CN113754756B

CN113754756B - An antibody that recognizes HLA-A0229-38TCR and uses thereof

Info

Publication number: CN113754756B
Application number: CN202111142425.2A
Authority: CN
Inventors: 吴显辉; 王晓娟; 陈亮; 汤小欣; 黄金燕; 栗红建
Original assignee: Shenzhen Prijin Biopharmaceutical Co ltd
Current assignee: Liyang Masai Bio Pharmaceutical Co ltd
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2022-11-01
Anticipated expiration: 2041-09-28
Also published as: CN113754756A

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

An antibody that recognizes HLA-A0229-38TCR and uses thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates tobase:Sub>A method for recognizing HLA-A02_29-38And uses thereof.

Background

Human Papilloma Virus (HPV) is an epitheliotropic virus, susceptible to infection of the Human epidermal and mucosal squamous epithelia, and is associated with the development of a variety of cancers (cervical, anal, penile, vaginal, vulvar, and head and neck, etc.) (Lancet Oncol,2010,11,781, clin Cancer res,2019,25, 1486), the mortality of head and neck Cancer associated with HPV infection has continued to rise over the past decade (CA Cancer J Clin,2021,71,7). There are several dozen subtypes of HPV virus (Virology, 2015,476,341), and these tumors are mainly associated with HPV16 infection (J Natl Cancer Inst,2015,107, djv086). The E6 protein encoded by HPV16, one of the major proteins in the viral life cycle, induces tumorigenesis and progression through a variety of mechanisms of action, such as inhibition of oncosuppressive proteins p53 and pRb, and repression of apoptosis (Cancer Sci,2007,98,1505); enhancing telomerase activity immortalizes host cells (Virus Res,2017, 231,50); induces the expression deletion of human histocompatibility antigen (HLA) (Clin Immunol,2005,115,295), is beneficial to the escape of tumor cells from the innate immune response of a host, thereby causing the generation, development, infiltration and metastasis of the tumor cells. HPV16-E6 belongs to tumor-specific antigens, is specifically expressed only in relevant tumor tissues and is not expressed in normal tissues (Nat Rev Cancer,2002,2,342, clin Immunol,2005,115, 295), so that the off-target rate can be reduced and the effectiveness and safety of treatment can be improved in immunotherapy targeting HPV16-E6 (Papilomavir Res,2018,5,46), which is an ideal target for HPV-related Cancer treatment.

T cell receptor genetically engineered T cell (TCR-T) therapy is the transduction of a TCR gene specific for a tumor antigen into normal T cells, capable of enhancing or re-conferring the ability of the T cells to recognize tumor antigens, specifically targeting and killing tumor cells (Science, 2016,352,1337 cells,2020, 9), is an important therapeutic approach in current adoptive cell therapy (Immunol Rev,2014,257,56). Relevant clinical trials currently in progress have demonstrated significant efficacy of TCR-T cell therapy in the treatment of melanoma, synovial cell sarcoma, myeloma and other malignancies (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 8 months at 2021, 461 clinical registration records related to TCR-T were available on clinicalters.gov website, suggesting that TCR-T has great potential and development value in tumor immunotherapy. TCR-T cell therapy against HPV16-E6 targets has been developed clinically at present (NCT 02280811, NCT03197025, NCT 03578406), and its effectiveness and safety have been preliminarily validated (Clin Cancer Res,2015,21,4431, j Clin oncol,2017,35,3009, j Clin oncol,2019, 37,2759), providing a new approach for the treatment of HPV-related cancers. With the development and research of related technologies, TCR-T therapy will gradually develop towards high efficiency, low toxicity and controllability, and can bring convenience of clinical application while improving curative effect and safety, thereby bringing hope of curing more tumor patients.

Disclosure of Invention

The invention providesbase:Sub>A method for effectively identifying HLA-A02_29-38TCR of target, T cell that use this TCR to transform through high-efficient recognition HLA-A02_29-38The target spot specifically kills tumor cells.

One of the technical schemes of the invention is as follows: a TCR, wherein CDR1, CDR2 and CDR3 of the α chain variable region of the TCR comprise the amino acid sequences set forth as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively;

or respectively comprises the amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9;

or, respectively, the amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 15.

