086). E6 protein encoded by HPV16, as one of the main proteins in the virus life cycle, can induce tumorigenesis and development through various mechanisms, such as inhibiting tumor suppressor proteins p53 and pRb, and inhibiting apoptosis (Cancer Sci, 2007, 98, 1505) ; Enhance telomerase activity to make host cells immortal (Virus Res, 2017, 231, 50); Induce the loss of human histocompatibility antigen (HLA) expression (Clin Immunol, 2005, 115, 295), which is beneficial for tumor cells to escape from the host's inherent The immune response leads to the occurrence, development, invasion and metastasis of tumor cells. HPV16-E6 is a tumor-specific antigen, which is only specifically expressed in relevant tumor tissues and not in normal tissues (Nat RevCancer, 2002, 2, 342; Clin Immunol, 2005, 115, 295), so HPV16-E6-targeted Immunotherapy can reduce its off-target rate and improve the efficacy and safety of treatment (Papillomavirus Res, 2018, 5, 46), which is an ideal target for HPV-related cancer treatment.

T cell receptor gene engineered T cells (TCR-T) therapy is to transduce tumor antigen-specific TCR genes into normal T cells, which can enhance or re-endow the T cells to recognize The ability of tumor antigens to specifically target and kill tumor cells (Science, 2016, 352, 1337; Cells, 2020, 9), is an important treatment method in current adoptive cell therapy (Immunol Rev, 2014, 257, 56) . The current relevant clinical trials have proved that TCR-T cell therapy has a significant effect in the treatment of malignant tumors such as melanoma, synovial cell sarcoma, and myeloma (Science, 2006, 314, 126; J Clin Oncol, 2011, 29, 917; Nat Med, 2015, 21, 914; Blood, 2017, 130, 1985; Cancer Discov, 2018, 8, 944). As of August 2021, there are 461 TCR-T-related clinical registration records on the clinicaltrials.gov website, indicating that TCR-T has great potential and development value in tumor immunotherapy. At present, clinical trials (NCT02280811, NCT03197025, NCT03578406) have carried out TCR-T cell therapy targeting HPV16-E6, and its effectiveness and safety have been preliminarily verified (Clin Cancer Res, 2015, 21, 4431; J Clin Oncol, 2017 , 35, 3009; J Clin Oncol, 2019, 37, 2759), providing a new way for the treatment of HPV-related cancers. With the development of related technologies and the continuous deepening of research, TCR-T therapy will gradually develop in the direction of high efficiency, low toxicity and controllability. More cancer patients bring hope of cure.

Contents of the invention

The present invention provides a TCR that effectively recognizes the HLA-A*02:01/E6 _29-38 target, and the T cells transformed by using the TCR can effectively recognize the HLA-A*02:01/E6 _29-38 target against tumors. Cell specific killing.

One of the technical solutions of the present invention is: a TCR, the CDR1, CDR2 and CDR3 of the α-chain variable region of the TCR respectively comprise the following sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 The amino acid sequence shown;

Or, respectively comprising the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9;

Alternatively, it comprises the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:15, respectively.

Preferably, CDR1, CDR2 and CDR3 of the β-chain variable region of the TCR comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;

Or, respectively comprising the amino acid sequences shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12;

Alternatively, it comprises the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

In a specific embodiment of the present invention, the CDR1, CDR2 and CDR3 of the α chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the CDR1, CDR2 and CDR3 of the β-chain variable region comprise the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

In another specific embodiment of the present invention, CDR1, CDR2 and CDR3 of the α-chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, and The CDR1, CDR2 and CDR3 of the β chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

In another specific embodiment of the present invention, CDR1, CDR2 and CDR3 of the α-chain variable region comprise the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 15, respectively, and The CDR1, CDR2 and CDR3 of the β-chain variable region respectively comprise the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

The α chain and/or β chain of the TCR described in the present invention preferably further comprise a framework region; wherein:

The framework region of the α chain is derived from germline TRAV, TRAJ and TRAC, wherein said TRAV is preferably TRAV14 or TRAV17, and said TRAJ is preferably TRAJ44 or TRAJ49;

The framework region of the β chain is derived from germline TRBV, TRBD, TRBJ and TRBC, the TRBV is preferably TRBV14, TRBV28 or TRBV20, the TRBD is preferably TRBD1, and the TRBJ is preferably TRBJ1-6, TRBJ1-4 or TRBJ2-7 , the TRBC is preferably TRBC1 or TRBC2.

In a preferred embodiment of the present invention, the α-chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23.

In another preferred embodiment of the present invention, the β-chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.

In a specific embodiment of the present invention, the α-chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 19, and the β-chain variable region of the TCR contains the amino acid sequence shown in SEQ ID NO: 20 amino acid sequence.

In another specific embodiment of the present invention, the α chain variable region contains the amino acid sequence shown in SEQ ID NO: 21, and the β chain variable region contains the amino acid sequence shown in SEQ ID NO: 22 .

In another specific embodiment of the present invention, the α-chain variable region contains the amino acid sequence shown in SEQ ID NO:23, and the β-chain variable region contains the amino acid sequence shown in SEQ ID NO:24 .

The TCRα chain of the TCR in the present invention preferably further comprises a constant region, and the constant region of the TCRα chain is preferably derived from human or mouse germline.

The TCRβ chain of the TCR in the present invention preferably further comprises a constant region, and the constant region of the TCRβ chain is preferably derived from human or mouse germline.

The constant region of TCRα chain derived from human germline preferably contains the sequence shown in SEQ ID NO:13.

The constant region of the TCRα chain derived from the murine germline preferably contains the amino acid sequence shown in SEQ ID NO:27.

The constant region of TCRβ chain derived from human germline preferably contains the sequence shown in SEQ ID NO:14 or SEQ ID NO:25.

