CN113754756A

CN113754756A - An antibody recognizing HLA-A02: 01/E629-38TCR and uses thereof

Info

Publication number: CN113754756A
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 Pregene Biopharma Co ltd
Current assignee: Liyang Masai Bio Pharmaceutical Co ltd
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2021-12-07
Anticipated expiration: 2041-09-28
Also published as: CN113754756B

Abstract

The invention discloses a method for identifying HLA-A02: 01/E6_29‑38And uses thereof. The CDR1, CDR2 and CDR3 of the alpha chain variable region of the TCR comprise the amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, or comprise the amino acid sequences shown as SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively, or comprise the amino acid sequences shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 15, respectively. The inventive TCRs and pMHC (HLA-A02: 01/TIHDIL)ECV) high affinity, K_DThe value can reach 4.2E-06M.

Description

An antibody recognizing HLA-A02: 01/E629-38TCR and uses thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for identifying HLA-A02: 01/E6_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 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), with a persistent increase in the mortality of head and neck cancers associated with HPV infection over the past decade (CA Cancer J Clin,2021,71, 7). There are several dozen subtypes of HPV virus (Virology,2015,476,341), and the above tumors are mainly associated with HPV16 infection (J Natl Cancer Inst,2015,107, djv 086). The E6 protein encoded by HPV16, one of the major proteins in the viral life cycle, can induce tumorigenesis and progression through multiple mechanisms of action, such as inhibition of oncosuppressors p53 and pRb, suppression of apoptosis (Cancer Sci,2007,98, 1505); enhancing telomerase activity immortalizes host cells (Virus Res,2017,231, 50); inducing 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 so as to cause the occurrence, 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 treatment effectiveness and safety can be improved in immunotherapy targeting HPV16-E6 (Papilomoavrius Res,2018,5,46), and the HPV-related Cancer antigen is an ideal target for HPV-related Cancer treatment.

T cell receptor genetically engineered T cell (TCR-T) therapy is a therapy that transduces a TCR gene specific for a tumor antigen into normal T Cells, can enhance or re-confer the ability of the T Cells to recognize the tumor antigen, specifically targets and kills the tumor Cells (Science,2016,352,1337; Cells,2020,9), and is an important therapeutic approach in current adoptive cell therapy (Immunol Rev,2014,257, 56). Relevant clinical trials currently conducted 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 aiming at HPV16-E6 targets is carried out clinically (NCT02280811, NCT03197025 and NCT03578406), the effectiveness and safety of the TCR-T cell therapy are preliminarily verified (Clin Cancer Res,2015,21, 4431; J Clin Oncol,2017,35, 3009; J Clin Oncol,2019,37,2759), and a new approach is provided for treating 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 provides a method for effectively identifying HLA-A02: 01/E6_29-38TCR of target point, T cell modified by the TCR for efficiently recognizing HLA-A02: 01/E6_29-38The target spot specifically kills the tumor cells.

One of the technical schemes of the invention is as follows: a TCR whose α chain variable region CDR1, CDR2 and CDR3 comprise the amino acid sequences shown as

SEQ ID NOs

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

Preferably, the CDR1, CDR2 and CDR3 of the beta chain variable region of said TCR comprise the amino acid sequences shown as 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, the CDR1, CDR2 and CDR3 of the alpha chain variable region comprise the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively, and the CDR1, CDR2 and CDR3 of the beta 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 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 comprises 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 TRAJ 49;

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 TRBV1, the TRBJ is preferably TRBJ1-6, TRBJ1-4 or TRBJ2-7, and the TRBC is preferably TRBC1 or TRBC 2.

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 an amino acid sequence as 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 of the present invention may further comprise 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 CD8⁺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 in 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 to pMHC (HLA-A02: 01/TIHDIILECV), K_DThe value can reach 4.2E-06M. For HLA-A02: 01⁺/HPV16-E6⁺The target cells (A375+ E6-1B3) have specific killing effect, and the killing effect is enhanced along with the increase of the effective target ratio; and to the other twoThe non-double positive target cells have no obvious killing effect. In addition, GFP transduced T cells had no apparent killing effect on A375+ E6-1B 3.