Preferably, CDR1, CDR2 and CDR3 of the beta chain variable region of the TCR comprise amino acid sequences as set forth in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively;

or respectively comprises the amino acid sequences shown as SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12;

or respectively comprises the amino acid sequences shown as SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18.

In a specific embodiment of the invention, CDR1, CDR2 and CDR3 of the alpha chain variable region comprise the amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and CDR1, CDR2 and CDR3 of the beta chain variable region comprise the amino acid sequences shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

In another specific embodiment of the invention, CDR1, CDR2 and CDR3 of the alpha chain variable region comprise the amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively, and CDR1, CDR2 and CDR3 of the beta chain variable region comprise the amino acid sequences shown as SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

In another specific embodiment of the invention, CDR1, CDR2 and CDR3 of the alpha chain variable region comprise the amino acid sequences shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:15, respectively, and CDR1, CDR2 and CDR3 of the beta chain variable region comprise the amino acid sequences shown as SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively.

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

the framework regions of the alpha chain are derived from the germline TRAV, TRAJ, and TRAC, wherein the TRAV is preferably TRAV14 or TRAV17, and the TRAJ is preferably TRAJ44 or TRAJ49;

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

In a preferred embodiment of the invention, the α chain variable region of the TCR comprises an amino acid sequence as shown in SEQ ID NO 19, 21 or 23.

In another preferred embodiment of the invention, the beta chain variable region of the TCR comprises the amino acid sequence shown in SEQ ID NO 20, SEQ ID NO 22 or SEQ ID NO 24.

In one embodiment of the invention, the α chain variable region of the TCR comprises the amino acid sequence shown in SEQ ID NO. 19 and the β chain variable region of the TCR comprises the amino acid sequence shown in SEQ ID NO. 20.

In another specific embodiment of the present invention, the alpha chain variable region comprises the amino acid sequence shown as SEQ ID NO. 21 and the beta chain variable region comprises the amino acid sequence shown as SEQ ID NO. 22.

In another specific embodiment of the present invention, the alpha chain variable region comprises the amino acid sequence shown as SEQ ID NO. 23 and the beta chain variable region comprises the amino acid sequence shown as SEQ ID NO. 24.

The TCR α chain of the TCRs of the invention preferably further comprises a constant region, preferably of human or murine origin.

The TCR β chain of the TCRs of the invention preferably further comprises a constant region, preferably the constant region of the TCR β chain is derived from human or murine germline.

The constant region of the TCR alpha chain derived from the human germline preferably comprises the sequence shown in SEQ ID NO 13.

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

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

In addition, the TCR α chains described herein can also include an extracellular domain and a transmembrane domain; preferably, the TCR α chain further comprises an intracellular sequence.

The TCR β chain may also comprise an extracellular domain and a transmembrane domain; preferably, the TCR β chain further comprises an intracellular sequence.

The second technical scheme of the invention is as follows: an isolated nucleic acid encoding a TCR according to any one of the embodiments of the invention.

The third technical scheme of the invention is as follows: a vector comprising a nucleic acid according to claim two, preferably a lentiviral vector; the nucleic acids encode a TCR α chain and a TCR β chain, respectively, in a single open reading frame, or in two different open reading frames.

The fourth technical scheme of the invention is as follows: a cell comprising a nucleic acid according to claim two or a vector according to claim three; preferably, the cell is a T cell or a stem cell, preferably a CD8 cell⁺T cells.

The fifth technical scheme of the invention is as follows: an isolated or non-naturally occurring cell, preferably a T cell, presenting a TCR as claimed in any one of the preceding claims.