The constant region of the TCR beta chain derived from the murine germline preferably contains the amino acid sequence shown in SEQ ID NO:28.

In addition, the TCRα chain in the present invention may also include an extramembrane region and a transmembrane region; preferably, the TCRα chain may also include an intracellular sequence.

The TCRβ chain can also include an extramembrane region and a transmembrane region; preferably, the TCRβ chain also includes an intracellular sequence.

The second technical solution of the present invention is: an isolated nucleic acid encoding the TCR described in the first technical solution of the present invention.

The third technical solution of the present invention is: a vector comprising the nucleic acid described in the second technical solution, the vector is preferably a lentiviral vector; the nucleic acid is in a single open reading frame, or in two different open reading frames The TCRα chain and the TCRβ chain are encoded respectively in the reading frame.

The fourth technical solution of the present invention is: a cell containing the nucleic acid as described in the second technical solution or the vector as described in the third technical solution; preferably, the cells are T cells or stem cells, and the The T cells are preferably CD8 ⁺ T cells.

The fifth technical solution of the present invention is: an isolated or non-naturally occurring cell presenting the TCR as described in one of the technical solutions, and the said cell is preferably a T cell.

The sixth technical solution of the present invention is: a pharmaceutical composition, which contains the TCR as described in the first technical solution or the cell described in the fourth technical solution; acceptable carrier.

The seventh technical solution of the present invention is: a TCR as described in one of the technical solutions, a cell as described in the fourth technical solution, or a pharmaceutical composition described in the sixth technical solution in the preparation of a drug for preventing and treating tumors related to HPV16 expression Preferably, the tumors include cervical cancer, oropharyngeal cancer, vaginal cancer, anal cancer, and penile cancer.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is:

The TCR of the present invention has high affinity with pMHC (HLA-A*02:01/TIHDIILECV), and the K _D value can reach 4.2E-06M. It has a specific killing effect on HLA-A*02:01 ⁺ /HPV16-E6 ⁺ target cells (A375+E6-1B3), and the killing effect is enhanced with the increase of the effect-to-target ratio; while the other two non-double positive No obvious killing effect on target cells. In addition, GFP-transduced T cells had no obvious killing effect on A375+E6-1B3.

Description of drawings

Figure 1 is the sorting process of HPV16-E6 _29-38 antigen-specific double positive monoclonal CD8 ⁺ T cells.

Figure 2A and Figure 2B are the anion exchange chromatography and SDS-PAGE electrophoresis diagrams of E63 TCR after renaturation.

Fig. 3A and Fig. 3B are gel filtration chromatography and SDS-PAGE electrophoresis diagrams after renaturation of E63 TCR.

Figure 4A and Figure 4B are the anion exchange chromatography and SDS-PAGE electrophoresis images of HLA-A*02:01/β2M/TIHDIILECV refolding; among them, the band with large molecular weight is HLA-A*02:01, and the band with small molecular weight The band is β2M, and the molecular weight of the TIHDIILECV polypeptide is too small to see the band on SDS-PAGE.

Figure 5A and Figure 5B are gel filtration chromatography and SDS-PAGE electrophoresis images of HLA-A*02:01/β2M/TIHDIILECV refolded.

Figure 6 is a Gel Shift map of E6-pMHC after biotinylation.

Figure 7 is a graph showing the results of the E63 TCR affinity test.

Figure 8 is a graph showing the positive rate of CD8 ⁺ T cells infected by E63 TCR lentivirus; wherein, E6con is the positive TCR control group, and GFP is the negative control group.

Figure 9A and Figure 9B are graphs of INF-γ release by E63 TCR on T2 cells loaded with E6 _29-38 or NY-ESO- _{1 157-165} polypeptide.

Figure 10E63 TCR release profile of INF-γ on tumor cell lines.

Fig. 11 is a tumor cell line LDH-specific killing experiment.

Fig. 12 is a graph showing the effect of C regions from different sources on the pairing rate of E63 TCR.

Figure 13A and Figure 13B are the INF-γ release graphs of E63 TCR on PBMC of 22 healthy people with different HLA-A types.

Figure 14 is the tumor growth curve of E63 TCR-T animal experiments.

Fig. 15 is the tumor body situation of mice after 26 days of administration of E63 TCR-T cells.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

Note: In the following examples, unless otherwise specified, all cell lines used were purchased from ATCC.

Example 1 Antigen-specific αβ-TCR cloning and gene sequence identification

The methods, reagents and consumables used in HPV16-E6 _29-38 (TIHDIILECV) antigen-specific CD8 ⁺ T cell TCR gene cloning mainly refer to Curr.Protoc.Immunol.2002,7,1; PLoS One.2011,6,e27930; OncoImmunology.2016, 5, e1175795; J Vis Exp.2011, 8, 3321; J Immunol Methods.2006, 310, 40; PLoS One.2014, 9, e110741 and its references. After CD8 + T cells were isolated from PBMC of HLA-A*02:01 genotype healthy volunteers by immunomagnetic bead negative selection method, ^{CD8 +} T cells were stimulated with EBV-B cells loaded with HPV16-E6 _29-38 peptide Cells, and then use PE-labeled HLA-A*02:01/HPV16-E6 _29-38 tetramer and APC-labeled anti-CD8 antibody to double-stain T cells, flow cytometry to obtain double-positive T cells, and The T cells were expanded and cultured to a certain number and then sorted again ( FIG. 1 ). After two rounds of stimulation culture and sorting, the double-positive T cells were monoclonal cultured by the limiting dilution method. The proliferated monoclonal T cells were detected by flow cytometry by HLA-A*02:01/HPV16-E6 _29-38 tetramer and anti-CD8 antibody double staining and sorted to obtain HPV16-E6 _29-38 antigen-specific monoclonal T cells.

