CN110016074B

CN110016074B - MAGE-A3 humanized T cell receptor

Info

Publication number: CN110016074B
Application number: CN201810015578.2A
Authority: CN
Inventors: 李懿; 陈琳
Original assignee: Guangzhou Institute of Biomedicine and Health of CAS
Current assignee: Guangzhou Institute of Biomedicine and Health of CAS
Priority date: 2018-01-08
Filing date: 2018-01-08
Publication date: 2021-03-30
Anticipated expiration: 2038-01-08
Also published as: CN110016074A

Abstract

The invention provides a humanised engineered T Cell Receptor (TCR) SRm1g13T having a binding human antigen MAGE-A3:112-120(KVAELVHFL) and Human Leukocyte Antigen (HLA) complex: the characteristics of pHLA (MAGE-A3); and the binding affinity of the SRm1g13t to the pHLA (MAGE-A3) complex is at least 2-fold greater than the binding affinity of the corresponding murine TCR SRm1 to the pHLA (MAGE-A3) complex. The invention also provides fusion molecules of such TCRs with therapeutic agents. Such TCRs can be used alone or in combination with a therapeutic agent.

Description

MAGE-A3 humanized T cell receptor

Technical Field

The present invention relates to the field of biotechnology, and more specifically to a humanized T Cell Receptor (TCR) capable of recognizing the human antigen pHLA (MAGE-a 3). The invention also relates to the preparation and use of said receptors.

Background

The current focus on T cell immunotherapy based on the expression of specific T Cell Receptors (TCRs) is increasing, and this immunotherapy is being held to hopefully eradicate tumors. This anti-tumor effect is mediated by the transferred TCR, which recognizes the peptide-human leukocyte antigen (pHLA) complex on the tumor cells, thereby allowing the T cells to recognize the tumor cells, which in turn cause the T cells to kill the tumor cells. Recent studies have shown that higher affinity TCRs can cause T cells to exert better anti-tumor effects. Clinical tests also show that the NY-ESO-1 specific T cells with improved affinity not only show safety, but also show good anti-tumor curative effect. These data all indicate that increasing T cell immune efficacy can start with increasing affinity.

MAGE-a3 is highly frequently expressed in human tumors, and its expression correlates positively with poor prognosis. Researchers have been able to obtain murine TCRs against the human antigen MAGE-A3 by immunizing transgenic mice for HLA-A02 with the short peptide of the human antigen MAGE-A3:112-120 (KVAELVHFL). This murine TCR exhibited good anti-tumor effects and was also studied in clinical trials (Chinnanamy, N., et al, J Immunol,2011.186(2): p.685-96.).

However, in order to prevent the development of autoimmune responses in humans, high affinity TCRs have been knocked out in the thymus, and it is very difficult to find high affinity TCRs in the human TCR repertoire. However, it was found that when T cells express high affinity TCRs, T cell activation is not assisted by the requirement of CD 8. However, mouse CD8 is not able to bind efficiently to human HLA α 3, and it is relatively much easier to find high affinity murine TCRs against human tumor antigens in HLA transgenic mice. And a large number of articles report that the mouse-derived TCR can play a good anti-tumor response, so that the high-affinity mouse-derived TCR has considerable advantages and potential to be applied to anti-tumor immunotherapy.

However, the murine TCR is immunogenic to humans and will generate host immune responses and antibodies against the murine TCR, which will most likely eliminate the returned T cells that have been transferred to the murine TCR. Researchers have now found that when a patient reinflates T cells that transfer murine TCRs against the human antigens p53 or gp100, antibodies against the murine TCRs are generated in the patient. It was also found that when the patient reinfused T cells transfected with murine TCR against the human antigen CEA, the patient produced antibodies against murine TCR 3-4 months after treatment, which antibodies also inhibited the ability of PBL expressing murine TCR to produce IFN- γ. Although the current data do not provide clear answers to the effect that antibodies against murine TCR may have on anti-tumor effect, the immunogenicity of murine TCR inevitably affects the body's production of antibodies, and this effect must be taken into account to prevent unwanted clinical application of murine TCR.

It is well known that the production of humanized antibodies has overcome the immunogenicity of murine antibodies and with considerable success, many humanized antibodies are now available on the market for therapeutic applications. Humanized antibodies are obtained by grafting Complementary Determining Regions (CDRs), which are mainly the murine CDRs, onto the framework of a human antibody variable region. Although CDR grafting retains the ability to recognize antigens, the affinity of such recognized antigens is often reduced. This phenomenon is probably due to the murine CDRs being grafted onto a human antibody framework such that the murine CDRs are conformationally altered.