Drawings

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

FIGS. 2A and 2B are the anion exchange chromatography and SDS-PAGE electrophoresis of E63TCR renaturation.

FIGS. 3A and 3B are gel filtration chromatography and SDS-PAGE electrophoresis of E63TCR renaturation.

FIGS. 4A and 4B are anion exchange chromatography and SDS-PAGE electrophoresis after HLA-A02: 01/beta 2M/TIHDIILECV renaturation; wherein the band with a large molecular weight is HLA-A02: 01, the band with a small molecular weight is beta 2M, the molecular weight of TIHDIILECV polypeptide is too small, and the band cannot be seen on SDS-PAGE.

FIGS. 5A and 5B are gel filtration chromatography and SDS-PAGE electrophoresis after HLA-A02: 01/. beta.2M/TIHDIILECV renaturation.

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

FIG. 7 is a graph of the results of the E63TCR affinity assay.

FIG. 8 shows E63TCR lentivirus infection CD8⁺A positive rate result graph of T cells; wherein E6con is a positive TCR control group, and GFP is a negative control group.

FIGS. 9A and 9B show E63TCR vs load E6_29-38Or NY-ESO-1_157-165T of a polypeptide₂INF- γ release profile of cells.

FIG. 10E63 INF- γ release profile of TCR versus tumor cell line.

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 TCR pairing ratio of E63.

FIGS. 13A and 13B are INF- γ release profiles of E63TCR on 22 different HLA-A type healthy human PBMCs.

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

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, 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⁺Methods, reagents and consumables adopted by T cell TCR gene cloning are mainly referred to curr.Protoc.Immunol.2002,7, 1; PLoS one.2011,6, e 27930; onco immunology.2016,5, e 1175795; j Vis exp.2011,8,3321; j Immunol methods.2006,310, 40; PLoS one.2014,9, e110741 and citations thereof. CD8 isolated from PBMC of healthy volunteers of HLA-A02: 01 genotype by immunomagnetic negative screening⁺After T cells, loaded with HPV16-E6_29-38Peptide-stretch EBV-B cell stimulation of CD8⁺T cells, then HLA-A02: 01/HPV16-E6 labeled with PE_29-38T cells are double-stained by tetramer and APC labeled anti-CD 8 antibody, 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 proliferated monoclonal T cells were passed through HLA-A02: 01/HPV16-E6_29-38Carrying out flow detection by double staining of tetramer and anti-CD 8 antibody and sorting 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 reverse-transcribed with the SMARTER RACE cDNA Amplification Kit (Clontech, product No. 634923) to obtain cDNA. Then using cDNA as template, PCR amplifying target gene, connecting to pUC19 vector, heat shock method converting E.coli-DH5 alpha, plate-coating and culturing overnight, picking single clone colony for identification and sequencing, sequencing to obtainThe obtained gene sequences are compared and analyzed in an IMGT database. Obtaining 3 HLA-A02: 01/HPV16-E6_29-38Antigen-specific TCRs, designated E63, E65, and E67 TCRs, respectively, have TCR α and β chain genotypes as shown in table 1.

TABLE 1 genotypes of TCR alpha and beta chains

Full-length sequence of α chain of E63 TCR:

full-length sequence of β chain of E63 TCR:

alpha chain of E65:

beta chain of E65:

alpha chain of E67:

beta chain 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 TCR V region of E6 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 E63TCR Gene

The alpha chain and beta chain genes of E63TCR are respectively connected to pET28a vector by using Nco I/Not I enzyme cutting sites, E.coli-BL21(DE3) is transformed by a heat shock method, after plating and culturing overnight, a monoclonal colony is picked up to be put into LB culture medium, and is subjected to shaking culture at 37 ℃ until OD is achieved₆₀₀When the concentration is 0.6-0.8, IPTG (1 mM) is added to induce the expression of the target protein, and after further culturing at 37 ℃ for 3 hours, the cells are centrifuged at 6000rpm for 10min to collect the cells.