The sixth technical scheme of the invention is as follows: a pharmaceutical composition comprising a TCR as claimed in any one of claims or a cell as claimed in any four of claims; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The seventh technical scheme of the invention is as follows: use of a TCR according to any one of the preceding claims, a cell according to claim four or a pharmaceutical composition according to claim six for the preparation of a medicament for the prevention or treatment of a tumor associated with HPV16 expression; preferably, the tumor includes cervical cancer, oropharyngeal cancer, vaginal cancer, anal cancer, penile cancer, and the like.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

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

The positive progress effects of the invention are as follows:

the TCRs of the invention have high affinity for pMHC (HLA-base:Sub>A × 02_DThe value can reach 4.2E-06M. For HLA-base:Sub>A 02⁺/HPV16-E6⁺The target cells (A375 + E6-1B 3) have specific killing effect, and the killing effect is enhanced along with the increase of the effective target ratio; but has no obvious killing effect on other two non-double positive target cells. In addition, GFP transduced T cells had no apparent killing effect on A375+ E6-1B 3.

Drawings

FIG. 1 is HPV16-E6_29-38Antigen-specific double positive monoclonal CD8⁺T cell sorting process.

FIGS. 2A and 2B are anion exchange chromatography and SDS-PAGE electrophoresis images of E63 TCR after renaturation.

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

FIGS. 4A and 4B are anion exchange chromatography and SDS-PAGE electrophoresis after HLA-A02/β 2M/TIHDIILECV renaturation; wherein, the band with large molecular weight is HLA-A02, the band with small molecular weight is beta 2M, the TIHDIILECV polypeptide has too small molecular weight, and the band can not be seen on SDS-PAGE.

FIGS. 5A and 5B are gel filtration chromatography and SDS-PAGE electrophoresis after HLA-A × 02.

FIG. 6 is a Gel Shift plot after biotinylation of E6-pMHC.

FIG. 7 is a graph of the results of the E63 TCR affinity assay.

FIG. 8 is a schematic representation of E63 TCR lentivirus infected CD8⁺A positive rate result chart of the T cells; wherein E6con is a positive TCR control group, and GFP is a negative control group.

FIGS. 9A and 9B show E63 TCR versus load E6_29-38Or NY-ESO-1_157-165T of a polypeptide₂INF-gamma release profile of cells.

FIG. 10E63 TCR INF- γ release profile for tumor cell lines.

FIG. 11 shows LDH-specific killing experiments of tumor cell lines.

FIG. 12 is a graph showing the effect of different source C regions on the E63 TCR match ratio.

FIGS. 13A and 13B are INF- γ release profiles of E63 TCR on 22 different HLA-A type healthy human PBMCs.

FIG. 14 is a graph of tumor growth in E63 TCR-T animals.

FIG. 15 shows the tumor size of mice 26 days after administration of E63 TCR-T cells.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

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

Example 1 antigen-specific α β -TCR cloning and Gene sequence identification

HPV16-E6_29-38(TIHDIILECV) antigen-specific CD8⁺T cell TCR gene clone adopts methods, reagents and consumables, mainly refer to curr.Protoc.Immunol.2002,7,1; PLoS one.2011,6, e27930; onco immunology.2016,5, e1175795; j Vis exp.2011,8,3321; j Immunol methods.2006,310,40; PLoS one.2014,9,e110741 and citations thereof. Was isolated from PBMCs of healthy volunteers of HLA-A x 02CD8⁺After T cells, loading with HPV16-E6_29-38Peptide-stretch EBV-B cell stimulation of CD8⁺T cells, then with PE-labeled HLA-base:Sub>A 02_29-38T cells are doubly stained by tetramer and APC marked anti-CD 8 antibodies, flow sorting is carried out to obtain double positive T cells, and the T cells are expanded and cultured to a certain number and then are sorted again (figure 1). After 2 rounds of stimulation culture and sorting, the double positive T cells were subjected to monoclonal culture by limiting dilution. The expanded monoclonal T cells were passed through HLA-A x 02_29-38Carrying out flow detection and sorting by double staining of tetramer and anti-CD 8 antibody to obtain HPV16-E6_29-38Antigen-specific monoclonal T cells.