Therefore, there is a need in the art to develop a method for humanizing a TCR from murine origin to obtain a humanized CDR with high affinity and low immunogenicity for use in the treatment of diseases such as tumors.

Disclosure of Invention

The present invention aims to provide a humanized TCR with higher affinity for the human antigen pHLA (MAGE-A3).

It is a further object of the invention to provide a method of making a humanized TCR of the type described above and the use of a humanized TCR of the type described above.

In a first aspect of the invention, there is provided a humanized T Cell Receptor (TCR) which has binding activity to the human antigen pHLA (MAGE-A3) and which comprises a TCR alpha chain variable domain and/or a TCR beta chain variable domain, and which has a higher affinity for the human antigen pHLA (MAGE-A3) than the murine TCR for the human antigen pHLA (MAGE-A3).

In another preferred embodiment, the humanized TCR has activity to bind to a complex of MAGE A3:112-120(KVAELVHFL) and HLA-A0201.

In another preferred embodiment, the humanized TCR further has a binding domain that binds MAGE a 12: 112, 120(KMAELVHFL), MAGE A2: (KMVELVHFL) or MAGE A6: (KVAKLVHFL) Activity of complexes with HLA-A0201.

In another preferred embodiment, the humanized TCR binds human antigen pHLA (MAGE-A3) with a dissociation constant that is 1.5-3 times the dissociation constant for binding of murine TCR to human antigen pHLA (MAGE-A3).

In another preferred embodiment, the variable domain of the TCR alpha chain comprises 3 CDR regions, and the reference sequence of the 3 CDR regions of the variable domain of the TCR alpha chain is as follows,

CDR1α：TIYSNPF，SEQ ID NO:5；

CDR2α：SFTDNKR，SEQ ID NO:6；

CDR3α：AFDTNAYKVI，SEQ ID NO:7；

and/or, the variable domain of the TCR beta chain comprises 3 CDR regions, the reference sequence of the 3 CDR regions of the variable domain of the TCR beta chain is as follows,

CDR1β：MSHET，SEQ ID NO:8；

CDR2β：SYDVDS，SEQ ID NO:9；

CDR3β：ASSSTNTEVF，SEQ ID NO:10；

wherein the amino acid sequence of any one of the above CDRs further comprises a derivative sequence optionally having at least one amino acid added, deleted, modified and/or substituted, and capable of retaining the binding affinity of human antigen pHLA (MAGE-A3).

In another preferred embodiment, the amino acid sequence of any of the above CDRs comprises a derivative CDR sequence with 1, 2 or 3 amino acids added, deleted, modified and/or substituted, and such that a derivative TCR comprising va and ν β comprising said derivative CDR sequence retains affinity for binding to human antigen pHLA (MAGE-a 3).

In another preferred embodiment, the ratio of the affinity of binding of the derivatized TCR to human antigen pHLA (MAGE-A3), F1, to the affinity of binding of the corresponding non-derivatized TCR to human antigen pHLA (MAGE-A3), F0 (F1/F0) is from 0.5 to 2, preferably from 0.7 to 1.5, and more preferably from 0.8 to 1.2.

In another preferred embodiment, the number of the amino acids to be added, deleted, modified and/or substituted is 1 to 5 (e.g., 1 to 3, preferably 1 to 2, and more preferably 1).

In another preferred embodiment, the ratio of the immunogenicity Z1 of the humanized TCR in humans to the immunogenicity Z0 of the non-humanized TCR (e.g., murine TCR) in humans (Z1/Z0) is 0 to 0.5, preferably 0 to 0.2, more preferably 0 to 0.05 (e.g., 0.001 to 0.05).

In a second aspect of the invention, there is provided a humanized T Cell Receptor (TCR) which has activity for binding to the human antigen pHLA (MAGE-A3) and which comprises a TCR alpha chain variable domain and/or a TCR beta chain variable domain,

the variable domain of the TCR alpha chain comprises 3 CDR regions, the reference sequence of the 3 CDR regions of the variable domain of the TCR alpha chain is as follows,

CDR1α：TIYSNPF，SEQ ID NO:5；

CDR2α：SFTDNKR，SEQ ID NO:6；

CDR3α：AFDTNAYKVI，SEQ ID NO:7；

CDR1β：MSHET，SEQ ID NO:8；

CDR2β：SYDVDS，SEQ ID NO:9；

CDR3β：ASSSTNTEVF，SEQ ID NO:10；

In another preferred embodiment, the humanized TCR has a higher affinity for the human antigen pHLA (MAGE-A3) than the murine TCR for the human antigen pHLA (MAGE-A3).