The cells were resuspended in a lysis solution (1 XPBS containing 0.5% Triton X-100), sonicated, and centrifuged at 12000rpm for 20 min. 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 E63TCR alpha chain inclusion bodies and 15mg beta chain inclusion bodies were each diluted in 5mL of 6M guanidine hydrochloride solution, and then TCR alpha chain and TCR beta chain were slowly added to the pre-cooled renaturation buffer in sequence (Science 1996,274,209; J.mol.biol.1999,285, 1831; Protein Eng.2003,16,707), and stirring was continued at 4 ℃ for 30 min. Adding the solution into dialysis bag, adding into 10 times volume of precooled deionized water, stirring, dialyzing for 8-12h, adding into precooled dialysate (pH 8.1,20mM Tris-HCl), dialyzing for 8-12h at 4 deg.C, 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 (5mL) 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 segmented manner, concentrating, sampling, and performing non-reducing SDS-PAGE (figure 2B), wherein the result shows that the purity of the target protein does not meet the requirement and further purification is needed. Performing gel filtration chromatography (FIG. 3A) on the concentrated protein sample by using superdex 7510/300, and performing non-reducing and reducing SDS-PAGE (FIG. 3B) electrophoresis detection on the sample, wherein the result shows that a band is formed in a non-reducing electrophoresis lane except 45kDa and no other obvious bands are formed; the reduced electrophoresis lane has two bands, namely an alpha chain and a beta chain of the E63TCR, and the purity of the two bands meets the requirements of subsequent experiments.

Example 3 preparation of biotinylated antigenic peptide-MHC (pMHC)

Reconstitution and purification of pMHC was carried out according to the NIH Tetramer Core Facility procedure. HPV16-E6 as described in on-line protocols_29-38The polypeptide solution, beta 2M and HLA-A02: 01 inclusion body solution were sequentially added with renaturation buffer (0.1M Tris-HCl,0.4M L-argine, 2mM EDTA,0.5mM oxidative glutathione and 5mM reductive glutathione, 0.2mM PMSF), stirred overnight at 4 ℃ the next morning and night, then the same amount of HLA-A02: 01 inclusion body solution was added thereto, stirred at 4 ℃ for 1-3 days, and then dialyzed 3 times against 10-fold volume of dialysate (pH 8.1,20mM Tris-HCl). The dialyzed protein sample was subjected to anion exchange chromatography using HiTrap Q HP (5mL), eluted linearly with an eluent (0-2M NaCl,20mM Tris pH 8.1), the eluted peaks were collected and pooled (FIG. 4A), and analyzed by reducing SDS-PAGE to clearly show two bands, HLA-A02: 01 and. beta.2M (FIG. 4B), and 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 7510/300) (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 verified by reaction with Streptavidin (SA), prepared as described in NIH Tetramer Core Facility and identified by Gel Shift purity. From the Gel Shift electropherogram (FIG. 6), 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 E63TCR was thus tested for affinity for pMHC (HLA-A02: 01/TIHDIILECV) as detailed in Table 3 and FIG. 7.

TABLE 3E 6 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 TOP) was usedO^TMCloning Kit, product No. K495510) packaging lentiviruses containing a gene encoding a TCR of interest. Packaging plasmids pMDLg/pRRE (adddge, product number k12251), pRSV-REV (adddge, product number 12253) and pMD2.G (adddge, product number 12259) are respectively mixed with shuttle plasmids containing target genes such as pLenti-E63, pLenti-E6con (the TCR gene sequence is from the patent number US9822162B2), pLenti-GFP (negative control) and the like according to the mass ratio of 4:2:1:1, and 293T cells in a logarithmic growth phase are transiently transfected by a transfection reagent PEI-MAX (Polyscience, product number 23966-1). The lentivirus-containing culture supernatant was collected 48-50 h 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. UFC905096) equipped with an Ultracel-50 filter. Lentivirus titer determination was performed on the concentrated samples, with reference to the p24 ELISA (Clontech, product No. 632200) kit instructions.

