CN114106144B

CN114106144B - TCR for identifying HLA-A 02/WT1 target and application thereof

Info

Publication number: CN114106144B
Application number: CN202010875385.1A
Authority: CN
Inventors: 吴显辉; 陈亮; 陈安安; 包朝乐萌; 卢菲; 栗红建
Original assignee: Liyang Masai Bio Pharmaceutical Co ltd
Current assignee: Liyang Masai Bio Pharmaceutical Co ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2024-01-26
Anticipated expiration: 2040-08-27
Also published as: CN114106144A

Abstract

The invention discloses a TCR capable of specifically recognizing HLA-A 02:01/WT1 targets and application thereof. The CDR1, CDR2 and CDR3 of the alpha chain variable region of the TCR respectively comprise sequences shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 or respectively comprise variants of the sequences shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; the variant has 4, 3 or less than 2 mutations compared to the pre-mutated sequence, and the variant retains at least the function of the pre-mutated sequence. The TCR of the invention has high affinity with HLA-A 02:01/RMFPNAPYL target, K _D The (M) value may even be up to 1.7. Mu.M. And these mutant transduced T cells activate specifically to target cells.

Description

TCR for identifying HLA-A 02/WT1 target and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a TCR for identifying HLA-A 02/WT1 targets and application thereof.

Background

The WT1 gene was originally recognized as a childhood kidney tumor (Wilms' tumor) and was named WT1 (Cell, 1990,60,509). It is located on human chromosome 11p13 and is a transcription factor comprising a zinc finger domain, capable of binding to GC-rich sequences, and is a transcriptional activator or receptor for many growth factor genes (J Med Invest,1999,46,130). The study of the relation between WT1 gene and tumor was first focused on acute Leukemia (Leukemia, 1992,6,405), and as the study was advanced, it was found that over-expression was observed in a series of solid tumors such as breast cancer, lung cancer, colorectal cancer, brain cancer, head and neck cancer, thyroid cancer, and the like, as well as in most hematological tumors (Exp Cell Res,2001,264,74;Jpn J Clin Oncol,2010,40,377). Later scholars also found that the WT1 gene exhibited more oncogenic functions in terms of function in tumors, and therefore the WT1 product became an attractive tumor associated antigen (Int J hemalo, 2001,73,177).

Given the broad expression of WT1 in tumor cells and limited expression in normal tissues, WT1 is a potential target for immunotherapy. Several studies have reported that WT1 has good immunogenicity and is capable of inducing the production of WT 1-specific Th cells in patients, releasing antibodies. Data also demonstrated that patients with MDS or AML were able to mount a cellular immune response against WT1 (Expert Rev Vaccines,2005,4,503). Several WT 1-associated immunogenic epitopes were reported successively with intensive studies targeting WT1, including epitopes of both HLA I and HLA II (Immunogenetics, 2000,51,99; blood,2000,95, 2198). With the discovery of the immunogenic epitope of WT1, immunotherapy targeting WT1 was also performed sequentially.

Recently, more and more experiments have demonstrated that adoptive T cell therapy is effective in clearing cells in vivo (Nat Immunol,2001,2,957), especially after the first clinical trial on melanoma by Rosenberg team demonstrated the feasibility of TCR-T (Science, 2006,314,126), by 2019, there have been more than 84 TCR-T immunotherapy clinical data registration records on the clinicaltris. Gov website, suggesting that TCR-T has great potential in tumor immunotherapy (Technol Cancer Res Treat,2019,18,1533033819831068). The safety of WT1 TCR-T and potential clinical efficacy in some tumors was demonstrated in phase I clinical trials conducted by Tawara (blood.2017, 130, 1985) and Greenberg team, with a particularly pronounced clinical efficacy after bone marrow transplantation in combination (Sci trans l med.2013,5,174ra27;Nat Med.2019,25,1064).

In summary, the TCR-T immune cell therapy method has obvious curative effect On diseases such as AML and the like aiming at Tumor related targets of WT1, in particular to HLA-A 02:01/RMFPNAPYL targets, and no toxic or side effect of On Target/Off-Tumor is observed. However, none of the TCRs used in these clinical studies, trials, has evidence of protein engineering and affinity optimization. Therefore, the prior art scheme has the problems of unstable expression and non-optimized affinity.

Disclosure of Invention

The invention aims to overcome the defect of lack of effective TCR in the prior art and provide a TCR for identifying HLA-A 02/WT1 targets and application thereof. The TCR of the invention has extremely high affinity and K _D The value can even reach 1.7 mu M.

One of the technical schemes of the invention is as follows: a TCR comprising CDR1, CDR2 and CDR3 of the α chain variable region of the TCR comprising the sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively, or comprising variants of the sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively;

the variant has 4, 3 or less than 2 mutations compared to the pre-mutated sequence, and the variant retains at least the function of the pre-mutated sequence.

Preferably, the sequence shown in SEQ ID NO.3 is obtained after substitution of one or more of positions 1,2, 4, 6 and 7 of the sequence shown in SEQ ID NO. 3.

More preferably, the 2 nd and 4 th positions of the sequence shown in SEQ ID NO.3 are replaced; even more preferably, bit 1 or bit 6 is replaced; most preferably, bit 7 is also replaced.

Wherein the 1-position is preferably replaced by asparagine, alanine, tryptophan or valine; preferably, the 2 nd position is replaced by proline, alanine, glutamic acid, phenylalanine, valine or threonine; the 4 th position is preferably replaced by valine or aspartic acid; the 6 th position is preferably replaced by methionine, arginine or glutamine; the 7 th position is preferably replaced by isoleucine;

in a specific embodiment of the invention, the amino acid sequence of the variant of the sequence shown as SEQ ID NO.3 is shown as any one of SEQ ID NO.4 to 18 in the sequence Listing.

In a specific embodiment of the invention, the amino acid sequence of the variant of the sequence shown as SEQ ID NO.2 is shown as SEQ ID NO.45 in the sequence Listing.

CDR1, CDR2 and CDR3 of the β chain variable region of the TCR of the present invention preferably comprise the sequences shown as SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21, respectively, or comprise variants of the sequences shown as SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21, respectively; the variant has less than 2 mutations compared to the pre-mutated sequence, and the variant retains at least the function of the pre-mutated sequence.

Preferably, the α and/or β chain of the TCR further comprises a framework region; the framework region of the alpha chain is preferably derived from the germline TRAV39 and the framework region of the beta chain is preferably derived from the germline TRBV7-9.