The total RNA of the obtained monoclonal T cells was extracted using the Quick-RNA ^TM MiniPrep kit (ZYMO research, product number R1050), and cDNA was obtained by reverse transcription of the RNA through the SMARTer RACE cDNA Amplification Kit (Clontech, product number 634923). Then, using cDNA as a template, the target gene was amplified by PCR and connected to the pUC19 vector, and transformed into E.coli-DH5α by heat shock method. After plating and culturing overnight, single clone colonies were picked for identification and sequencing. The gene sequence obtained by sequencing was in IMGT database for comparative analysis. A total of three HLA-A*02:01/HPV16-E6 _29-38 antigen-specific TCRs were obtained, named E63, E65, and E67 TCRs, respectively, and their TCR α and β chain genotypes are shown in Table 1.

Table 1. Genotypes of TCR α and β chains

The full-length sequence of the alpha chain of E63 TCR:

The full-length sequence of the β chain of E63 TCR:

Alpha chain of E65:

Beta chain of E65:

Alpha chain of E67:

Beta chain of E67:

Among the above sequences: the underlined sequence is the signal peptide region, the black bold sequence is Vα (variable region of α chain) or Vβ (variable region of β chain), and the sequence marked in gray is Cα (constant region of α chain) Or Cβ (the constant region of the β chain), the underlined sequence in italics is the transmembrane intracellular region, and the sequence marked in bold and underlined is the CDR sequence.

According to the rules of the IMGT database, the amino acid sequence of the CDR region, the unique key sequence of the E6 TCR V region, is shown in Table 2:

Table 2. Amino acid sequences of α and β chain CDR regions of E6 TCR V region

Example 2 Expression and purification of E63 TCR gene

The α-chain and β-chain genes of E63 TCR were respectively connected to the pET28a vector using Nco I/Not I restriction sites, and transformed into E.coli-BL21(DE3) by heat shock method. In LB medium, shake culture at 37°C until OD ₆₀₀ =0.6-0.8, add IPTG at a final concentration of 1mM to induce the expression of the target protein, continue to culture at 37°C for 3h, and then centrifuge at 6000rpm for 10min to collect the bacteria.

The cells were resuspended in lysate (1×PBS containing 0.5% Triton X-100), ultrasonically disrupted, and then centrifuged at 12000 rpm for 20 min. Discard the supernatant, resuspend the precipitate with the lysate until there are no visible particles, centrifuge at 12000rpm for 10min, repeat the above operation 2-3 times, dissolve the precipitate with 6M guanidine hydrochloride solution, collect the supernatant after centrifuging at 12000rpm for 10min, the supernatant is Purified inclusion bodies. Inclusion bodies were quantified by BCA method.

Dilute 20mg of E63 TCRα chain inclusion body and 15mg of β chain inclusion body in 5mL of 6M guanidine hydrochloride solution, then slowly add TCRα chain and TCRβ chain into the pre-cooled refolding buffer (Science 1996, 274, 209; J. Mol. Biol. 1999, 285, 1831; Protein Eng. 2003, 16, 707), stirring continuously at 4°C for 30 min. Put the solution into a dialysis bag, place in 10 times the volume of pre-cooled deionized water, stir and dialyze for 8-12 hours, then place in pre-cooled dialysate (pH8.1, 20mM Tris-HCl) and dialyze at 4°C for 8-12 hours , Repeat 2-3 times.

The solution in the dialysis bag was taken out, and after high-speed centrifugation for 10min to remove precipitates and air bubbles, anion exchange chromatography was performed through HiTrap Q HP (5mL), and linear elution was performed with eluent (0-2M NaCl, 20mM Tris pH 8.1) (Fig. 2A). The elution peaks containing the target protein components were collected in sections, concentrated and then sampled for non-reducing SDS-PAGE electrophoresis (Figure 2B). The results showed that the purity of the target protein did not meet the requirements and further purification was required. The concentrated protein samples were subjected to gel filtration chromatography (Fig. 3A) using superdex 75 10/300, and samples were taken for non-reducing and reducing SDS-PAGE electrophoresis detection (Fig. 3B). There is one band nearby, and no other obvious impurity bands; there are two bands in the reducing electrophoresis lane, which are the α chain and β chain of E63 TCR, and their purity meets the requirements of subsequent experiments.

Example 3 Preparation of Biotinylated Antigen Peptide-MHC (pMHC)

The renaturation and purification of pMHC were prepared according to the method of NIH Tetramer Core Facility. According to online protocols, HPV16-E6 _29-38 polypeptide solution, β2M and HLA-A*02:01 inclusion body solution were sequentially added to refolding buffer (0.1M Tris-HCl, 0.4M L-arginine, 2mM EDTA, 0.5mM oxidized glutathione and 5 mM reduced glutathione, 0.2mM PMSF), stirred overnight at 4°C, and added the same amount of HLA-A*02:01 inclusion body solution in the morning and evening of the next day After stirring at 4°C for 1-3 days, dialyze three times in 10 volumes of dialysate (pH 8.1, 20 mM Tris-HCl). The dialyzed protein sample was subjected to anion-exchange chromatography with HiTrap Q HP (5mL), linearly eluted with the eluent (0-2M NaCl, 20mMTris pH 8.1), and the eluted peaks were collected and combined (Figure 4A). In SDS-PAGE electrophoresis analysis, two bands of HLA-A*02:01 and β2M were clearly visible (Figure 4B), while the molecular weight of the HPV16-E6 _29-38 polypeptide was too small to see the bands in the gel map. The eluted peaks containing pMHC components were concentrated, further purified by gel filtration chromatography (Superdex 75 10/300) (Figure 5A), and then detected by reducing SDS-PAGE electrophoresis. It can be seen from the electropherogram that the purity Better pMHC complexes (Fig. 5B). The pMHC complex was biotinylated with the recombinase BirA (Protein Expr.Purif.2012, 82, 162; J.Bacteriol.2012, 194, 1113.), and then streptavidin (SA) was added for reaction verification. The reaction system According to the method of NIH Tetramer Core Facility, the preparation and the purity identification of Gel Shift were carried out. From the Gel Shift electrophoresis diagram (Figure 6), the biotinylation of the E6-pMHC complex was successfully prepared.