Using Quick-RNA^TMTotal RNA from which the monoclonal T cells were obtained was extracted with MiniPrep Kit (ZYMO research, product No. R1050), and the RNA was reverse-transcribed with the SMARTER RACE cDNA Amplification Kit (Clontech, product No. 634923) to obtain cDNA. And then, taking the cDNA as a template, amplifying a target gene by PCR, connecting the target gene to a pUC19 vector, converting E.coli-DH5 alpha by a heat shock method, plating and culturing overnight, selecting a monoclonal colony for identification and sequencing, and comparing and analyzing a gene sequence obtained by sequencing in an IMGT database. A total of 3 HLA-A02/HPV 16-E6 were obtained_29-38Antigen-specific TCRs, designated E63, E65 and E67 TCRs, have TCR α and β chain genotypes as shown in table 1.

TABLE 1 genotypes of TCR alpha and beta chains

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

e63 The beta chain full length sequence of the TCR:

alpha chain of E65:

beta strand of E65:

alpha chain of E67:

beta strand of E67:

in the above sequence: the underlined sequences are signal peptide regions, the black bold sequences are V α (variable region of α chain) or V β (variable region of β chain), the gray-scale sequences are C α (constant region of α chain) or C β (constant region of β chain), the italic underlined sequences are transmembrane intracellular regions, and the bold underlined sequences are CDR sequences.

According to the rules of the IMGT database, the amino acid sequences of the CDR regions of the unique key sequences of the V region of the E6 TCR are shown in table 2:

TABLE 2.E6 TCR V region alpha and beta chain CDR region amino acid sequences

Example 2 expression and purification of the E63 TCR Gene

E63 Of TCRsConnecting alpha chain and beta chain genes to pET28a vector by adopting Nco I/Not I enzyme cutting sites respectively, transforming E.coli-BL21 (DE 3) by a heat shock method, plating for overnight culture, picking a monoclonal colony to LB culture medium, and carrying out shake culture at 37 ℃ until OD is reached₆₀₀And (4) =0.6-0.8, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 1mM to induce the expression of the target protein, continuously culturing at 37 ℃ for 3h, and centrifuging at 6000rpm for 10min to collect thalli.

The cells were resuspended in a lysis solution (1 XPBS containing 0.5% Triton X-100), sonicated, and centrifuged at 12000rpm for 20min. Discarding the supernatant, resuspending the precipitate with lysis solution until no macroscopic particles exist, centrifuging at 12000rpm for 10min, repeating the above operations for 2-3 times, dissolving the precipitate with 6M guanidine hydrochloride solution, centrifuging at 12000rpm for 10min, and collecting the supernatant, wherein the supernatant is the purified inclusion body. The inclusion bodies were quantified by BCA assay.

20mg of E63 TCR α chain inclusion bodies and 15mg of β chain inclusion bodies were diluted in 5mL of 6M guanidine hydrochloride solution, respectively, and then the TCR α chain and the TCR β chain were slowly added to the pre-cooled renaturation buffer in this order (Science 1996,274, 209, J.mol.biol.1999,285,1831 protein Eng.2003,16, 707), and stirring was continued at 4 ℃ for 30min. Adding the solution into dialysis bag, adding into 10 times volume of pre-cooled deionized water, stirring, dialyzing for 8-12 hr, adding into pre-cooled dialysate (pH 8.1,20mM Tris-HCl), dialyzing at 4 deg.C for 8-12 hr, and repeating for 2-3 times.

The solution in the dialysis bag was taken out, centrifuged at high speed for 10min to remove precipitates and air bubbles, and then subjected to anion exchange chromatography by HiTrap Q HP (5 mL) and eluted linearly with an eluent (0-2M NaCl,20mM Tris pH 8.1) (FIG. 2A). And collecting elution peaks containing target protein components in a sectional manner, concentrating the elution peaks, sampling and performing non-reducing SDS-PAGE electrophoresis (figure 2B), wherein the results show that the purity of the target protein does not meet the requirement and further purification is needed. Performing gel filtration chromatography on the concentrated protein sample by using superdex 75/300 (figure 3A), and performing non-reducing and reducing SDS-PAGE (figure 3B) electrophoretic detection on the sample, wherein the result shows that a band is formed in a non-reducing electrophoretic lane except for the position near 45kDa, and other obvious bands are not formed; the reduced electrophoresis lane has two bands, namely an alpha chain and a beta chain of the E63 TCR, and the purity of the bands meets the requirements of subsequent experiments.