More preferably, the amino acid sequences of the FRs 1 to 4 of the alpha chain are respectively shown in SEQ ID nos. 23 to 26 or variants thereof, and/or the amino acid sequences of the FRs 1 to 4 of the beta chain are respectively shown in SEQ ID nos. 23 to 26 or variants thereof; wherein the amino acid sequence of the FR1 variant of the alpha chain is preferably shown in SEQ ID NO.38, the amino acid sequence of the FR3 variant of the alpha chain is preferably shown in SEQ ID NO.39, and the amino acid sequence of the FR4 variant of the alpha chain is preferably shown in SEQ ID NO. 40; the amino acid sequence of the FR1 variant of the beta chain is preferably shown as SEQ ID NO.41, the amino acid sequence of the FR2 variant of the beta chain is preferably shown as SEQ ID NO.42, the amino acid sequence of the FR3 variant of the beta chain is preferably shown as SEQ ID NO.43, and the amino acid sequence of the FR4 variant of the beta chain is preferably shown as SEQ ID NO. 44.

In one embodiment of the invention, the TCR is a scTCR in which the tcra variable region and the tcra variable region are linked by a linker; the amino acid sequence of the linker is preferably shown as SEQ ID NO.22 in the sequence Listing.

The TCR alpha chain and/or TCR beta chain of a TCR as described above preferably further comprises a constant region, preferably derived from germline TRAC; and/or the constant region of the TCR β chain is preferably derived from germline TRBC2.

More preferably, the TCR α chain and/or TCR β chain of the TCR further comprises an outer membrane region and a transmembrane region; even more preferably, the TCR a chain and/or TCR β chain of the TCR further comprises an intracellular sequence. The intracellular sequences may be conventional in the art.

The second technical scheme of the invention is as follows: a nucleic acid encoding a TCR as described above.

The third technical scheme of the invention is as follows: a vector comprising a nucleic acid as described above, preferably a lentiviral vector; the nucleic acid encodes the TCR alpha chain and the TCR beta chain in a single open reading frame, or in two different open reading frames, respectively.

The technical scheme of the invention is as follows: a cell comprising a nucleic acid as described above or a vector as described above; preferably, the cells are T cells or stem cells, 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 described above.

The sixth technical scheme of the invention is as follows: a pharmaceutical composition comprising a TCR as described above or a cell as described above; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The seventh technical scheme of the invention is as follows: use of a TCR as described above, a cell as described above, or a pharmaceutical composition as described above, in the manufacture of a medicament for the prevention or treatment of a tumor associated with WT1 expression; preferably, the tumor comprises breast cancer, lung cancer, carcinoma of large intestine, brain cancer, thyroid cancer and head and neck cancer.

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

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

The invention has the positive progress effects that:

the TCR of the invention has high affinity with HLA-A 02:01/RMFPNAPYL target, K _D The (M) value may even be up to 1.7. Mu.M. And these protrusionsVariant transduced T cells activate specifically to target cells.

Drawings

FIG. 1 is a sorting of double positive T cell clones.

FIG. 2 shows in vitro specificity analysis of monoclonal T cells.

FIGS. 3A and 3B are scTCR purification anion exchange chromatography and electrophoresis.

FIGS. 4A and 4B are scTCR purified molecular sieve chromatograms and electrophoreses.

FIGS. 5A and 5B are graphs of anion exchange chromatography and electrophoresis of mutant A7B 0.

FIGS. 6A and 6B are molecular sieve chromatograms and electrophoreses of mutant A7B 0.

FIGS. 7A and 7B are pMHC anion exchange chromatography and electrophoresis.

FIGS. 8A and 8B are pMHC molecular sieve chromatograms and electrophoreses.

FIG. 9 is an A15B0 affinity test.

FIG. 10 shows lentiviral infection CD8 ⁺ Infection efficiency of T cells.

FIG. 11 shows the detection of INF-gamma release by T2 cell polypeptides by WT1 specific TCR function.

FIG. 12 shows the LDH-specific killing of tumor cell lines of WT 1-specific TCRs.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

MHC molecules belong to the immunoglobulin superfamily and may be class I or class II MHC molecules. It is specific for antigen presentation and different individuals have different MHC's that present different short peptides of a protein antigen to the surface of their respective Antigen Presenting Cells (APC). Human MHC is commonly referred to as an HLA gene or HLA complex.

T Cell Receptor (TCR), the only receptor for specific antigen presented on the Major Histocompatibility Complex (MHC). In the immune system, direct physical contact of T cells with APCs is initiated by binding of antigen-specific TCR and pMHC complexes, and then interaction of T cells with other cell membrane surface molecules of both APCs occurs, which causes a series of subsequent cell signaling and other physiological responses, thereby allowing T cells of different antigen specificities to exert immune effects on their target cells.

TCRs are functional units of antigen recognition by T lymphocytes, belonging to the immunoglobulin superfamily, whose coding chains include four chains, α, β, γ and δ, and are glycoproteins on the surface of cell membranes in which the α/β or γ/δ chains exist in heterodimeric form. Peripheral blood 95% of TCRs are heterodimers composed of two polypeptide chains, α and β. Recombinant TCRs may also consist of a single TCR β chain or TCR α chain, which has been shown to be capable of binding to an antigen peptide-MHC molecule (WO 2005/113595).

In a broad sense, each of the α and β chains comprises a variable region, a linking region, and a constant region, and the β chain also typically comprises a short variable region between the variable region and the linking region, but the variable region is often considered part of the linking region. Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are chimeric in a framework structure (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, wherein CDR3 is recombined from the variable region and the linking region, known as the hypervariable region. The α and β chains of TCRs are generally regarded as having two "domains" each, i.e., a variable domain and a constant domain, the variable domain being composed of linked variable and linking regions. The sequence of the TCR constant domain can be found in published databases of the international immunogenetic information system (IMGT), for example the constant domain sequence of the α chain of a TCR molecule is TRAC (also known as TRAC x 01) and the constant domain sequence of the β chain of a TCR molecule is TRBC1 (also known as TRBC1 x 01) or TRBC2 (also known as TRBC2 x 01). In addition, the α and β chains of TCRs also contain transmembrane and cytoplasmic regions, which are short.

In addition, the alpha chain consists of a TRAV, TRAJ, TRAC rearrangement in the germ line gene; the β chain consists of TRBV, TRBD, TRBJ, TRBC rearrangements in germ line genes, and after re-shooting of different V (D) J, random insertions of different numbers of nucleotides form a variable region (N) into complementarity determining region 3 (CDR 3) upon V-J (or V-D and D-J) ligation, such random insertions rendering TCR α and β chain sequences highly diverse; the TCR rearrangement of different cloned T lymphocytes varies in CDR3 length and base sequence, which is the region of the TCR that specifically recognizes an antigen, which determines the specificity of the TCR.