Embodiment 4 affinity test

Biacore is an instrument for detecting affinity based on surface plasmon resonance (surface plasmon resonance, SPR) technology. In this experiment, using Biacore T200, the biotinylated pMHC was first coupled to the CM5 chip, and then its binding and dissociation constants with different TCRs were detected to calculate the _KD value. Based on this, the affinity between E63 TCR and pMHC (HLA-A*02:01/TIHDIILECV) was tested, see Table 3 and Figure 7 for details.

Table 3. E6 TCR affinity K _D value

It should be noted that: as known to those skilled in the art, SPR technology is currently one of the most commonly used and reliable methods for determining affinity, but it involves protein quantification, chip age, instrument status, etc., and experiments between different batches may vary. A certain error, the error value can even reach 3 to 5 times; and the present invention uses the same batch of experiments carried out by the same protein quantification, the same chip and the same instrument, so the data can be used to compare the size of the affinity, But specific numerical values do not constitute a limitation to the protection scope of the present invention.

Example 5 TCR lentivirus preparation and transduction of CD8 ⁺ T cells

1) TCR lentiviral packaging

The third-generation lentivirus packaging system (Invitrogen, pLenti6/V5 DirectionalTOPO ^™ Cloning Kit, product number K495510) was used to package the lentivirus containing the gene encoding the target TCR. Combine packaging plasmids pMDLg/pRRE (addgene, product number k12251), pRSV-REV (addgene, product number 12253), pMD2.G (addgene, product number 12259) with shuttle plasmids containing target genes such as pLenti-E63, pLenti- E6con (the TCR gene sequence is derived from patent No. US9822162B2), pLenti-GFP (negative control), etc. were mixed at a mass ratio of 4:2:1:1, and transfection reagent PEI-MAX (Polyscience, product number 23966-1 ) transiently transfected 293T cells in logarithmic growth phase. After 48–50 hours of transfection, the medium supernatant containing lentivirus was collected, and after centrifugation and a 0.45 μm filter to remove cell debris, the Amicon Ultra-15 centrifugal filter equipped with an Ultracel-50 filter membrane (Merck Millipore, product number UFC905096) the supernatant was concentrated. The concentrated sample was subjected to lentiviral titer determination, and the steps were referred to the p24ELISA (Clontech, product number 632200) kit instructions.

2) TCR lentivirus transduction of CD8 ⁺ T cells

CD8 ⁺ T cells were isolated from PBMC of healthy volunteers, inoculated in 48-well plates with RPMI 1640 complete medium containing 10% FBS and 100IU/mL IL-2, 1× ¹⁰⁶ cells per well, and added Anti-CD3/CD28 antibody-coupled magnetic beads are used to stimulate activated CD8 ⁺ T cells, which are cultured overnight in a cell incubator. After being stimulated overnight, add E63, E6con or GFP lentivirus at a ratio of MOI=5, and centrifuge at 900g for 1 hour at 32°C. After the infection, the lentiviral infection solution was removed, the cells were cultured for 3 days, and the anti-CD3/CD28 antibody-coupled magnetic beads were removed with a magnet. After that, the cells were counted every two days, and fresh complete medium was replaced or added to maintain the cell density at 1-2×10 ⁶ cells/mL. On the ninth day of cell culture, flow cytometry and positive rate analysis of T cells were performed by double staining with HLA-A*02:01/HPV16-E6 _29-38 tetramer and anti-CD8 antibody. The results are shown in Figure 8, the positive rate of E63 TCR-T cells was 48.4%, the positive rate of E6con TCR-T cells was 59%, and the positive rate of GFP TCR-T cells was 67.4%.

Example 6 In vitro functional specificity analysis of E63 TCR—ELISPOT method to detect INF-γ release from polypeptide-loaded T ₂ cells

6000Pro-Fβ,Bio-Sys)对孔板进行分析。In this example, ELISPOT assay was used to analyze the release of INF-γ factor by E63 TCR under the stimulation of T2 cells loaded with specific or non _- specific polypeptides. The effector cells in this example are CD8 ⁺ T cells transduced with E63, E6con and GFP lentiviruses in Example 5. The target cells in this example are T _{2 cells loaded with different concentrations of polypeptides, and the T 2 cells} were mixed with seven gradient concentrations (10 ^-11 , 10 ^-10 , 10 ^-9 , 10 ^-8 , 10 ^-7 , 10 ^-6 , 10 ^-5 M) HPV16-E6 _29-38 polypeptide or 10 ^-6 M NY-ESO-1 _157-165 polypeptide, mixed evenly, placed in a 37°C incubator for 4 hours, centrifuged, washed once with 1×PBS, Cells were resuspended in RPMI1640 medium containing 10% FBS for the next step of plating. Follow-up experiments were performed according to the instructions of the Human INF-γ ELISPOT Set kit (BD biosciences, product number 551849). Add 4×10 ³ double-positive effector cells/well and 4×10 ⁴ target cells/well into the ELISPOT well plate, culture system 200 μL per well, and place the well plate in a cell culture incubator for overnight incubation. After incubation, wash according to the instructions of the kit, then add BCIP/NBT solution to develop for 5–15 minutes, wash the well plate with deionized water, and finally turn the well plate upside down, and let the plate dry naturally at room temperature. Enzyme-linked immunospot analyzer (

6000Pro-Fβ, Bio-Sys) to analyze the orifice plate.