In another preferred embodiment, the humanized TCR binds to human antigen MAGE-A3 with a dissociation constant that is 1.5-3 times the dissociation constant for murine TCR binding to human antigen MAGE-A3.

In another preferred embodiment, the framework region of the TCR α chain variable domain comprises 3 FR regions, and the reference sequences of the 3 FR regions are as follows:

FR1α：GDSVTQTEGLLNVPEGLPVSINCTYQ，SEQ ID NO:21；

FR2α：LFWYRQDPGKSPRLLLK，SEQ ID NO:22；

FR3α：TEHQRFHATLHKSDSSFHLQIERIQPNDSGTYFC，SEQ ID NO:23；

and/or, the framework region of the variable domain of the TCR β chain comprises 3 FR regions, the reference sequences of the 3 FR regions are as follows:

FR1β：DMKITQTPRYLIVKTGENVTLECGQD，SEQ ID NO:24；

FR2β：MYWYRQDPGQGLQLIYI，SEQ ID NO:25；

FR3β：NSEGDIPKRYRVSRKKREHFSLRIDSVKTSDSALYLC，SEQ ID NO:26；

wherein the amino acid sequence of any of the above FRs further comprises a derivative sequence optionally having at least one amino acid added, deleted, modified and/or substituted, and capable of retaining the binding affinity of the human antigen pHLA (MAGE-A3).

In another preferred embodiment, the sequence of the FR region of the framework region of the TCR α chain variable domain is derived from TRAV18 x 01.

In another preferred embodiment, the sequence of the FR region of the framework region of the TCR β chain variable domain is derived from TRBV28 x 01.

In another preferred embodiment, the amino acid sequence of any of the above-mentioned FRs comprises a derivative FR sequence comprising 1, 2 or 3 amino acids added, deleted, modified and/or substituted, and such that the derivative TCR comprising V.alpha.and V.beta.of said derivative FR sequence retains affinity for binding to the human antigen pHLA (MAGE-A3).

In another preferred embodiment, the T cell receptor is selected from the group consisting of:

(a) a T cell receptor having the TCR alpha chain variable domain represented by SEQ ID No. 1 (sequence of SRm1a g13T) or a variant thereof;

(b) a T cell receptor having the TCR β chain variable domain represented by SEQ ID No. 2 (sequence of SRm1b g13T) or a variant thereof;

(c) a T cell receptor having (a) a TCR α chain variable domain of said T cell receptor and (b) a TCR β chain variable domain of a T cell receptor; and

(d) a T cell receptor which competes with the T cell receptor of any one of (a) to (c) for binding to the humanized T cell receptor of human antigen pHLA (MAGE-A3).

In another preferred embodiment, the TCR comprises a TCR alpha chain variable domain which is an amino acid sequence having at least 90% sequence identity to SEQ ID No. 1, and a TCR beta chain variable domain; and/or the TCR β chain variable domain is identical to SEQ ID NO:2 has at least 90% sequence identity.

In another preferred embodiment, the TCR is represented in SEQ ID NO:1, and the mutated amino acid residue positions comprise: one or more of L (11), N (12), V (13), P (14), S (20), I (21), R (43), D (45), P (46), G (47), K (48), R (75), D (84), I (91), E (92), R (93), I (94), P (96), N (97), G (100), T (101) and F (103), wherein the amino acid residue number adopts the number shown in SEQ ID NO: 1; and/or the TCR is as set out in SEQ ID NO:2, and the mutated amino acid residue positions comprise one or more of I (4), T (7), V (13), K (14), T (15), T (20), Q (48), R (75), R (90), I (91), V (94), S (97), D (98), S (99), A (100), L (101) and L (103), wherein the amino acid residue numbers adopt the numbers shown in SEQ ID NO: 2.

In another preferred embodiment, the soluble TCR is an α β heterodimer comprising both a TCR α chain constant region and a TCR β chain constant region that are human.

In another preferred embodiment, the soluble TCR is an α β heterodimer comprising the FR region of TCR α chain TRAV18 x 01 and the FR region of TCR β chain TRBV28 x 01.

In another preferred embodiment, the soluble TCR is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC 101 or TRBC2 01.

In another preferred embodiment, the α chain amino acid sequence of the TCR is SEQ ID NO:17 and/or the beta chain amino acid sequence of the TCR is SEQ ID NO 18.

In another preferred embodiment, the TCR is soluble.

In another preferred embodiment, the TCR is single chain.

In another preferred embodiment, the TCR is formed by linking an α chain variable domain to a β chain variable domain via a peptide linker.