In addition: in addition to the mutations explicitly stated, there are also conservative modifications or conservative substitutions or substitutions known in the art. By conservative modifications or conservative substitutions or substitutions are meant that amino acids in a protein are substituted for other amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, rigidity, etc.), such that changes can be made frequently without altering the biological activity of the protein. Those skilled in The art know that in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) molecular μ lar Biology of The Gene, the Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Common conservative substitutions of amino acids are as follows:

EXAMPLE 1 cloning specific CD8 ⁺ T cells, obtaining heterodimeric TCR sequences (acquisition and identification of wild-type TCR)

Methods, reagents and consumables for T cell cloning are mainly referenced curr.protoc.immunol.2002,7,1; PLoS One,2011,6, e27930; onco Immunology 2016,5, e1175795 and references thereto. Stimulation of HLA-A 02:01 genotype healthy volunteers' CD8 with EBV (EBV) transduced B cells (EBV-B) (J Vis exp.2011,8,3321) (EBV virus: ATCC product number VR-1492) loaded with WT1 short peptide (RMFPNAPYL) ⁺ T cells. Among them, the monoclonal T cell culture method is mainly referred to the work of the relevant literature (J Immunol methods.2006,310,40;PLoS One.2014,9,e110741). Marked with PEThe noted short peptide-HLA tetramer (MBL, product number TS-M047-1) and APC-labeled anti-CD 8 antibody (Biolegend, product number 301014) were used to sort double positive T cells. T cells stimulated with RMFPNAPYL short peptide were expanded to a number and sorted. After multiple times of stimulation culture and sorting, the monoclonal culture is carried out by adopting limiting dilution. The expanded monoclonal T cells were stained with tetramers, and the double positive cloned cells were detected by multiple sorting with Tetramer, anti-CD 8 antibody, as shown in FIG. 1. Monoclonal T cell growth to 1X 10 ⁵ Sorting more than the second time with tetra mer and anti-CD 8 antibody, adding positive cells (monoclonal T cells) into RNA extraction kit Quick-RNA ^TM The lysate in MiniPrep (ZYMO research, product number R1050) is blown evenly and then stored at-80 ℃ for standby.

In order to clone the TCR gene from the monoclonal T cells for further in vitro activity studies, mRNA from the monoclonal T cells was extracted for reverse transcription of cDNA, from which the TCR gene was cloned and lentiviral vectors were constructed. Thawing the selected monoclonal T cell lysate of example 1, and using the kit Quick-RNA according to the instructions ^TM Total RNA of T cells was extracted from MiniPrep (ZYMO research, product No. R1050). Finally, 10 mu L Tricine-EDTA buffer (SMART RACE cDNA amplification kit) is used for eluting, and RNA is stored at-80 ℃ for later use. The cDNA was synthesized using a SMART RACE cDNA amplification kit from clontech. The target gene was amplified by PCR according to the instructions and then ligated to pUC19 vector (attached to SMART RACE cDNA amplification kit), E.coli DH 5. Alpha. Plates were transformed, and the single clone was picked up for sequencing. The sequencing results were compared in the IMGT database, with TCR alpha and beta chains gemline being TRAV39 and TRBV7-9, respectively.

Full length of alpha chain of wild type TCR:

beta chain full length of wild type TCR:

according to IMGT regulations, the amino acid sequence of the CDR regions, which are the unique key sequences of the V region of wild-type TCRs, are as follows:

alpha chain CDR1-TTSDR (SEQ ID NO. 1)

Alpha chain CDR2-LLSNGAV (SEQ ID NO. 2)

Alpha chain CDR3-GGGADGLT (SEQ ID NO. 3)

Beta chain CDR1-SEHNR (SEQ ID NO. 19)

Beta chain CDR2-FQNEAQ (SEQ ID NO. 20)

Beta chain CDR3-ASSLYGGGDTQY (SEQ ID NO. 21)

Alpha chain FR1-ELKVEQNPLFLSMQEGKNYTIYCNYS (SEQ ID NO. 23)

Alpha chain FR2-LYWYRQDPGKSLESLFV (SEQ ID NO. 24)

Alpha chain FR3-KQEGRLMASLDTKARLSTLHITAAVHDLSATYFC (SEQ ID NO. 25)

Alpha chain FR4-FGKGTHLIIQP (SEQ ID NO. 26)

Beta chain FR1-DTGVSQDPRHKITKRGQNVTFRCDPI (SEQ ID NO. 27)

Beta chain FR2-LYWYRQTLGQGPEFLTY (SEQ ID NO. 28)

Beta chain FR3-SRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLC (SEQ ID NO. 29)

Beta chain FR4-FGPGTRLTVL (SEQ ID NO. 30).

The FR 1-FR 4 sequences of the alpha chain and the FR 1-FR 4 sequences of the beta chain, respectively, are bolded and underlined.

The full length sequence of the alpha chain or the beta chain is as follows: the underlined part is the signal peptide sequence, the unlabeled sequence is V.alpha (variable region of alpha chain) or V.beta (variable region of beta chain), the bolded part is C.alpha (constant region of alpha chain) or C.beta (constant region of beta chain) sequence, the italic part is extracellular linker, the italic and the underlined part is transmembrane region and intracellular sequence.

The amino acid sequences A0 and B0 (where "a" and "B" represent the alpha and beta chains, respectively, and "0" represents the unmutated) were obtained after removal of the transmembrane and intracellular sequences (italicized and underlined portions), respectively, of the alpha and beta chain lengths.

Example 2 in vitro specificity analysis of monoclonal T cells

For the purpose ofAnalysis of in vitro Activity of WT1 tetramer-positive monoclonal in Experimental example 1 monoclonal T cells were analyzed with ELISPOT assay and load 10 ^-6 Release of INF- γ after co-culture of antigen presenting cells with M concentration of specific or non-specific polypeptide. WT1 monoclonal T cells showed the strongest activity against WT1 polypeptides (FIG. 2), with no apparent activity against other HLA-A.02 binding polypeptides, such as NY-ESO-1 polypeptides or no polypeptide-containing medium.

Example 3 stability engineering of scTCR Domain for wild type TCR prepared in example 1

The variable region sequences of V.alpha.and V.beta.obtained in example 1 (full length of alpha chain and full length of beta chain) were linked by Linker amino acid sequences to obtain wild-type scTCR (hereinafter abbreviated as scTCR-wt), respectively, which was very unstable in structure and required stability optimization for affinity optimization (PNAS, 1999,96,5651;Nat Biotechnol.2000,18,754;Front.Oncol.2015,4,1;WO2016124142) and inclusion body renaturation (cloning, expression and purification procedures of scTCR-wt were performed according to conventional methods in the art).