2) TCR Lentiviral transduction of CD8⁺T cells

CD8 isolated 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 placed in a cell culture box for overnight culture. After overnight stimulation, E63, E6con or GFP lentivirus was added at an MOI of 5 and centrifuged at 900g for 1h at 32 ℃. 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, by HLA-A02: 01/HPV16-E6_29-38And carrying out double staining on the tetramer and an 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 6E 63TCR in vitro functional specificity assay-ELISPOT assay for detecting Supported polypeptide T₂INF-gamma release from cells

This example analyzes T of E63TCR in the load of specific or non-specific polypeptides by 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 are 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 in a37 ℃ incubator for 4h, centrifuged, washed 1 times with 1 XPBS, and the cells were resuspended in RPMI 1640 medium containing 10% FBS for further plating. The subsequent experimental procedures were performed according to the Human INF-gamma ELISPOT Set kit instructions (BD biosciences, product No. 551849). Will be 4X 10³Double positive effector cells/well and 4X 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 5-15 min, 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 had significant activity, whereas the E63 and E6con TCR-T cells were loaded with non-specific NY-ESO-1_157-165T of₂None of the cells had significant activity (fig. 9B). T loaded with E6 or NY-ESO-1 peptide fragment₂The cells can not stimulateGFP TCR-T cells release INF-gamma factor. Taken together, the E63TCR function was close to that of the positive control E6con and was specific for HLA-A02: 01/HPV16-E6_29-38Has specificity.

EXAMPLE 7 construction of E6 transgenic A375 monoclonal cells

To construct HLA-A02: 01⁺/HPV16-E6⁺Double positive target cell line for E63TCR function verification, E6 full-peptide fragment gene was ligated to the corresponding shuttle plasmid by the method of example 5, and lentivirus packaging was performed, followed by transduction to A375 tumor cells (HLA-A02: 01)⁺/HPV16-E6^-) A375 polyclonal cell having the E6 gene integrated into the chromosome was obtained. To obtain A375 monoclonal cells stably expressing E6 gene, A375+ E6 polyclonal cells were isolated and cultured in 96-well plates containing 0.5-1 candidate cells 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 4, the copy number of the E6 gene of the a375+ E6 monoclonal cell numbered 1B3, which will be used for in vitro functional validation of E63TCR and animal experiments, was 19.23.

TABLE 4A 375+ E6-1B3 cell E6 Gene copy number test results

Full-length sequence of HPV 16-E6:

MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINCQKPLCPEEKQRHLDKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL(SEQ ID NO:26)

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

Example 8E 63TCR in vitro functional validation-ELISPOT method for detecting INF-Gamma Release from tumor cell lines

This example analyzes the release of INF-gamma factor from E63TCR 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 9E 63TCR in vitro functional validation-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 target cell lysis, a 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 plated in a 96-well round bottom plate at 200. mu.L per well, and the cells were replaced with 5% FBS-only RPMI 1640 medium at plating time, and the plate was placed in a cell culture incubator and cultured for 24 hours. According to CytoTox

Non-Radioactive cytotoxin Assay kit (Promega, product number G1780), adding lysate into blank hole of culture medium and maximum self-releasing hole of tumor target cell, placing in cell culture box, incubatingAnd culturing for 45 min. 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: the percentage of cytotoxicity, i.e.,% LDH release (experimental group release-amount of tumor cells released by themselves-amount of TCR-T cells released by themselves)/(maximum amount of tumor cells released by themselves) is 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 effect on the double positive tumor target cell A375+ E6-B3, with the cytotoxicity percentages being close, 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

The α and β chains of the E63TCR 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 E63TCR in T cells, the C regions of the 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: 01/HPV16-E6_29-38And carrying out double staining on the tetramer and an 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 same as the humanized 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 E63TCR in the T cell, which indicates that the alpha chain and the beta chain of the E63TCR have better pairing efficiency.

Alpha chain sequence of E63TCR (murine C region):

beta chain sequence of E63TCR (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 11E 63TCR in vitro functional validation-healthy human PBMC specific INF-Gamma Release

To investigate the safety of the E63TCR, PBMCs of 22 healthy persons were subjected to safety investigation using the ELISPOT method of example 6. 22 healthy human PBMCs (FIG. 13A) contained 4 HLA-A02: 01 and 18 non-HLA-A02: 01 typing. As shown in FIG. 13B, INF- γ release response showed no significant response of E63 TCR-T cells with healthy human PBMC.