The results are shown in Figure 9A, T ₂ cells loaded with HPV16-E6 _29-38 peptide strongly stimulated E63 TCR-T cells to release INF-γ in a peptide concentration-dependent manner, and the trend was consistent with the positive control E6con. When the loading concentration was greater than 10 ^-7 M, the release of INF-γ from E63 and E6con TCR-T cells reached the peak, and there was no significant difference between them. 1G4 TCR specifically recognizing NY-ESO-1 _157-165 had significant activity, while E63 and E6con TCR _- T cells had no significant activity against T2 cells loaded with non-specific NY-ESO-1 _157-165 (Fig. 9B) . Neither T2 cells loaded with E6 nor NY-ESO-1 peptide could stimulate GFP TCR _- T cells to release INF-γ factor. In summary, the function of E63 TCR is close to that of the positive control E6con, and it has specificity for the recognition of HLA-A*02:01/HPV16-E6 _29-38 .

Example 7 Construction of E6 transgenic A375 monoclonal cells

In order to construct HLA-A*02:01 ⁺ /HPV16-E6 ⁺ double positive target cell line for E63 TCR function verification, the E6 full peptide gene was connected to the corresponding shuttle plasmid by the method in Example 5, and lentiviral Packaging, and then transducing into A375 tumor cells (HLA-A*02:01 ⁺ /HPV16-E6 ^- ), initially obtaining A375 polyclonal cells with E6 gene integrated in the chromosome. In order to obtain A375 monoclonal cells that can stably express the E6 gene, the A375+E6 polyclonal cells were isolated and cultured in a 96-well plate by the limiting dilution method, and each well contained 0.5–1 candidate cell. After the amplification of the candidate cells was completed, the copy number of the E6 gene in the cells was measured by the fluorescent quantitative PCR method. As shown in Table 4, the E6 gene copy number of A375+E6 monoclonal cell numbered 1B3 is 19.23, and this cell will be used for in vitro functional verification of E63 TCR and animal experiments.

Table 4. Detection results of E6 gene copy number in A375+E6-1B3 cells

HPV16-E6 full-length sequence:

MHQKRTAMFQDPQERPRKLPQLCTELQT TIHDIILECV YCKQQLLRREVYDFAFR DLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQ KPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCRSSRTRRETQL (SEQ ID NO: 26)

Among the above sequences: the underlined sequence is HPV16-E6 _29-38 peptide.

Example 8 In vitro function verification of E63 TCR—ELISPOT method to detect INF-γ release in tumor cell lines

In this example, ELISPOT assay was used to analyze the release of INF-γ factor by E63 TCR under the stimulation of different tumor cell lines. The effector cells in this example are CD8 ⁺ T cells transduced with E63, E6con and GFP lentiviruses in Example 5. The tumor target cells in this example are A375, A375+E6-1B3, CaSki, C33A, SiHa and Hela cells, respectively. As described in Example 6, sequentially add 4×10 ³ positive effector cells/well and 4×10 ⁴ tumor target cells/well into the ELISPOT well plate, culture system 200 μL per well, and place the well plate in a cell culture incubator for incubation overnight. After incubation, wash and develop the ELISPOT well plate according to the method in Example 6, and finally analyze the well plate with an enzyme-linked immunospot analyzer.

The results are shown in Figure 10, E63 and E6con TCR-T cells exhibited strong stimulating activity on the double-positive tumor target cell A375+E6-1B3, and the ability of the two to release INF-γ factor was equivalent. Other tumor cells could not stimulate E63 and E6conTCR-T cells to release INF-γ. GFP TCR-T cells were inactive against all tumor target cells.

Example 9 In vitro functional verification of E63 TCR - LDH-specific killing of tumor cell lines

Non-Radioactive Cytotoxicity Assay试剂盒说明书(Promega,产品号G1780)，在培养基空白孔与肿瘤靶细胞最大自释放孔中加入裂解液，置于细胞培养箱中孵育45min。孵育结束后，每孔取50μL上清与50 μL LDH检测液混匀，室温避光孵育30min后，加入终止液，并于490nm波长下读板。按照计算公式对数据进行处理分析：细胞毒性百分比％，即LDH释放百分比％＝(实验组释放量–肿瘤细胞自释放量–TCR-T细胞自释放量)/(肿瘤细胞最大释放量-肿瘤细胞自释放量)*100％。计算时，各组LDH释放量数值均减去培养基本底光吸收值。The function of killing target cells by effector cells is evaluated by quantitatively measuring LDH released after target cells are lysed. For specific experimental schemes, refer to Eur.J Immunol.1993, 23, 3217. The effector cells in this example are CD8 ⁺ T cells transduced with E63, E6con and GFP lentiviruses in Example 5. The tumor target cells in this example are A375 and A375+E6-1B3 cells. Add 3×10 ⁴ double-positive effector cells/well and 1×10 ⁴ tumor target cells/well into a 96-well round bottom plate in turn, and culture system is 200 μL per well, and the cells are replaced with RPMI containing only 5% FBS when plating 1640 medium, the well plate was placed in a cell culture incubator for 24 hours. According to CytoTox

Instructions for Non-Radioactive Cytotoxicity Assay kit (Promega, product number G1780), add lysate to the blank well of the medium and the largest self-release well of tumor target cells, and incubate in a cell culture incubator for 45 minutes. After the incubation, mix 50 μL of supernatant and 50 μL of LDH detection solution from each well, incubate at room temperature in the dark for 30 min, add stop solution, and read the plate at a wavelength of 490 nm. The data were processed and analyzed according to the calculation formula: percentage of cytotoxicity, i.e. LDH release percentage%=(experimental group release amount-tumor cell self-release amount-TCR-T cell self-release amount)/(tumor cell maximum release amount-tumor cell Self-release amount)*100%. When calculating, the value of LDH release in each group was subtracted from the basic light absorption value of the culture.