In another preferred embodiment, the TCR comprises (a) all or part of a TCR α chain, excluding the transmembrane domain; and (b) all or part of a TCR β chain, excluding the transmembrane domain;

and (a) and (b) each comprise a functional variable domain, or comprise a functional variable domain and at least a portion of the TCR chain constant domain.

In another preferred embodiment, the cysteine residues form an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.

In another preferred embodiment, the cysteine residues forming the artificial disulfide bond in the TCR are substituted at one or more groups of sites selected from the group consisting of:

thr48 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser57 of TRBC2 × 01 exon 1;

thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1;

tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01;

thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1;

ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1;

arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1;

pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; and

tyr10 and TRBC1 × 01 of exon 1 of TRAC × 01 or Glu20 of exon 1 of TRBC2 × 01.

In another preferred embodiment, the TCR comprises an artificial interchain disulfide bond between the α chain variable region and the β chain constant region.

In another preferred embodiment, the cysteine residues that form the artificial interchain disulfide bond in the TCR replace one or more groups of sites selected from the group consisting of:

amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01;

amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01;

amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or

Amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01.

In another preferred embodiment, the TCR comprises an alpha chain variable domain and a beta chain variable domain and all or part of the beta chain constant domain, excluding the transmembrane domain, but which does not comprise an alpha chain constant domain, the alpha chain variable domain of the TCR forming a heterodimer with the beta chain.

In another preferred embodiment, the TCR has a conjugate attached to the C-or N-terminus of the alpha and/or beta chain.

In another preferred embodiment, the conjugate that binds to the T cell receptor is a detectable label, a therapeutic agent, a PK modifying moiety or a combination of any of these. Preferably, the therapeutic agent is an anti-CD3, anti-PDL 1, anti-PD 1 or anti-CD 28 antibody.

In a third aspect of the invention there is provided a multivalent TCR complex comprising at least two TCR molecules, and at least one of which is a TCR according to the first or second aspect of the invention.

In a fourth aspect of the invention, there is provided a nucleic acid molecule comprising a nucleic acid sequence encoding a TCR according to any one of the first or second aspects of the invention, or a complement thereof.

In another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence encoding the variable domain of the TCR α chain SEQ ID NO: 3.

in another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 4.

in another preferred embodiment, the nucleic acid molecule comprises the nucleotide sequence encoding the TCR α chain SEQ ID NO:19 and/or a nucleic acid sequence comprising the nucleotide sequence encoding a TCR β chain SEQ ID NO: 20.

in a fifth aspect of the invention, there is provided a vector comprising a nucleic acid molecule as described in the fourth aspect of the invention.

In another preferred embodiment, the vector is a viral vector.

In another preferred embodiment, the vector is a lentiviral vector.

In a sixth aspect of the invention, there is provided a host cell comprising a vector according to the fifth aspect of the invention or a chromosome having integrated therein an exogenous nucleic acid molecule according to the fourth aspect of the invention.

In another preferred embodiment, the cell is a T cell or a stem cell.

In a seventh aspect of the invention, there is provided an isolated cell expressing a TCR according to any one of the first or second aspects of the invention.

In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of the first or second aspects of the invention, or a TCR complex according to the third aspect of the invention, or a cell according to the seventh aspect of the invention.

In a ninth aspect of the invention, there is provided the use of a T cell receptor according to any one of the first or second aspects of the invention, a TCR complex according to the third aspect of the invention or a cell according to the seventh aspect of the invention in the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.

In a tenth aspect of the invention, there is provided a method of treating a disease comprising administering to a subject in need thereof a TCR according to any one of the first or second aspects of the invention, or a TCR complex according to the third aspect of the invention, or a cell according to the seventh aspect of the invention, or a pharmaceutical composition according to the eighth aspect of the invention.

Preferably, the disease is a tumor. Preferably the tumour comprises lung cancer, breast cancer, melanoma, liver cancer, squamous cell carcinoma.

In an eleventh aspect of the invention there is provided a method of preparing a T cell receptor according to any one of the first or second aspects of the invention, comprising the steps of:

(i) culturing a host cell according to the sixth aspect of the invention so as to express a T-cell receptor according to any one of the first or second aspects of the invention;

(ii) isolating or purifying said T cell receptor.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 sequence of a stability optimized humanized TCR (SRm1g13 t). (A) SRm1a alpha chain of murine MAGE-A3 TCR; SRm1a g13 t. alpha chain of stability optimized humanized MAGE-A3 TCR; TRAV18 x 01, alpha chain of the most similar human TCR in IMGT after sequence alignment; (B) SRm1b beta chain of murine MAGE-A3 TCR; SRm1b g13t beta chain of stability optimised humanized MAGE-A3 TCR; TRBV28 × 01, β chain of the most similar human TCR in IMGT after sequence alignment; blue, site of humanization.