Example 12E 63TCR animal experiments demonstrated-E6 transgenic A375 melanoma allografting

The experimental animal germ line of the present example is a B-NDG mouse (SPF grade, jiangsu gene biotechnology limited, baiosaichu), female, 4-6 weeks old. During the experiment, the experimental animals are arranged in the center of SPF animal, 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 groups 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 each group of administration, IL-2 was intraperitoneally injected at 5 ten thousand IU/mouse for 5 days. The tumor diameter was measured on the divided day, and then the major diameter (a) and the minor diameter (b) of the tumor were measured every 3 days 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 detail in FIGS. 14 and 15, and the E63 TCR-T cells can effectively kill tumor cells A375+ E6-1B3 and inhibit the growth of tumors. The tumor growth curves of the GFP TCR-T group were not significantly changed from those of the Model Control group.

SEQUENCE LISTING

<110> Shenzhen Puruijin biopharmaceutical industry Co., Ltd

<120> a TCR for recognizing HLA-A02: 01/E629-38 and use thereof

<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

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<223> E65 alpha chain CDR1

<400> 7

Thr Ser Ile Asn Asn

1 5

<210> 8

<211> 7

<212> PRT

<213> Artificial Sequence

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<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

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<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

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<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. A TCR which comprises the amino acid sequences shown as SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 of CDR1, CDR2 and CDR3 of the α chain variable region of the TCR;

2. A TCR as claimed in claim 1 wherein the CDR1, CDR2 and CDR3 of the β chain variable region of the TCR comprise the amino acid sequences shown as SEQ ID NOs 4, 5 and 6, respectively;

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

preferably:

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;

alternatively, 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;

alternatively, 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.

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

4.A TCR as claimed in claim 3 wherein the alpha chain variable region comprises an amino acid sequence as set out in SEQ ID NO 19, SEQ ID NO 21 or SEQ ID NO 23; and/or the beta-chain variable region comprises an amino acid sequence shown as SEQ ID NO. 20, SEQ ID NO. 22 or SEQ ID NO. 24;

preferably:

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

or the alpha chain variable region contains an amino acid sequence shown as SEQ ID NO. 21, and the beta chain variable region contains an amino acid sequence shown as SEQ ID NO. 22;

or the alpha chain variable region comprises an amino acid sequence shown as SEQ ID NO. 23, and the beta chain variable region comprises an amino acid sequence shown as SEQ ID NO. 24.

5. A TCR as claimed in any of claims 1 to 4 wherein the TCR α chain and/or TCR β chain of the TCR further comprises a constant region, preferably the constant region of the TCR α chain is of human or murine origin; and/or the constant region of the TCR β chain is preferably derived from a 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 comprises the sequence shown as SEQ ID NO. 14 or SEQ ID NO. 25;

6. A TCR as claimed in claim 5 wherein the TCR α chain and/or TCR β chain of the TCR further comprises an extracellular domain and a transmembrane domain; preferably, the TCR α chain and/or TCR β chain of the TCR further comprises an intracellular sequence.

7. An isolated nucleic acid encoding a TCR as claimed in any one of claims 1 to 6.

8. A vector, preferably a lentiviral vector, comprising the nucleic acid of claim 7; 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.

9. A cell comprising the nucleic acid of claim 7 or the vector of claim 8; preferably, the cell is a T cell or a stem cell, preferably CD8⁺T cells.

10. An isolated or non-naturally occurring cell, preferably a T cell, presenting a TCR as claimed in any one of claims 1 to 6.

11. A pharmaceutical composition comprising a TCR as claimed in any one of claims 1 to 6 or a cell as claimed in claim 9 or 10; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

12. Use of a TCR according to any one of claims 1 to 6, a cell according to claim 9 or 10 or a pharmaceutical composition according to claim 11 in the manufacture of a medicament for the prevention or treatment of a tumour associated with HPV16 expression; preferably, the tumor comprises cervical cancer, oropharyngeal cancer, vaginal cancer, anal cancer, and penile cancer.