For the beta-turn structure of scTCR-wt, mutations contributing to the stability of the beta-turn structure were introduced according to the preference of different amino acid side chains for different positions of the beta-turn. Due to the removal of the cα and cβ domains, hydrophobic amino acids at some positions in the vα and vβ domains are exposed on the surface, reducing amino acid mutations of the hydrophobic nature of the surface, resulting in a stable sctcr_x0 sequence. The scTCR-wt and scTCR-X0 genes were synthesized directly and then cloned into the pET28a vector by the Nco I/Not I endonuclease.

sctcr_x0 amino acid sequence (CDR regions in bold, lower case linker):

alpha chain variable region

Wherein the amino acid sequences of FR 1-FR 4 are respectively shown as SEQ ID NO.38, SEQ ID NO.24, SEQ ID NO.39 and SEQ ID NO. 40.

The amino acid sequence of CDR2 is shown in SEQ ID NO. 45.

Linker:GSADDAKKDAAKKDGKS(SEQ ID NO.22)

Beta chain variable region

Wherein: the amino acid sequences of FR 1-FR 4 are shown as SEQ ID NO. 41-44 respectively.

sctcr_x0 base sequence (CDR regions in bold, lower case linker):

alpha chain variable region:

Linker：

GGTAGCGCCGATGATGCAAAAAAGGATGCCGCAAAAAAGGACGGTAAAAGT(SEQ ID NO.34)

beta chain variable region:

inclusion body expression, protein renaturation, purification and affinity testing of scTCR-X0 were performed as described in example 5, example 6 and example 8, and the results are shown in fig. 3A, 3B and fig. 4A, 4B.

Example 4 phage display

Inserting the gene of the engineered RMF4 scTCR into a phage display vector by adopting an Nco I/Not I enzyme cutting site, taking the gene as a phage display library construction template, designing discontinuous mutation primers for constructing a library for a variable region CDR3 alpha/CDR 3 beta, and electrotransferring the library into TG1 competence for the subsequent 2-3 rounds of phage screening. The whole process of phage selection is mainly described in two articles, nature Protocols,2007,2,3001 and Nat. Biotech.2005,23,349, a doctor paper of the Cardiff University pharmaceutical system (Liddy S.2013, molecular engineering of high affinity T-cell receptors for bispecific therapeutics), in addition to the traditional handbook of molecular biology.

(a) Phage selection

Library bacteria were cultured to OD using 2 XYT medium containing ampicillin and glucose ₆₀₀ =0.4 to 0.5, a proper amount of M13KO7 helper phage (NEB, product number N0315S) was infected for 1h, 2 xyt medium containing ampicillin and kanamycin was changed, and packaged overnight at 26 ℃. Bacterial culture was centrifuged at high speed, the supernatant was precipitated using a PEG/NaCl solution, and after centrifugation again, the supernatant was discarded and resuspended in 1mL PBS to obtain phage solution. After blocking the phage solution with milk, the phage solution is incubated with proper amount of biotinylated pMHC and streptavidin magnetic beads to form phage-TCR-pMHC-magnetic bead complex, and the magnetic beads are washed 3-5 times with 0.1% PBST (v/v) and PBS, respectively. The phage released by pancreatin digestion was infected with TG1 in the logarithmic growth phase, plated, and incubated upside down at 30 ℃ overnight. Repeating the above procedures for the second and third rounds of screening.

(b) Phage ELISA

Referring to the workflow described in Nature Protocols,2007,2,3001, the first day, phage 200 μl was packed in 96-well deep well plates and 96-well ELISA plates (1 μg/well) were coated with streptavidin. The next day ELISA plate was incubated with 100. Mu.L of pMHC (0.5. Mu.g/well), 400. Mu.L of 3% milk PBS, 100. Mu.L of milk blocked phage, and 100. Mu. L M13-HRP antibody (product No. 11973-MM05T-H,1:10000 dilution) for 1H at room temperature, with each addition of plate solution and 3 washes of 0.1% PBST (v/v). The reaction was then stopped with TMB chromogenic solution (Sigma, product number T4444) and 1M sulfuric acid for 90 seconds, and the absorbance (OD) ₄₅₀ ). Select OD ₄₅₀ The corresponding mutant is obtained by DNA sequencing analysis of the monoclonal with the value of more than 0.20.

Example 5 Gene cloning and inclusion body expression

The mutation sites obtained in example 3 were introduced into A0 of TCR as shown in Table 1 to obtain the variable regions of amino acid sequences A1 to A16 ("A" represents an alpha chain, "B" represents a beta chain, "A" with different numbers represents an "alpha chain" containing different mutations; in addition, in the following examples, heterodimers composed of TCR alpha chain and TCR beta chain are abbreviated as AmB0; where m is an integer of 0 to 16).

The mutation sites occurring on CDR3α and CDR3β are as follows in table 1 (wherein the bolded amino acids are mutated amino acids):

TABLE 1 mutation site display

The specific expression process is as follows:

the TCR alpha-chain (A0-A16), beta-chain (B0), scTCR, HLA-A 02:01 and beta 2M genes and RMFPNAPYL are simultaneously renatured to form a pMHC complex; where HLA-A 02:01 UniProt ID was P01892 and β2M UniProt ID was P61769) were cloned into pET28a vector (from Novagen) using the Nco I/Not I cleavage site, E.coli BL21 (DE 3) (from NEB), and the single clone was picked up in LB medium and shake-cultured at 37℃to OD ₆₀₀ =0.6 to 0.8, and IPTG was then added to a final concentration of 1mm and incubation was continued for 3 hours at 37 ℃. Centrifuging at 6000rpm for 10min, collecting thallus, and storing at-20deg.C.

EXAMPLE 6 inclusion body purification, renaturation and purification

The cells were resuspended in lysate (PBS of 0.5% Triton X100), sonicated, and centrifuged at 12000rpm for 20 minutes. Discarding supernatant, re-suspending the precipitate with lysate until no macroscopic particles are present, centrifuging at high speed for 10min, repeating the above operation for 2-3 times, dissolving the precipitate with 6M guanidine hydrochloride solution, centrifuging at high speed for 10min, collecting supernatant, and freeze-preserving at-80deg.C.

20mg of TCR alpha chain and 15mg of beta chain (prepared in example 5) were each diluted in 5mL of 6M guanidine hydrochloride solution. The TCR alpha chain and TCR beta chain were slowly added in sequence to a pre-chilled renaturation buffer (Science 1996,274,209;J.Mol.Biol.1999,285,1831;Protein Eng.2003,16,707) and stirred and mixed for 30 minutes at 4 ℃. Then adding the mixture into a dialysis bag, putting the dialysis bag into 10 times of pre-cooled deionized water, and stirring and dialyzing for 8-12 hours. The dialysis is repeated for 2 to 3 times at 4 ℃ for 4 to 8 hours in precooled dialysis external liquid (mainly for maintaining the pH value, but the pH value is not fixed, and the pI value of surrounding protein can be adjusted to obtain better renaturation effect).