Example 3 biotinylated antigenic peptide-MHC (pMHC) preparation

Reconstitution and purification of pMHC was carried out according to the NIH Tetramer Core Facility procedure. HPV16-E6 according to in-line protocols_29-38Polypeptide solution, β 2M and HLA-A x 02 solution were added sequentially to renaturation buffer (0.1M Tris-HCl,0.4M L-argine, 2mM EDTA,0.5mM oxidized glutathione and 5mM reduced glutathione, 0.2mM PMSF), stirred overnight at 4 deg.C, then added the same amount of HLA-A02 inclusion solution the next morning and evening, and after stirring for 1-3 days at 4 deg.C, dialyzed 3 times against 10-fold volume of dialysate (pH 8.1,20mM Tris-HCl). The dialyzed protein samples were subjected to anion exchange chromatography using HiTrap Q HP (5 mL), eluted linearly with eluent (0-2M NaCl,20mM Tris pH 8.1), and the eluted peaks were collected and pooled (FIG. 4A), and analyzed by reducing SDS-PAGE, two bands of HLA-A02 and β 2M (FIG. 4B) were clearly visible, while HPV16-E6_29-38The molecular weight of the polypeptide was too small and no bands were visible in the gel. The peak eluted containing the pMHC fraction was concentrated and further purified by gel filtration chromatography (Superdex 75/10/300) (FIG. 5A), followed by detection by reducing SDS-PAGE electrophoresis to give a pMHC complex of better purity (FIG. 5B). The pMHC complexes were biotinylated with the recombinase BirA (Protein expr. Purif.2012,82,162 j. Bacteriol.2012,194, 1113.), and then Streptavidin (SA) was added for reaction validation, the reaction system was prepared and Gel Shift purity identified according to the method of NIH primer Core Facility. From the Gel Shift electropherogram (FIG. 6), the biotinylation of the E6-pMHC complex was successfully prepared.

Example 4 affinity assay

Biacore is an instrument for detecting affinity based on Surface Plasmon Resonance (SPR) technology. In this experiment, using Biacore T200, biotinylated pMHC was first coupled to a CM5 chip and then measured for binding dissociation constants to different TCRs, and K was calculated_DThe value is obtained. The E63 TCR was tested accordingly for affinity to pMHC (HLA-base:Sub>A × 02/TIHDIILECV) as detailed in table 3 and fig. 7.

TABLE 3 E6 TCR affinity K_DValue of

It should be noted that: as known by persons in the art, the SPR technology is one of the most common and reliable methods for measuring affinity at present, but protein quantification, chip freshness, instrument state and the like are involved, experiments in different batches have certain errors, and the error value can even reach 3-5 times; the present invention is a batch experiment performed by using the same protein quantification, the same chip and the same instrument, so that the data can be used for comparing the affinity, but the specific value does not limit the protection scope of the present invention.

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

1) TCR Lentiviral packaging

A third generation lentiviral packaging system (Invitrogen, pLenti6/V5 directive TOPO) was used^TMCloning Kit, product number K495510) packaging lentiviruses containing the gene encoding the TCR of interest. Packaging plasmids pMDLg/pRRE (adddge, product number k 12251), pRSV-REV (adddge, product number 12253), pMD2.G (adddge, product number 12259) were respectively mixed with shuttle plasmids containing a target gene such as pLenti-E63, pLenti-E6 con (the TCR gene sequence is derived from patent number US9822162B 2), pLenti-GFP (negative control) and the like in a mass ratio of 4. The lentivirus-containing culture supernatant was collected 48-50h after transfection and after centrifugation and removal of cell debris by 0.45 μm filter, the supernatant was concentrated using Amicon Ultra-15 centrifugal filter (Merck Millipore, product No. UFC 905096) equipped with an Ultracel-50 filter. Lentivirus titer determination was performed on the concentrated samples, according to the protocol of the p24 ELISA (Clontech, product number 632200) kit.