The results are shown in Figure 11, E63 and E6con TCR-T cells showed obvious killing effect on double-positive tumor target cells A375+E6-B3, and the cytotoxic percentages of the two were close, which were 35.56±5.86% and 34.93±4.72%, respectively. E63 and E6con TCR-T cells had no obvious killing effect on A375, similar to the negative control GFP TCR-T cells.

Example 10 In vitro functional verification of E63 TCR—detection of the positive rate of E63 and E6conTCR-T cells using the amino acid sequences of human and mouse C regions

The α chain and β chain of E63 TCR adopt the amino acid sequence of the human C region, while the α chain and β chain of the positive control E6con TCR adopt the amino acid sequence of the murine C region. In order to compare the effects of C region amino acid sequences from different sources on the expression of E63 TCR in T cells, the C regions of E63 and E6con TCR were exchanged, and finally four different TCR combinations were obtained. These four TCRs were respectively transduced into CD8 ⁺ T cells, and HLA-A*02:01/HPV16-E6 _29-38 tetramer and anti-CD8 antibody were used for double staining, and the T cells were detected by flow cytometry and positive rate analysis.

As shown in Fig. 12, the positive rate of E63 can reach 70.5% for the same human C region sequence, while the positive rate of E6con is 27.1%, which is 43.4% lower than that of E63. When the C region is replaced with the mouse amino acid sequence, the positive rate of E63 is 85.7%, which is an increase of 15.2%; the positive rate of E6con, which is also the mouse C region, is 78.1%, which is 51% higher than that of the E6con of the human C region. . It can be seen that the human or mouse C region has little effect on the expression rate of E63 TCR in T cells, indicating that the α chain and β chain of E63 TCR have better pairing efficiency.

Alpha chain sequence of E63 TCR (murine C region):

β chain sequence of E63 TCR (murine C region):

Alpha chain sequence of E6con TCR (human C region):

β chain sequence of E6con TCR (human C region):

In the above sequence: the sequence marked in black is signal peptide and Vα (variable region of α chain) or Vβ (variable region of β chain), and the sequence marked in gray is Cα (constant region of α chain) or Cβ (constant region of β chain) region), the italic underlined sequence is the transmembrane intracellular region.

Example 11 In vitro functional verification of E63 TCR—healthy human PBMC-specific INF-γ release

In order to check the safety of E63 TCR, the ELISPOT method in Example 6 was used to check the safety of PBMCs from 22 healthy people. The 22 cases of healthy PBMC ( FIG. 13A ) included 4 cases of HLA-A*02:01 type and 18 cases of non-HLA-A*02:01 type. As shown in FIG. 13B , the INF-γ release response showed that E63 TCR-T cells had no obvious response to PBMC from healthy people.

Example 12 E63 TCR animal experiment verification—E6 transgenic A375 melanoma allograft

The experimental animal strain of this example is B-NDG mouse (Biocytogen Jiangsu Gene Biotechnology Co., Ltd., SPF grade), female, 4-6 weeks old. During the experiment, the experimental animals were placed in an SPF animal center, and all technical indicators met the technical requirements of GB14925-2010 barrier environment. Animals in this study will eat feed that meets national standards and is within the validity period, and their drinking water will be sterilized by filtration or high-temperature and high-pressure sterilization in an animal drinking pure water system, and will be ingested freely from animal drinking bottles. The experimental animals were adaptively fed for 1 week before subsequent experiments.

B-NDG mice qualified for adaptive observation were selected to inoculate A375+E6-1B3 tumor cells subcutaneously, and the cell density was adjusted to 1-2×10 ⁷ cells/mL, and each mouse was inoculated with 0.2 mL. Observed every day after inoculation, when the average tumor volume was about 100mm ³ , they were divided into 3 groups, namely Model Control group, E63 TCR-T group and GFP TCR-T group, with 8 animals in each group. The E63 TCR-T group was given the dose of 4× ¹⁰⁸ positive T cells/kg, the GFP TCR-T group was given the same dose of T cells, and the model control group was given the same volume of vehicle. The mice in each group were administered by tail vein injection. After administration, each group was intraperitoneally injected with IL-2, 50,000 IU/rat, for 5 consecutive days. The tumor diameter was measured on the day of grouping, and the long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper every 3 days, and the tumor volume (Tumor Volume) was calculated according to the formula 1/ ² ×a×b2, and the tumor growth curve was drawn. After 26 days of administration, the animals were euthanized, and the tumors were taken to take pictures.

See Figure 14 and Figure 15 for the tumor growth curves and tumor volumes of mice in each group. E63 TCR-T cells can effectively kill tumor cells A375+E6-1B3 and inhibit tumor growth. There was no significant change in the tumor growth curve of the GFP TCR-T group compared with the Model Control group.

SEQUENCE LISTING

<110> Shenzhen Puruijin Biological Pharmaceutical Co., Ltd.