The solution in the dialysis bag was poured out, centrifuged at high speed for 10 minutes to remove precipitates and air bubbles, and subjected to anion exchange chromatography by HiTrap Q HP (5 mL), eluting linearly from 0 to 2M NaCl,20mM Tris pH 8.1. The elution peaks were collected and pooled to concentrate the fractions containing the target protein, and a band around 45kD was present in the non-reducing SDS-PAGE electrophoresis, but the purity was not satisfactory and further purification was required. The concentrated protein samples were subjected to molecular sieve chromatography using superdex 75/300. A high-concentration band is arranged near 45kD in non-reducing SDS-PAGE electrophoresis detection, and two bands, namely an alpha-chain and a beta-chain, are detected by adopting reducing SDS-PAGE electrophoresis, and the purity is about 90%. Specifically, as shown in fig. 5A, 5B, and 6A and 6B (purification results are shown only for A7B0 in this case, since the molecular weights of A0 to a16 are the same and the molecular weights of B1 to B4 are the same).

EXAMPLE 7 biotinylated antigen peptide-MHC (pMHC) preparation

Renaturation and purification of pMHC, prepared according to the method of NIH Tetramer Core Facility. Taking HLA-A 02:01/RMFPNAPYL as an implementation representative example, according to the online protocols, adding a polypeptide solution and beta 2M inclusion body solution of HLA-A 02:01 into renaturation buffer (0.1M Tris-HCl,0.4M L-arginine,2mM EDTA;0.5mM oxidized glutathione and 5mM reduced glutathione, 0.2mM PMSF) in sequence, stirring overnight at 4 ℃, and adding the inclusion body solution of the same amount of HLA-A 02:01 in the morning and evening respectively, and stirring for 1-3 days at 4 ℃. Then dialyzed 3 times in 10 volumes 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 0-2M NaCl,20mM Tris pH 8.1 solution, and the eluted peaks were collected and analyzed by SDS-PAGE electrophoresis, with two bands of purer HLA-A x 02:01 and β2m, but RMFPNAPYL having a too small molecular weight and no band seen in the gel. The eluted peaks containing the pMHC fractions were pooled and concentrated, further purified by gel filtration chromatography (Superdex 75/300), and then detected by SDS-PAGE electrophoresis, as seen from the electropherograms, to give a pMHC complex of better purity. Biotinylation (Protein Expr.Purif.2012,82,162;J.Bacteriol.2012,194,1113) was performed with a recombinase BirA (BPS Bioscience product, product number 70031), and specific experimental methods such as reaction system and the like were performed according to the method of NIH Tetramer Core Facility, and Gel Shift purity was identified. From Gel Shift electrophoretogram, purity meets the requirement. The results are shown in detail in FIGS. 7A, 7B and FIGS. 8A and 8B.

Example 8 affinity test

The Octet is an instrument for detecting affinity by adopting SPR technology, and according to the optical interference technology of a biological film layer based on an optical fiber biosensor, the kinetic parameters among interaction molecules are detected, the kinetics and the affinity analysis are carried out, and the binding dissociation constant is calculated. In this experiment we used SA sensor to immobilize biotinylated pMHC, detect its binding dissociation constant to different TCRs, calculate K _D Values. HLA-A.times.02:01/RMFPNAPYL affinity was tested using A15B0 as an example representative, see FIG. 9 for details. Table 2 below is a summary of one arrangement of binding affinities for the TCR mutant of RMF4 and HLA-A 02:01/RMFPNAPYL.

The description is as follows: as known by those skilled in the art, the SPR technology is one of the most commonly used and reliable methods for determining affinity at present, but involves protein quantification, chip freshness, instrument state and the like, and experiments between 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 comparison of the affinity between the data can be performed, but the specific numerical values do not limit the scope of the present invention.

TABLE 2 WT1 TCR affinity

EXAMPLE 9 lentivirus preparation and infection of CD8 ⁺ T cell

(a) WT1 TCR lentiviral packaging.

Using the third generation lentiviral packaging System (Invitrogen, pLenti6/V5 direct TOPO) ^TM Cloning Kit, product number K495510) packages lentiviruses containing the gene encoding the desired TCR. Plasmids pLenti-WT1 TRA-2A-TRB-IRES-NGFR and pLenti-eGFP were mixed with packaging plasmids pMDLg/pRRE (adedge, product number k 12251), pRSV-REV (adedge, product number 12253) and pMD2.G (adedge, product number 12259) in a 4:2:1:1 ratio, respectively, and 293T cells (purchased from ATCC, product number CRL-3216) in the logarithmic growth phase were transiently transfected, and the transfection reagent PEI-MAX (Polyscience, product number 23966-1) to plasmid (40. Mu.g/15 cm dish) in a ratio of 2:1 (volume/mass ratio), respectively, and the specific procedures were performed as described. Culture supernatants containing packaged lentiviruses were collected on days 3 and 4. To further concentrate the lentiviruses, the collected culture supernatant was centrifuged at 3000rpm for 15min and filtered through a 0.45. Mu.M filter (Merck Millipore) to remove debris. Finally, the mixture was concentrated to 1mL by a 50kD molecular weight cut-off concentration tube (Merck Millipore), and frozen at-80℃after packaging. Pseudovirus samples were taken for virus titer assays, and the procedure was referred to p24 ELISA (Clontech, product No. 632200) kit instructions.

(b) Transduction of primary CD8 with lentiviruses containing WT 1-specific T cell receptor genes ⁺ T cell

Enrichment of CD8 from blood of healthy volunteers by negative isolation ⁺ T cells (antibody-conjugated magnetic beads were purchased from Miltenyi Biotec). Cells were counted and seeded 1X 10 per well in 48-well plates ⁶ The individual cells, each well containing 0.5mL of 100IU/mL IL-2 (Peprotech, product No. AF-200-02) of RPMI1640 complete medium (10% FBS), were incubated overnight for stimulation with pre-washed anti-CD 3/CD28 antibody-coated beads (life technologies, product No. 11452D).