FIG. 2 is a graph of inclusion bodies after purification of alpha, beta strands for SRm1 and SRm1g13 t. (A) The different TCRs were expressed as gene expression profiles of inclusion bodies, and these sequences were cloned into pET-28a vector. V α variable region of the α chain of the TCR, C α: constant region of the α chain of the TCR, V β: the variable region of the beta chain of the TCR, C.beta.the constant region of the beta chain of the TCR. (B) And (C) SDS-PAGE of alpha, beta chain purified inclusion bodies of SRm1 and SRm1g13t after dilution by fold. (B) The alpha and beta chain purified inclusion bodies of SRm1 were diluted 10, 20 and 40 times with urea, respectively, and then the Protein marker (M) was run on SDS-PAGE, the alpha chain inclusion bodies of SRm1 were diluted 10, 20 and 40 times, and the beta chain inclusion bodies of SRm1 were diluted 10, 20 and 40 times, respectively, and the Protein marker (Protein marker) was run on Lane 1-3, and the Lane 4-6. (C) SRm1g13t purified α, β chain inclusion bodies were diluted 5, 10, 20, 40 fold with urea, respectively, and run on SDS-PAGE, M: protein marker (Protein marker); lanes (Lane)1-4, SRm1g13t diluted 40, 20, 10, 5 fold with α chain inclusion bodies, lanes (Lane) 5-8: beta-chain inclusion bodies of SRm1 were diluted 40, 20, 10, 5 fold.

FIG. 3SRm1 and SRm1g13t TCR and its ligand pHLA. (A) Elution profile of in vitro renatured SRm1 through molecular sieves. After the inclusion body expression extraction, SRm1 was renatured in vitro, sample exchange column purification was performed first using AKTA purifier, and the collected sample was eluted again using PBS through molecular sieves. (B) Elution profile of in vitro renatured SRm1g13t through molecular sieves. After the inclusion body expression extraction, SRm1g13t was renatured in vitro, and the sample was first purified by an AKTA purification apparatus using a column exchanger, and the collected sample was eluted again by PBS through a molecular sieve. (C) Expression of the TCR ligand pHLA complex was purified and biotinylated for detection. The pHLA in vitro renaturation sample is finally biotinylated, passed through a molecular sieve and eluted with PBS. (D) And collecting the eluted pHLA, concentrating, and finally verifying the biotinylation effect. Lane (Lane) M protein marker (protein marker), Lane (Lane)1 streptavidin, Lane (Lane)2 pHLA, Lane (Lane)3-5 streptavidin and pHLA in a molar ratio: 1: 1,1: 4,1: 8.

FIG. 4 the affinity between the different TCRs and the ligand pHLA (MAGE-A3). (A) The binding of SRm1 and (B) SRm1g13t TCR recognizes that pHLA (MAGE-A3) is analyzed by Biacore SPR. CM5 Biacore chip was SA coated, biotinylated pHLA (MAGE-A3) was then bound to the chip, different concentrations of SRm1 and SRm1g13T TCR were subjected to dissociation of the binding, and finally the binding constant k was analyzed using Biacore T200 analysis software_aDissociation constant k_dAnd a binding dissociation constant K_D。

FIG. 5SRm1 and SRm1g13t TCR transfection CD3⁺Positive rate of assay after T cells. (A) mRNA expression profiles for SRm1 and SRm1g13t TCR. (B) SRm1 and SRm1g13t TCR transfection CD3⁺Positive rate of T cells. CD3 transfected with SRm1 and SRm1g13t TCR⁺T cells were stained with anti-CD3 and anti-mouse TCR Beta chain (anti-mouse TCR Beta chain) antibodies, respectively, and then subjected to flow analysis. (C) SRm1 and SRm1g13t TCR transfection CD3⁺CD8 in T cells⁺Proportion of T cells and ability to bind pHLA tetramerizationThe ratio of the volumes. CD3 transfected with SRm1 and SRm1g13t TCR⁺T cells were stained with anti-CD8 antibody and pHLA tetramer, respectively, and then subjected to flow analysis.

FIG. 6 CD3 of SRm1 and SRm1g13t TCRs⁺Specific analysis of T cells. (A) And (B) CD3 transfected with SRm1 and SRm1g13t TCR⁺T cells and different target cells are incubated for 24h, and the release of IFN-gamma is detected by using an ELISpot detection kit.