2) TCR Lentiviral transduction of CD8⁺T cells

Isolation of CD8 from PBMC of healthy volunteers⁺T cells, seeded in 48-well plates at 1X 10 per well in RPMI 1640 complete medium containing 10% FBS and 100IU/mL IL-2⁶Adding anti-CD 3/CD28 antibody coupled magnetic beads for stimulating and activating CD8⁺T cells are cultured in a cell culture box overnight. After overnight stimulation, E63, E6con or GFP lentivirus was added at MOI =5 and centrifuged at 32 ℃ at 900g for 1h. After infection, the lentivirus infection solution was removed, cells were cultured for 3 days, and the anti-CD 3/CD28 antibody coupled magnetic beads were removed with a magnet. Thereafter, the cells were counted every two days, and the cell density was maintained at 1-2X 10 by replacing or adding fresh complete medium⁶cells/mL. On day 9 of cell culture, the cells were cultured by HLA-A x 02_29-38And carrying out double staining on the tetramer and the anti-CD 8 antibody, and carrying out flow detection and positive rate analysis on the T cells. As shown in FIG. 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 E63 TCR in vitro functional specificity assay-detection of Supported polypeptide T by the ELISPOT method₂INF-gamma release from cells

This example analyzes the T of E63 TCR in the load of specific or non-specific polypeptides by the ELISPOT assay₂Release of INF-gamma factor upon cell stimulation. The effector cells of this example were CD8 transduced with E63, E6con and GFP lentiviruses of example 5⁺T cells. The target cells of this example were T loaded with different concentrations of the polypeptide₂Cells of the general formula₂The cells were subjected to 7 gradient concentrations (10)^-11、10^-10、10^-9、10^-8、10^-7、10^-6、10^-5M)HPV16-E6_29-38Polypeptide or 10^-6NY-ESO-1 of M_157-165The polypeptide was mixed well, incubated at 37 ℃ for 4h, centrifuged, washed 1 times with 1 XPBS, and the cells were resuspended in 10% FBS-containing RPMI 1640 medium for further plating. The subsequent experimental procedures were performed according to the Human INF-gamma ELISPOT Set kit instructions (BD biosciences, product number 551849). Will be 4X 10³Double positive effector cells/well and4×10⁴target cells/well were added to ELISPOT well plates at 200 μ L per well culture system and the plates were placed in a cell incubator for overnight incubation. And after the incubation is finished, washing according to the kit instruction, then adding a BCIP/NBT solution for development for 5-15min, washing the pore plate by using deionized water, and finally reversely buckling the pore plate to naturally dry the plate at room temperature. Using an enzyme-linked immunospot analyzer (

6000Pro-F β, bio-Sys) were performed on the well plates.

The results are shown in FIG. 9A, loaded with HPV16-E6_29-38T of peptide fragment₂The cells strongly stimulated the E63 TCR-T cells to release INF-gamma and was polypeptide concentration dependent with a trend consistent with the positive control E6 con. When the loading concentration is more than 10^-7INF- γ release by M, E63 and E6con TCR-T cells peaked and there was no significant difference between the two. Specific recognition of NY-ESO-1_157-165The 1G4 TCR has significant activity, while the E63 and E6con TCR-T cells are loaded with nonspecific NY-ESO-1_157-165T of₂None of the cells had significant activity (fig. 9B). T loaded with E6 or NY-ESO-1 peptide fragment₂None of the cells stimulated GFP TCR-T cells to release INF-gamma factor. Taken together, the E63 TCR function was close to that of the positive control E6con and for HLA-A02_29-38Has specificity.

EXAMPLE 7 construction of E6 transgenic A375 monoclonal cells

To construct HLA-base:Sub>A 02⁺/HPV16-E6⁺Double positive target cell lines for E63 TCR functional validation, the E6 whole-peptide fragment gene was ligated to the corresponding shuttle plasmid by the method of example 5, and lentivirus packaging was performed, followed by transduction tobase:Sub>A 375 tumor cells (HLA-base:Sub>A × 01⁺/HPV16-E6^-) Thus, a375 polyclonal cell having the E6 gene integrated into the chromosome was obtained. In order to obtain A375 monoclonal cells stably expressing the E6 gene, A375+ E6 polyclonal cells were isolated and cultured in a 96-well plate containing 0.5 to 1 candidate cell per well by limiting dilution. And after the candidate cells are amplified, determining the copy number of the E6 gene in the cells by adopting a fluorescent quantitative PCR method. As shown in table 4The copy number of E6 gene of A375+ E6 monoclonal cell numbered 1B3 was 19.23, and the cell was used for in vitro functional verification of E63 TCR and animal experiments.