After overnight stimulation, concentrated lentiviruses expressing WT1 wild-type or GFP gene were added at moi=10 and centrifuged at 32 ℃ for 1 hour at 900 g. Lentiviral infection solution was removed after infection and cells resuspended in RPMI1640 complete medium containing 100IU/mL IL-2, 37 ℃/5% CO ₂ Culturing under the condition for 3 days. Thereafter, cells were counted every two days, replaced or fresh medium containing 100IU/mL IL-2 was added, and the cells were maintained at 1X 10 ⁶ -2×10 ^6- Individual cells/ml. Cell infection efficiency was analyzed by flow cytometry on day 3 of transduction (fig. 10). The results showed that WT1 TCR transduced T cells double-stained WT1 tetramer and anti-CD 8 antibody, A0B0 positive rate of 20.887%, a12B0 of 21.0%; positive control 1G4 transduced T cells (WT 1 TCR) double stained WT1 tetramer and anti-CD 8 antibody with a positive rate of 46.3% (APC-H represents CD8 antibody staining and R-PE-H represents tetramer (i.e. antigen peptide recognized TCR) staining).

Example 10 validation of WT1 specific TCR function-ELISPOT protocol detection T ₂ INF-gamma release by cellular polypeptide loading

The experimental protocol uses IFN-gamma production as an indicator of T cell activation, and detects the specific activation response of TCR-transduced T cells to target cells.

The target cells for this assay are T ₂ Cells and H226 cells, effector cells were CD8 expressing WT1 TCR as analyzed by flow cytometry in example 49 ⁺ T cells (cell name, HLA class, and WT1 antigen expression are shown in Table 3) and expressed as CD8 in the same volunteer ⁺ T cells served as negative control effector cells. In example 9, T cells transduced with anti-CD 3/CD28 coated beads and lentiviral expressing WT1 TCR gene were expanded in RPMI1640 medium containing 10% FBS of 100IU/mL IL-2 until 9-12 days post transduction, and then the cells were placed in experimental medium (10% FBS RPMI 1640) and centrifuged at 300g for 10 minutes at room temperature for washing. The cells were then resuspended in assay medium at 2X the desired final concentration. Negative control effector cells were treated in the same manner.

Table 3 cell name, HLA typing and WT1 antigen expression

Cell name	HLA typing	WT1 antigen
			NCI H226	24:02；01:01	+
NCI H226-A0201	Over-expression A02:01;24:02;01:01	+
			NCI H226-A0203	Over-expression A02:03;24:02;01:01	+
T ₂	A02:01；A02:01	-
			CD8 ⁺ T cell	A02:03；A11:01	-

PVDF ELISPOT 96-well plates (Merck Millipore, product number MSIPS 4510) were coated according to the manufacturer's instructions and incubated overnight at 4 ℃. After removing the excess capture antibody by pre-plate washing, the wells were blocked with PBS containing 10% fbs for 2 hours at room temperature and the liquid was removed.

The components of the assay were added sequentially to an ELISPOT well plate: (1) 20000 target cells (T) ₂ Cell)/well; (2) 1350 WT1 TCR CD8 ⁺ Double positive T cells, or CD8 ⁺ Negative control effector cells; (3) 20 microliters 10 ^-5 Molar/liter of TCR-related short peptide (final concentration 10 ^-6 Moles/liter). All experimental groups were in duplicate.

Incubation of the well plate overnight (37 ℃ C./5% CO) ₂ ) The next dayThe assay was performed according to the instructions of the human IFN-. Gamma.ELISPOT PVDF-enzyme kit (BD, product No. 551849), and finally developed with BCIP/NBT solution for 5-15 minutes. During this period the spots of the developed well plate were routinely examined and the optimal time for termination of the reaction was determined. The plates were rinsed with double distilled water to stop development and dried completely at room temperature.

As shown in FIG. 11, T2 cells loaded with WT1 short peptide and H226 cells expressing A0201 strongly stimulated T cells to release INF-gamma to form significantly more spots, whereas H226 cells, H226 cells expressing A0201 without WT1 short peptide, H226 cells expressing A0203 without polypeptide and H226 cells loaded with non-specific short peptide (NY-ESO-1) did not stimulate T cells to release INF-gamma to form significantly more spots.

Example 11 validation of WT 1-specific TCR function-tumor cell line LDH-specific killing

The test is 51 ^Cr Colorimetric surrogate assay to release cytotoxicity assay, quantitative determination of Lactate Dehydrogenase (LDH) released after cell lysis, protocol reference (eur. J immunol.1993,23,3217), specific detection methods were performed according to kit instructions (Promega, G1780). LDH released in the medium was detected using a 30 minute coupled enzyme reaction in which LDH converts one tetrazolium salt (INT) to red formazan (formazan). The amount of red product produced is proportional to the number of cells lysed. The 490nm absorbance data can be collected using a standard 96-well plate reader.

After the end of the Elispot assay, lentivirus infection of T cells was performed on day 13.

(1) And (2) loading polypeptide: target cell (2X 10) ⁵ Per mL) and gradient dilution of the polypeptide (final concentration 10 ^-8 ，10 ^-7 ，10 ^-6 M) mixing, incubating for 4 hours in a 37-degree incubator, centrifuging, washing away unbound polypeptide, and using the cells for the next inoculation.

(2) Inoculating cells: tumor cells 1×10 ⁴ Holes; A12B0 and A0B0 are 3×10 ⁴ Well (effective target ratio 1:3). 200. Mu.L of culture system per well, cells were replaced with RPMI1640 medium containing only 5% FBS at the time of inoculation. The cells were exposed to 37 degrees 5% CO ₂ Culturing in an incubator for 24 hours. (Note: H226-A0201-WT 1-1)E-8 indicates that H226-A0201 cells are loaded with 10 ^-8 M WT1 polypeptide, H226-A0201-1E-7 indicates that H226-A0201 cells are 10 loaded ^-7 M polypeptide, H226-A0201-1E-6 indicates that H226-A0201 cells are loaded with 10 ^-6 M polypeptide).

(3) LDH reaction color development: the assay was performed according to the instructions provided by the manufacturer of the killing assay kit (Promega, G1780) and the plate was read at 490nm light waves over 1 hour.

The calculation formula is as follows: percent cytotoxicity, i.e., percent LDH release = (experimental group release-tumor cell self-release-TCRT cell self-release)/(tumor cell maximum release-tumor cell self-release) ×100%. In this figure, the ordinate 1 is 100%. When calculating, the LDH release amount value of each group is subtracted by the background light absorption value of the culture medium.

The experimental results are collated in FIG. 12, which shows CD8 expressing the high affinity TCR mutant A12B0 ⁺ T cells have a stronger killing effect than the wild type.