<120> A TCR for recognizing HLA-A*02:01/E629-38 and its application

<130> P20014623C

<160> 28

<170> PatentIn version 3.5

<210> 1

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 alpha chain CDR1

<400> 1

Thr Ser Asp Gln Ser Tyr Gly

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 alpha chain CDR2

<400> 2

Gln Gly Ser Tyr Asp Glu Gln Asn

1 5

<210> 3

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 alpha chain CDR3

<400> 3

Ala Met Arg Glu Asn Thr Gly Thr Ala Ser Lys Leu Thr

1 5 10

<210> 4

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 beta chain CDR1

<400> 4

Ser Gly His Asp Asn

1 5

<210> 5

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 beta chain CDR2

<400> 5

Phe Val Lys Glu Ser Lys

1 5

<210> 6

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 beta chain CDR3

<400> 6

Ala Ser Ser Ala Trp Gly His Gln Asn Ser Pro Leu His

1 5 10

<210> 7

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 alpha chain CDR1

<400> 7

Thr Ser Ile Asn Asn

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 alpha chain CDR2

<400> 8

Ile Arg Ser Asn Glu Arg Glu

1 5

<210> 9

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 alpha chain CDR3

<400> 9

Ala Thr Asp Ala Phe Gly Asn Gln Phe Tyr

1 5 10

<210> 10

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 beta chain CDR1

<400> 10

Met Asp His Glu Asn

1 5

<210> 11

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 beta chain CDR2

<400> 11

Ser Tyr Asp Val Lys Met

1 5

<210> 12

<211> 13

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 beta chain CDR3

<400> 12

Ala Ser Ser Leu Trp Gly Arg Val Val Glu Lys Leu Phe

1 5 10

<210> 13

<211> 89

<212> PRT

<213> Artificial Sequence

<220>

<223> Constant region of TCRα chain

<400> 13

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser

1 5 10 15

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn

20 25 30

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val

35 40 45

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp

50 55 60

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

65 70 75 80

Ile Pro Glu Asp Thr Phe Phe Pro Ser

85

<210> 14

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> Constant region of TCR beta chain

<400> 14

Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala

<210> 15

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> E67 alpha chain CDR3

<400> 15

Ala Met Arg Glu Ala Arg Arg Ser Gln Phe Tyr

1 5 10

<210> 16

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> E67 beta chain CDR1

<400> 16

Asp Phe Gln Ala Thr Thr

1 5

<210> 17

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> E67 beta chain CDR2

<400> 17

Ser Asn Glu Gly Ser Lys Ala

1 5

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> E67 beta chain CDR3

<400> 18

Ser Ala Ala Leu Gly Ser Tyr Glu Gln Tyr

1 5 10

<210> 19

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 alpha chain variable region

<400> 19

Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe Val Gln Glu Lys

1 5 10 15

Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser Asp Gln Ser Tyr

20 25 30

Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu Met Ile Phe Leu

35 40 45

Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr Glu Gly Arg Tyr

50 55 60

Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn Leu Val Ile Ser

65 70 75 80

Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys Ala Met Arg Glu

85 90 95

Asn Thr Gly Thr Ala Ser Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu

100 105 110

Gln Val Thr Leu Asp

115

<210> 20

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> E63 beta chain variable region

<400> 20

Glu Ala Gly Val Thr Gln Phe Pro Ser His Ser Val Ile Glu Lys Gly

1 5 10 15

Gln Thr Val Thr Leu Arg Cys Asp Pro Ile Ser Gly His Asp Asn Leu

20 25 30

Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe Leu Leu His

35 40 45

Phe Val Lys Glu Ser Lys Gln Asp Glu Ser Gly Met Pro Asn Asn Arg

50 55 60

Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys Val Gln

65 70 75 80

Pro Ala Glu Leu Glu Asp Ser Gly Val Tyr Phe Cys Ala Ser Ser Ala

85 90 95

Trp Gly His Gln Asn Ser Pro Leu His Phe Gly Asn Gly Thr Arg Leu

100 105 110

Thr Val Thr

115

<210> 21

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 alpha chain variable region

<400> 21

Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser Ile Gln Glu Gly

1 5 10 15

Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser Ile Asn Asn Leu

20 25 30

Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val His Leu Ile Leu

35 40 45

Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg Leu Arg Val Thr

50 55 60

Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile Thr Ala Ser Arg

65 70 75 80

Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp Ala Phe Gly Asn

85 90 95

Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val Ile Pro Asn

100 105 110

<210> 22

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> E65 β chain variable region

<400> 22

Asp Val Lys Val Thr Gln Ser Ser Arg Tyr Leu Val Lys Arg Thr Gly

1 5 10 15

Glu Lys Val Phe Leu Glu Cys Val Gln Asp Met Asp His Glu Asn Met

20 25 30

Phe Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu Ile Tyr Phe

35 40 45

Ser Tyr Asp Val Lys Met Lys Glu Lys Gly Asp Ile Pro Glu Gly Tyr

50 55 60

Ser Val Ser Arg Glu Lys Lys Glu Arg Phe Ser Leu Ile Leu Glu Ser

65 70 75 80

Ala Ser Thr Asn Gln Thr Ser Met Tyr Leu Cys Ala Ser Ser Leu Trp

85 90 95

Gly Arg Val Val Glu Lys Leu Phe Phe Gly Ser Gly Thr Gln Leu Ser

100 105 110

Val Leu

<210> 23

<211> 115

<212> PRT

<213> Artificial Sequence

<220>

<223> E67 alpha chain variable region

<400> 23

Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe Val Gln Glu Lys

1 5 10 15

Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser Asp Gln Ser Tyr

20 25 30

Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu Met Ile Phe Leu

35 40 45

Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr Glu Gly Arg Tyr

50 55 60

Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn Leu Val Ile Ser

65 70 75 80

Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys Ala Met Arg Glu

85 90 95

Ala Arg Arg Ser Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val

100 105 110

Ile Pro Asn

115

<210> 24

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> E67 beta chain variable region

<400> 24

Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly

1 5 10 15

Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr

20 25 30

Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala

35 40 45

Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys

50 55 60

Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr

65 70 75 80

Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala

85 90 95

Ala Leu Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr

<210> 25

<211> 129

<212> PRT

<213> Artificial Sequence

<220>

<223> Constant region of TCR beta chain

<400> 25

Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro

1 5 10 15

Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys

50 55 60

Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu

65 70 75 80

Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys

85 90 95

Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp

100 105 110

Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg

115 120 125

Ala

<210> 26

<211> 158

<212> PRT

<213> Artificial Sequence

<220>

<223> HPV16-E6 full-length sequence

<400> 26

Met His Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro

1 5 10 15

Arg Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp

20 25 30

Ile Ile Leu Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu

35 40 45

Val Tyr Asp Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly

50 55 60

Asn Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile

65 70 75 80

Ser Glu Tyr Arg His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu

85 90 95

Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile Asn

100 105 110

Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp Lys

115 120 125

Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys Met

130 135 140

Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu

145 150 155

<210> 27

<211> 82

<212> PRT

<213> Artificial Sequence

<220>

<223> TCR alpha chain constant region sequence (mouse)