TABLE 4A 375C E6-1B3 cell E6 gene copy number detection results

HPV16-E6 full-length sequence:

MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFR DLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQ KPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL(SEQ ID NO:26)

in the above sequence: the underlined marker sequence is HPV16-E6_29-38A peptide fragment.

Example 8 in vitro functional validation of E63 TCR-detection of INF-Gamma Release from tumor cell lines by the ELISPOT method

This example analyzes the release of INF-gamma factor from E63 TCR upon stimulation with different tumor cell lines by the ELISPOT assay. The effector cells of this example were CD8 transduced with E63, E6con and GFP lentiviruses of example 5⁺T cells. The tumor target cells of this example were A375, A375+ E6-1B3, caSki, C33A, siHa and Hela cells, respectively. 4X 10 are successively introduced as described in example 6³Positive effector cells/well and 4X 10⁴Each tumor target cell/well was added to an ELISPOT well plate at 200 μ L per well culture system, and the well plate was placed in a cell incubator for overnight incubation. After the incubation, the ELISPOT plates were washed and developed according to the method of example 6, and finally analyzed with an elisa spot analyzer.

The results are shown in FIG. 10, where E63 and E6con TCR-T cells exhibited strong stimulatory activity on the double positive tumor target cell A375+ E6-1B3, and the ability of both to release INF-gamma factor was comparable. None of the other tumor cells stimulated the release of INF-gamma from E63 and E6con TCR-T cells. GFP TCR-T cells were not significantly active against all tumor target cells.

Example 9 in vitro functional validation of E63 TCR-LDH-specific killing of tumor cell lines

The function of killing target cells by effector cells was assessed by quantitative determination of LDH released after lysis of target cells, and the specific experimental protocol reference eur.j immunol.1993,23,3217. The effector cells of this example were CD8 transduced with E63, E6con and GFP lentiviruses of example 5⁺T cells. The tumor target cells of this example were A375 and A375+ E6-1B3 cells. Sequentially mixing 3 × 10⁴Double positive effector cells/well and 1X 10⁴Each tumor target cell/well was placed in a 96-well round bottom plate, 200. Mu.L per well of the culture system, the cells were replaced with RPMI 1640 medium containing only 5% FBS at the time of plating, and the well plate was placed in a cell incubator and cultured for 24 hours. According to CytoTox

Non-Radioactive cytoxicity Assay kit (Promega, product number G1780), adding lysate into blank holes of culture medium and maximum self-releasing holes of tumor target cells, and incubating in a cell culture box for 45min. After incubation, 50. Mu.L of supernatant was mixed with 50. Mu.L of LDH detection solution per well, incubated at room temperature in the dark for 30min, and then stop buffer was added and the plate was read at 490 nm. And (3) processing and analyzing the data according to a calculation formula: percentage of cytotoxicity, i.e.,% LDH release (= (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 background light absorption value of the culture medium is subtracted from the LDH release amount value of each group.

The results are shown in FIG. 11, where E63 and E6con TCR-T cells exhibited significant killing on the double positive tumor target cell A375+ E6-B3 with similar cytotoxicity percentages, 35.56. + -. 5.86% and 34.93. + -. 4.72%, respectively. E63 and E6con TCR-T cells did not significantly kill A375, similar to the negative control GFP TCR-T cells.

Example 10 in vitro functional validation of E63 TCR-detection of Positive Rate of E63, E6con TCR-T cells Using amino acid sequences of the human and murine C region

E63 The α and β chains of the TCR employed the amino acid sequence of the human C region, while the α and β chains of the positive control E6con TCR employed the amino acid sequence of the murine C region. To compare the effect of the amino acid sequences of the C regions from different sources on the expression of E63 TCR in T cells, the C regions of E63 and E6con TCRs were interchanged, resulting in 4 different TCR combinations. These four TCRs were transduced to CD8, respectively⁺T cells, using HLA-A02_29-38And carrying out double staining on the tetramer and the anti-CD 8 antibody, and carrying out flow detection and positive rate analysis on the T cells.