FIG. 7 CD3 of SRm1 and SRm1g13t TCRs⁺Killing ability of T cells was compared. CD3 transfected with SRm1 and SRm1g13t TCR⁺T cells were incubated with different target cells for 24h, with CD3⁺The number of T cells was 7500, the effective target ratio was 5: 1, then after 24h detection was performed with LDH detection kit.

FIG. 8SRm1 and SRm1g13t TCRs conferring CD3⁺The ability of T cells to release factors. CD3+ T cells transfected with SRm1 and SRm1g13T TCR were co-cultured overnight with NCI-H1299-A2 cells, and these CD3+ T cells were then examined for intracellular IFN-. gamma.IL-2, TNF. alpha. levels by intracellular staining, the upper right corner showing intracellular IFN-. gamma.IL-2, TNF. alpha. levels in cells positive for CD 3.

Detailed Description

The present inventors have made extensive and intensive studies and have selected a murine TCR against the human antigen MAGE-A3 (e.g., MAGE-A3:112-120(KVAELVHFL, SEQ ID NO: 27)), and constructed a humanized TCR based on the principle of humanized antibodies and the non-immunogenicity and advantages of murine constant regions. The humanized TCR is prepared by grafting CDR regions of a murine TCR onto the framework of the variable region of the corresponding human TCR. And considering the conformational stability after CDR grafting, the present inventors also introduced point mutations to stabilize those important interfaces by computer modeling the structure. The results show that the murine TCR against the human antigen MAGE-a3 can be humanised and shows better affinity after humanisation (e.g. a binding constant of 2.3 times that of the murine TCR before engineering). Will CD3⁺The T cell transferred into humanized TCR can retain the specificity of the recognition antigen, and compared with the murine TCR, the T cell shows higher capability of releasing IFN-gamma, TNF alpha and IL-2, and simultaneously, the T cell has the capability of killing tumors compared with the murine TCRThere was no significant difference in the source TCR. The invention also provides nucleic acid molecules encoding the TCRs and vectors comprising the nucleic acid molecules. In addition, the invention provides cells that transduce a TCR of the invention. On the basis of this, the present invention has been completed.

Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.

Term(s) for

MHC molecules

MHC molecules are proteins of the immunoglobulin superfamily, which may be MHC class I or class II molecules. Therefore, it is specific for antigen presentation, different individuals have different MHC, and different short peptides in one protein antigen can be presented on the cell surface of respective APC. Human MHC is commonly referred to as an HLA gene or HLA complex.

T Cell Receptor (TCR)

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

TCRs are cell membrane surface glycoproteins that exist as heterodimers from either the α chain/β chain or the γ chain/δ chain. In 95% of T cells the TCR heterodimer consists of α and β chains, while 5% of T cells have TCRs consisting of γ and δ chains. Native α β heterodimeric TCRs have an α chain and a β chain, which constitute subunits of an α β heterodimeric TCR. Broadly, each of the α and β chains comprises a variable region, a linker region and a constant region, and the β chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered to be part of the linker region. Each variable region comprises 3 CDRs (complementarity determining regions) CDR1, CDR2 and CDR3, which are chimeric in framework structures (framework regions). The CDR regions determine the binding of the TCR to the pMHC complex, where CDR3 is recombined from variable and connecting regions, referred to as hypervariable regions. The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domain being made up of linked variable regions and linking regions. The sequences of TCR constant domains can be found in public databases of the international immunogenetic information system (IMGT), such as the constant domain sequence of the α chain of the TCR molecule is "TRAC 01", the constant domain sequence of the β chain of the TCR molecule is "TRBC 1 01" or "TRBC 2 01". In addition, the α and β chains of the TCR also comprise a transmembrane region and a cytoplasmic region, the cytoplasmic region being very short.

The TCR may be described using the international immunogenetics information system (IMGT). Native α β heterodimeric TCRs have an α chain and a β chain. In a broad sense, each chain comprises a variable region, a linker region and a constant region, and the beta chain also typically contains a short diversity region between the variable region and the linker region, but the diversity region is often considered part of the linker region. The TCR connecting region is defined by the unique TRAJ and TRBJ of IMGT, and the TCR constant region is defined by the TRAC and TRBC of IMGT.

Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2, and CDR3, chimeric in a framework sequence. In the IMGT nomenclature, the different numbers of TRAV and TRBV refer to different types of V α and V β, respectively. In the IMGT system, the α chain constant domain has the following symbols: TRAC 01, wherein "TR" denotes a T cell receptor gene; "A" represents an alpha chain gene; c represents a constant region; ". 01" indicates allele 1. The beta-strand constant domain has the following symbols: TRBC1 x 01 or TRBC2 x 01, wherein "TR" denotes a T cell receptor gene; "B" represents a beta chain gene; c represents a constant region; ". 01" indicates allele 1. The constant region of the alpha chain is uniquely defined, and in the form of the beta chain, there are two possible constant region genes, "C1" and "C2". The constant region gene sequences of the TCR alpha and beta chains can be obtained by those skilled in the art from published IMGT databases.

The α and β chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain. The variable domain is composed of linked variable regions and linked regions. Thus, in the description and claims of this application, the "TCR α chain variable domain" refers to the linked TRAV and TRAJ regions, and likewise the "TCR β chain variable domain" refers to the linked TRBV and TRBD/TRBJ regions. The 3 CDRs of the TCR α chain variable domain are CDR1 α, CDR2 α and CDR3 α, respectively; the 3 CDRs of the TCR β chain variable domain are CDR1 β, CDR2 β and CDR3 β, respectively. The framework sequences of the TCR variable domains of the invention may be murine or human, preferably human. The constant domain of the TCR comprises an intracellular portion, a transmembrane region, and an extracellular portion. To obtain soluble TCRs, in order to determine the affinity between the TCR and the KVAELVHFL-HLA-a 0201 complex, the inventive TCR preferably does not comprise a transmembrane region. More preferably, the amino acid sequence of the TCR of the invention refers to the extracellular amino acid sequence of the TCR.

In a preferred embodiment of the invention, a T Cell Receptor (TCR) according to the invention comprises a TCR alpha chain variable domain comprising CDR1 alpha, CDR2 alpha, and CDR3 alpha, and a TCR beta chain variable domain.

Mouse-derived TCR

In the present invention, the murine TCR refers to a TCR in a TCR library derived from a mouse. Since the murine TCR repertoire does not knock out TCRs against human antigens during thymic development, it is relatively easy to obtain TCRs against human antigens. Meanwhile, since mouse CD8 cannot be effectively combined with human HLA alpha 3, it is easier to find high affinity mouse TCR against human tumor antigen in HLA transgenic mice. However, the murine TCR is immunogenic to humans and will generate host immune responses and antibodies against the murine TCR, which will most likely eliminate the transfected T cells from the murine TCR.

In the present invention, the terms "polypeptide of the invention", "TCR of the invention", "T cell receptor of the invention" are used interchangeably.

Natural interchain disulfide bond and artificial interchain disulfide bond

A set of disulfide bonds, referred to herein as "native interchain disulfide bonds," exist between the C α and C β chains of the membrane proximal region of native TCRs. In the present invention, the artificially introduced interchain covalent disulfide bond whose position is different from that of the natural interchain disulfide bond is referred to as an "artificial interchain disulfide bond".

For convenience of description, the amino acid sequences of TRAC 01 and TRBC1 × 01 or TRBC2 × 01 are position-numbered in the order from the N-terminus to the C-terminus, such as TRBC1 × 01 or TRBC2 × 01, and the 60 th amino acid is P (proline) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2 × 01 exon 1 Pro60 in the invention, or TRBC1 × 01 or TRBC2 × 01 exon 1, and as TRBC1 × 01 or TRBC2 × 01, and the 61 st amino acid is Q (glutamine) in the order from the N-terminus to the C-terminus, and thus it may be described as TRBC1 × 01 or TRBC2, and as glbc 8201 or TRBC 8536. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. If an amino acid in TRAV, the position listed in IMGT is numbered 46, it is described herein as the 46 th amino acid of TRAV, and so on. In the present invention, the sequence position numbers of other amino acids are specifically described.

Tumor(s)

The term "tumor" is meant to include all types of cancer cell growth or carcinogenic processes, metastatic or malignantly transformed cells, tissues or organs, regardless of the type of pathology or the stage of infection. Examples of tumors include, but are not limited to: solid tumors, soft tissue tumors, and metastatic lesions. Examples of solid tumors include: malignancies of different organ systems, such as sarcomas, squamous carcinomas of the lung and cancers. For example: infected prostate, lung, breast, lymph, gastrointestinal (e.g., colon), and genitourinary tract (e.g., kidney, epithelial cells), pharynx. Squamous carcinoma of the lung includes malignant tumors, such as, for example, most cancers of the colon, rectum, renal cell, liver, lung, small cell, small intestine and esophagus. Metastatic lesions of the above-mentioned cancers can likewise be treated and prevented using the methods and compositions of the present invention.