SEQUENCE LISTING

<110> Shenzhen Prigin biological pharmaceutical Co., ltd

<120> TCR recognizing HLA-A 02/WT1 target and use thereof

<130> P20012985C

<160> 45

<170> PatentIn version 3.5

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain CDR1

<400> 1

Thr Thr Ser Asp Arg

1 5

<210> 2

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain CDR2

<400> 2

Leu Leu Ser Asn Gly Ala Val

1 5

<210> 3

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain CDR3

<400> 3

Gly Gly Gly Ala Asp Gly Leu Thr

1 5

<210> 4

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 4

Asn Pro Gly Val Asp Gly Leu Thr

1 5

<210> 5

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 5

Ala Ala Gly Asp Asp Gly Leu Thr

1 5

<210> 6

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 6

Gly Phe Gly Val Asp Gly Leu Thr

1 5

<210> 7

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 7

Gly Glu Gly Val Asp Gly Leu Thr

1 5

<210> 8

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 8

Ser Pro Gly Asp Asp Gly Leu Thr

1 5

<210> 9

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 9

Val Val Gly Val Asp Gly Leu Thr

1 5

<210> 10

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> AEGVDGLT

<400> 10

Ala Glu Gly Val Asp Gly Leu Thr

1 5

<210> 11

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 11

Gly Val Gly Asp Asp Gly Leu Thr

1 5

<210> 12

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 12

Gly Glu Gly Asp Asp Gly Leu Thr

1 5

<210> 13

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 13

Gly Glu Gly Val Asp Met Leu Thr

1 5

<210> 14

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 14

Gly Glu Gly Asp Asp Met Leu Thr

1 5

<210> 15

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 15

Gly Thr Gly Asp Asp Arg Leu Thr

1 5

<210> 16

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 16

Gly Thr Gly Asp Asp Gln Leu Thr

1 5

<210> 17

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 17

Gly Thr Gly Asp Asp Met Ile Thr

1 5

<210> 18

<211> 8

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR3 variants

<400> 18

Gly Glu Gly Asp Asp Gln Leu Thr

1 5

<210> 19

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain CDR1

<400> 19

Ser Glu His Asn Arg

1 5

<210> 20

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain CDR2

<400> 20

Phe Gln Asn Glu Ala Gln

1 5

<210> 21

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain CDR3

<400> 21

Ala Ser Ser Leu Tyr Gly Gly Gly Asp Thr Gln Tyr

1 5 10

<210> 22

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> joint

<400> 22

Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp Gly Lys

1 5 10 15

Ser

<210> 23

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain FR1

<400> 23

Glu Leu Lys Val Glu Gln Asn Pro Leu Phe Leu Ser Met Gln Glu Gly

1 5 10 15

Lys Asn Tyr Thr Ile Tyr Cys Asn Tyr Ser

20 25

<210> 24

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain FR2

<400> 24

Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu Phe

1 5 10 15

Val

<210> 25

<211> 34

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain FR3

<400> 25

Lys Gln Glu Gly Arg Leu Met Ala Ser Leu Asp Thr Lys Ala Arg Leu

1 5 10 15

Ser Thr Leu His Ile Thr Ala Ala Val His Asp Leu Ser Ala Thr Tyr

20 25 30

Phe Cys

<210> 26

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> alpha chain FR4

<400> 26

Phe Gly Lys Gly Thr His Leu Ile Ile Gln Pro

1 5 10

<210> 27

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain FR1

<400> 27

Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly

1 5 10 15

Gln Asn Val Thr Phe Arg Cys Asp Pro Ile

20 25

<210> 28

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain FR2

<400> 28

Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr

1 5 10 15

Tyr

<210> 29

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain FR3

<400> 29

Ser Arg Leu Leu Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser

1 5 10 15

Phe Ser Thr Leu Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met

20 25 30

Tyr Leu Cys

35

<210> 30

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain FR4

<400> 30

Phe Gly Pro Gly Thr Arg Leu Thr Val Leu

1 5 10

<210> 31

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0 alpha chain variable region

<400> 31

Met Ala Glu Leu Lys Val Glu Gln Asn Pro Thr Asn Leu Ser Val Pro

1 5 10 15

Glu Gly Ala Asn Val Thr Ile Tyr Cys Asn Tyr Ser Thr Thr Ser Asp

20 25 30

Arg Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu

35 40 45

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

50 55 60

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

65 70 75 80

Ala Thr Pro Asp Leu Ser Ala Thr Tyr Phe Cys Gly Gly Gly Ala Asp

85 90 95

Gly Leu Thr Phe Gly Ala Gly Thr Asn Leu Thr Ile Gln

100 105

<210> 32

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0. Beta. Chain variable region