<400> 27

Ile Gln Asn Pro Glu Pro Ala Val Tyr Gln Leu Lys Asp Pro Arg Ser

1 5 10 15

Gln Asp Ser Thr Leu Cys Leu Phe Thr Asp Phe Asp Ser Gln Ile Asn

20 25 30

Val Pro Lys Thr Met Glu Ser Gly Thr Phe Ile Thr Asp Lys Thr Val

35 40 45

Leu Asp Met Lys Ala Met Asp Ser Lys Ser Asn Gly Ala Ile Ala Trp

50 55 60

Ser Asn Gln Thr Ser Phe Thr Cys Gln Asp Ile Phe Lys Glu Thr Asn

65 70 75 80

Ala Thr

<210> 28

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> TCR beta chain constant region sequence (mouse)

<400> 28

Glu Asp Leu Arg Asn Val Thr Pro Pro Lys Val Ser Leu Phe Glu Pro

1 5 10 15

Ser Lys Ala Glu Ile Ala Asn Lys Gln Lys Ala Thr Leu Val Cys Leu

20 25 30

Ala Arg Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn

35 40 45

Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Ala Tyr Lys

50 55 60

Glu Ser Asn Tyr Ser Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala

65 70 75 80

Thr Phe Trp His Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe

85 90 95

His Gly Leu Ser Glu Glu Asp Lys Trp Pro Glu Gly Ser Pro Lys Pro

100 105 110

Val Thr Gln Asn Ile Ser Ala Glu Ala Trp Gly Arg Ala

115 120 125

Claims (26)

1. base:Sub>A TCR which specifically recognizes HLA-base:Sub>A x 02_29-38The amino acid sequences of CDR1, CDR2 and CDR3 of the alpha chain variable region of the TCR are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the beta chain variable region of the TCR are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

2. A TCR as claimed in claim 1 wherein the α chain and/or β chain of the TCR further comprises a framework region;

the framework regions of the alpha chain are derived from the germline TRAV, TRAJ and TRAC;

the framework regions of the beta chain are derived from the germline TRBV, TRBD, TRBJ and TRBC.

3. A TCR as claimed in claim 2 wherein said TRAV is TRAV14.

4.A TCR as claimed in claim 2 wherein said TRAJ is TRAJ44.

5. A TCR as claimed in claim 3 wherein the TRBV is TRBV14.

6. A TCR as claimed in claim 3 wherein the TRBD is TRBD1.

7. A TCR as claimed in claim 3 wherein the TRBJ is TRBJ1-6.

8. A TCR as claimed in claim 3 wherein the TRBC is TRBC1.

9. A TCR as claimed in claim 2,

the alpha chain variable region comprises an amino acid sequence shown as SEQ ID NO. 19, and the beta chain variable region comprises an amino acid sequence shown as SEQ ID NO. 20.

10. A TCR as claimed in any one of claims 1 to 9 wherein the TCR α chain and/or TCR β chain of the TCR further comprises a constant region.

11. A TCR as claimed in claim 10 wherein the constant region of the TCR α chain is derived from the human or murine germline and/or the constant region of the TCR β chain is derived from the human or murine germline.

12. A TCR as claimed in claim 11 wherein the constant region of a TCR α chain derived from the human germline comprises the sequence set out in SEQ ID No. 13;

the constant region of the TCR alpha chain derived from the murine germline comprises the amino acid sequence shown as SEQ ID NO 27;

the constant region of the TCR beta chain derived from the human germline comprises the sequence shown as SEQ ID NO. 14 or SEQ ID NO. 25;

the constant region of the TCR β chain derived from the murine germline comprises the amino acid sequence shown in SEQ ID NO 28.

13. A TCR as claimed in claim 10 wherein the TCR α chain and/or TCR β chain of the TCR further comprises an extracellular domain and a transmembrane domain.

14. A TCR as claimed in claim 13 wherein the TCR α chain and/or TCR β chain of the TCR further comprises intracellular sequences.

15. An isolated nucleic acid encoding a TCR as claimed in any one of claims 1 to 14.

16. A vector comprising the nucleic acid of claim 15, wherein said nucleic acid encodes TCR α chain and TCR β chain, respectively, in a single open reading frame, or in two different open reading frames.

17. The vector of claim 16, wherein the vector is a lentiviral vector.

18. A cell comprising the nucleic acid of claim 15 or the vector of claim 16 or 17.

19. The cell of claim 18, wherein the cell is a T cell or a stem cell.

20. The cell of claim 19, wherein said T cell is CD8⁺T cells.

21. An isolated or non-naturally occurring cell presenting a TCR comprising any one of claims 1 to 14.

22. The cell of claim 21, wherein the cell is a T cell.

23. A pharmaceutical composition comprising a TCR as claimed in any one of claims 1 to 14 or a cell as claimed in any one of claims 18 to 22.

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

25. Use of a TCR as claimed in any one of claims 1 to 14, a cell as claimed in any one of claims 18 to 22 or a pharmaceutical composition as claimed in claim 23 or 24 in the manufacture of a medicament for the prevention or treatment of a tumour associated with HPV16 expression.

26. The use of claim 25, wherein said neoplasm comprises cervical cancer, oropharyngeal cancer, vaginal cancer, anal cancer, and penile cancer.

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