As shown in FIG. 12, the positive rate of E63 was 70.5% and the positive rate of E6con was 27.1% lower than that of E63 by 43.4% when the sequence was the human C region sequence. When the C region is replaced by a murine amino acid sequence, the positive rate of E63 is 85.7 percent, which is increased by 15.2 percent; the positive rate of E6con which is also the murine C region is 78.1 percent, which is 51 percent higher than that of E6con which is the human C region. Therefore, the human or mouse C region has small influence on the expression rate of the E63 TCR in the T cell, which indicates that the alpha chain and the beta chain of the E63 TCR have better pairing efficiency.

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

e63 Beta chain sequence of TCR (murine C region):

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

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

in the above sequence: the black marker sequence is the signal peptide and either V α (variable region of α chain) or V β (variable region of β chain), the gray marker sequence is C α (constant region of α chain) or C β (constant region of β chain), and the italicized underlined marker sequence is the transmembrane intracellular region.

Example 11 in vitro functional validation of E63 TCR-healthy human PBMC specific INF-Gamma Release

To investigate the safety of the E63 TCR, PBMC of 22 healthy persons were investigated for safety using the ELISPOT method of example 6. 22 healthy human PBMCs (fig. 13A) contained 4 HLA-base:Sub>A 02, 18 non-HLA-base:Sub>A 02. As shown in FIG. 13B, INF- γ release response showed no significant response of E63 TCR-T cells with PBMCs of healthy humans.

Example 12 Experimental validation of E63 TCR animals-E6 transgenic A375 melanoma allografting

The experimental animal germ line of this example was a B-NDG mouse (Chinesota gene Biotechnology Co., ltd., SPF grade), female, 4 to 6 weeks old. During the test, the experimental animals are arranged in the center of SPF animals, and all technical indexes meet the technical requirements of GB14925-2010 barrier environment. The animals in the study can eat the feed with the quality meeting the national standard and within the validity period, and drinking water of the feed is subjected to filtration sterilization treatment or high-temperature high-pressure sterilization treatment by an animal drinking pure water system and is freely taken by an animal drinking bottle. The experimental animals are adaptively raised for 1 week and then subjected to subsequent experiments.

Selecting B-NDG mice qualified for adaptive observation, inoculating A375+ E6-1B3 tumor cells subcutaneously, and adjusting the cell density to 1-2 × 10⁷one/mL, 0.2mL per inoculation. Observations were made daily after inoculation at an average tumor volume of 100mm³The animals were divided into 3 groups, i.e., model Control group, E63 TCR-T group and GFP TCR-T group, each of which had 8 animals. E63 TCR-T group at dose 4X 10⁸One positive T cell/kg was administered, the GFP TCR-T group was given the same dose of T cells, and the model control group animals were given an equal volume of vehicle. Administration to each group of mice was tail vein injection. After administration, IL-2,5 ten thousand IU/mouse was intraperitoneally injected for 5 days. When groupingTumor diameter was measured every 3 days, and thereafter, the major diameter (a) and the minor diameter (b) of the tumor were measured with a vernier caliper according to the formula 1/2 × a × b²Tumor Volume (Tumor Volume) was calculated and Tumor growth curves were plotted. Animals were euthanized 26 days after dosing, and tumor bodies were photographed.

The tumor growth curves and tumor bodies of the mice in each group are shown in FIG. 14 and FIG. 15, and the E63 TCR-T cells can effectively kill tumor cells A375+ E6-1B3 and inhibit the growth of tumors. The tumor growth curve of the GFP TCR-T group was not significantly changed from that of the Model Control group.

SEQUENCE LISTING

<110> Shenzhen Puruijin biopharmaceutical industry Co., ltd

<120 >base:Sub>A TCR recognizing HLA-A02

<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 TCR alpha 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 beta 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> constant region sequence of TCR alpha chain (murine)

<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> constant region sequence of TCR beta chain (murine)

<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

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.