<400> 32

Asp Thr Gly Val Ser Gln Asp Pro Thr Asn Ile Thr Val Pro Arg Gly

1 5 10 15

Ala Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu

20 25 30

Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr Tyr

35 40 45

Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg

50 55 60

Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln

65 70 75 80

Arg Val Glu Pro Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu

85 90 95

Tyr Gly Gly Gly Asp Thr Gln Tyr Phe Gly Pro Gly Thr Gln Leu Thr

100 105 110

Val Asn

<210> 33

<211> 327

<212> DNA

<213> Artificial Sequence

<220>

<223> scTCR-X0 alpha chain variable region

<400> 33

atggctgaac tgaaagttga acagaatccg accaatctga gcgtgccgga aggcgcaaat 60

gtgaccatct attgcaatta tagcaccacc agtgatcgcc tgtattggta tcgtcaggat 120

ccgggtaaaa gcctggaaag cctgtttgtt ctgctgagca atggcgccac caaacaggaa 180

ggccgcctgc aggccagcct ggataccaaa gcccgtctga gcaccctgca tattaccgca 240

gcaaccccgg atctgagtgc cacctatttt tgtggtggtg gtgcagatgg tctgaccttt 300

ggtgccggca ccaatctgac cattcag 327

<210> 34

<211> 51

<212> DNA

<213> Artificial Sequence

<220>

<223> base sequence of linker

<400> 34

ggtagcgccg atgatgcaaa aaaggatgcc gcaaaaaagg acggtaaaag t 51

<210> 35

<211> 342

<212> DNA

<213> Artificial Sequence

<220>

<223> scTCR-X0. Beta. Chain variable region

<400> 35

gacaccggcg tgagtcagga tccgaccaat attaccgtgc cgcgcggtgc aaatgttacc 60

tttcgttgtg atccgattag tgaacataat cgcctgtatt ggtaccgcca ggatccgggc 120

cagggtccgg aatttctgac ctattttcag aatgaagccc agctggaaaa aagtcgtctg 180

ctgagtgatc gttttagcgc cgaacgtccg aaaggcagct ttagtaccct ggaaattcag 240

cgcgtggaac cgggcgatag cgccatgtat ctgtgcgcca gcagtctgta tggtggcggt 300

gacacccagt attttggccc gggcacccag ctgaccgtta at 342

<210> 36

<211> 267

<212> PRT

<213> Artificial Sequence

<220>

<223> full alpha chain length of wild-type TCR

<400> 36

Met Lys Lys Leu Leu Ala Met Ile Leu Trp Leu Gln Leu Asp Arg Leu

1 5 10 15

Ser Gly Glu Leu Lys Val Glu Gln Asn Pro Leu Phe Leu Ser Met Gln

20 25 30

Glu Gly Lys Asn Tyr Thr Ile Tyr Cys Asn Tyr Ser Thr Thr Ser Asp

35 40 45

Arg Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu

50 55 60

Phe Val Leu Leu Ser Asn Gly Ala Val Lys Gln Glu Gly Arg Leu Met

65 70 75 80

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

85 90 95

Ala Val His Asp Leu Ser Ala Thr Tyr Phe Cys Gly Gly Gly Ala Asp

100 105 110

Gly Leu Thr Phe Gly Lys Gly Thr His Leu Ile Ile Gln Pro Tyr Ile

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys

210 215 220

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

225 230 235 240

Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe

245 250 255

Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 37

<211> 312

<212> PRT

<213> Artificial Sequence

<220>

<223> beta chain full Length of wild type TCR

<400> 37

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr

20 25 30

Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His

35 40 45

Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe

50 55 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu

65 70 75 80

Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu

85 90 95

Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala

100 105 110

Ser Ser Leu Tyr Gly Gly Gly Asp Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

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

130 135 140

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

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp

180 185 190

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

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

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

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 38

<211> 28

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0 alpha chain FR1

<400> 38

Met Ala Glu Leu Lys Val Glu Gln Asn Pro Thr Asn Leu Ser Val Pro

1 5 10 15

Glu Gly Ala Asn Val Thr Ile Tyr Cys Asn Tyr Ser

20 25

<210> 39

<211> 34

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0 alpha chain FR3

<400> 39

Lys Gln Glu Gly Arg Leu Gln Ala Ser Leu Asp Thr Lys Ala Arg Leu

1 5 10 15

Ser Thr Leu His Ile Thr Ala Ala Thr Pro Asp Leu Ser Ala Thr Tyr

20 25 30

Phe Cys

<210> 40

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0 alpha chain FR4

<400> 40

Phe Gly Ala Gly Thr Asn Leu Thr Ile Gln

1 5 10

<210> 41

<211> 26

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0. Beta. Chain FR1

<400> 41

Asp Thr Gly Val Ser Gln Asp Pro Thr Asn Ile Thr Val Pro Arg Gly

1 5 10 15

Ala Asn Val Thr Phe Arg Cys Asp Pro Ile

20 25

<210> 42

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0. Beta. Chain FR2

<400> 42

Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Pro Glu Phe Leu Thr

1 5 10 15

Tyr

<210> 43

<211> 38

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0. Beta. Chain FR3

<400> 43

Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg Phe Ser Ala Glu Arg Pro

1 5 10 15

Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln Arg Val Glu Pro Gly Asp

20 25 30

Ser Ala Met Tyr Leu Cys

35

<210> 44

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> scTCR-X0. Beta. Chain FR4

<400> 44

Phe Gly Pro Gly Thr Gln Leu Thr Val Asn

1 5 10

<210> 45

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> CDR2 variants

<400> 45

Leu Leu Ser Asn Gly Ala Thr

1 5

Claims

1. A TCR characterized in that CDR1, CDR2 and CDR3 of the α chain variable region of the TCR are the sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively, or CDR3 is a variant of the sequence shown in SEQ ID No. 3; the amino acid sequence of the variant with the sequence shown as SEQ ID NO.3 is shown as any one of SEQ ID NO. 5-7 or 9-18 in a sequence table;

the CDR1, CDR2 and CDR3 of the beta chain variable region of the TCR are the sequences shown as SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 respectively.

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

3. A TCR as claimed in claim 2 wherein the framework region of the α chain is derived from germline TRAV39 and the framework region of the β chain is derived from germline TRBV7-9.

4. A TCR as claimed in claim 2 wherein the amino acid sequences of FR 1-4 of the alpha chain are shown in SEQ ID nos. 23-26 or variants thereof, respectively, and/or the amino acid sequences of FR 1-4 of the beta chain are shown in SEQ ID nos. 23-26 or variants thereof, respectively.

5. A TCR as claimed in claim 4 wherein the amino acid sequence of the FR1 variant of the α chain is as shown in SEQ ID No.38, the amino acid sequence of the FR3 variant of the α chain is as shown in SEQ ID No.39, and the amino acid sequence of the FR4 variant of the α chain is as shown in SEQ ID No. 40; the amino acid sequence of the FR1 variant of the beta chain is shown as SEQ ID NO.41, the amino acid sequence of the FR2 variant of the beta chain is shown as SEQ ID NO.42, the amino acid sequence of the FR3 variant of the beta chain is shown as SEQ ID NO.43, and the amino acid sequence of the FR4 variant of the beta chain is shown as SEQ ID NO. 44.

6. A TCR as claimed in claim 1 wherein the TCR is a scTCR in which the tcra variable region and the tcra variable region are linked by a linker.

7. A TCR as claimed in claim 6 wherein the linker has an amino acid sequence as shown in SEQ ID No.22 of the sequence listing.

8. A TCR as claimed in any one of claims 1 to 7 wherein the TCR α chain and/or TCR β chain of the TCR further comprises a constant region.

9. A TCR as claimed in claim 8 wherein the constant region of the TCR α chain is derived from germline TRAC; and/or the constant region of the TCR β chain is derived from germline TRBC2.

10. A TCR as claimed in any one of claims 1 to 7 wherein the TCR α chain and/or TCR β chain of the TCR further comprises an outer membrane region and a transmembrane region.

11. A TCR as claimed in any one of claims 1 to 7 wherein the TCR α chain and/or TCR β chain of the TCR further comprises an intracellular sequence.

12. A nucleic acid encoding a TCR as claimed in any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. The vector of claim 13, wherein the vector is a lentiviral vector; the nucleic acid encodes the TCR alpha chain and the TCR beta chain in a single open reading frame, or in two different open reading frames, respectively.

15. A cell comprising the nucleic acid of claim 12 or the vector of claim 13.

16. The cell of claim 15, wherein the cell is a T cell or a stem cell.

17. The cell of claim 16, wherein said T cell is CD8 ⁺ T cells.

18. A pharmaceutical composition comprising a TCR as claimed in any one of claims 1 to 11 or a cell as claimed in any one of claims 15 to 17.

19. The pharmaceutical composition of claim 18, further comprising a pharmaceutically acceptable carrier.

20. Use of a TCR as claimed in any one of claims 1 to 11, a cell as claimed in any one of claims 15 to 17 or a pharmaceutical composition as claimed in claim 18 or 19 in the manufacture of a medicament for the prevention and treatment of a tumour associated with WT1 expression; the tumor is breast cancer or lung cancer.