Detailed Description

TCR molecules

During antigen processing, antigens are degraded within cells and then carried to the cell surface by MHC molecules. T cell receptors are capable of recognizing peptide-MHC complexes on the surface of antigen presenting cells. Accordingly, in a first aspect the invention provides a TCR molecule capable of binding to the MAGE-A3:112-120(KVAELVHFL) and HLA-A0201 complexes. Preferably, the TCR molecule is isolated or purified. The α and β chains of the TCR each have 3 Complementarity Determining Regions (CDRs).

In a preferred embodiment of the invention, the humanized TCR has activity to bind to a complex of MAGE A3:112-120(KVAELVHFL) and HLA-A0201.

In another preferred embodiment of the invention, the humanized TCR further has a binding domain that binds MAGE a 12: 112, 120(KMAELVHFL), MAGE A2: (KMVELVHFL) or MAGE A6: (KVAKLVHFL) Activity of complexes with HLA-A0201.

In a preferred embodiment of the invention, the 3 complementarity determining regions of the TCR α chain variable domain are:

CDR1α：TIYSNPF，SEQ ID NO:5

CDR2α：SFTDNKR，SEQ ID NO:6

CDR3α：AFDTNAYKVI，SEQ ID NO:7

the 3 complementarity determining regions of the TCR β chain variable domain are:

CDR1β：MSHET，SEQ ID NO:8

CDR2β：SYDVDS，SEQ ID NO:9

CDR3β：ASSSTNTEVF，SEQ ID NO:10

chimeric TCRs can be prepared by embedding the above-described amino acid sequences of the CDR regions of the invention into any suitable framework. One skilled in the art can design or synthesize a TCR molecule with the corresponding function based on the CDR regions disclosed herein, so long as the framework structure is compatible with the CDR regions of the TCR of the invention. Thus, the TCR molecules of the invention are those which comprise the above-described α and/or β chain CDR region sequences and any suitable framework structure. The TCR α chain variable domain of the invention is an amino acid sequence having at least 90%, preferably 95%, more preferably 98% sequence identity to SEQ ID No. 1; and/or the TCR β chain variable domain of the invention is a variant of SEQ ID NO:2, or a variant thereof, and 2 amino acid sequences having at least 90%, preferably 95%, more preferably 98% sequence identity.

In a preferred embodiment of the invention, the framework region of the variable domain of the TCR α chain comprises 3 FR regions, the reference sequences of the 3 FR regions are as follows:

FR1α：GDSVTQTEGLLNVPEGLPVSINCTYQ，SEQ ID NO:21；

FR2α：LFWYRQDPGKSPRLLLK，SEQ ID NO:22；

FR3α：TEHQRFHATLHKSDSSFHLQIERIQPNDSGTYFC，SEQ ID NO:23；

FR1β：DMKITQTPRYLIVKTGENVTLECGQD，SEQ ID NO:24；

FR2β：MYWYRQDPGQGLQLIYI，SEQ ID NO:25；

FR3β：NSEGDIPKRYRVSRKKREHFSLRIDSVKTSDSALYLC，SEQ ID NO:26；

Preferably, the amino acid sequence of the variable domain of the TCR alpha chain (SRm1a g13t) of the invention is SEQ ID NO 1GDSVTQTEGLLNVPEGLPVSINCTYQTIYSNPFLFWYRQDPGKSPRLLLKSFTDNKRTEHQRFHATLHKSDSSFHLQIERIQPNDSGTYFCAFDTNAYKVIFGKGTHLHVLP；

And/or the amino acid sequence of the variable domain of the TCR beta chain (SRm1b g13t) of the invention is SEQ ID NO 2DMKITQTPRYLIVKTGENVTLECGQDMSHETMYWYRQDPGQGLQLIYISYDVDSNSEGDIPKRYRVSRKKREHFSLRIDSVKTSDSALYLCASSSTNTEVFFGPGTRLTVV。

In a preferred embodiment of the invention, the TCR molecules of the invention are heterodimers consisting of α and β chains. In particular, in one aspect the α chain of the heterodimeric TCR molecules comprises a variable domain and a constant domain, the α chain variable domain amino acid sequence comprising CDR1(SEQ ID NO: 5), CDR2(SEQ ID NO: 6) and CDR3(SEQ ID NO:7) of the above-described α chain. Preferably, the TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the amino acid sequence of the α chain variable domain of the TCR molecule is SEQ ID NO 1. In another aspect, the β chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain, and the β chain variable domain amino acid sequence comprises CDR1(SEQ ID NO:8), CDR2(SEQ ID NO: 9), and CDR3(SEQ ID NO:10) of the above-described β chain. Preferably, the TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 2. More preferably, the amino acid sequence of the β chain variable domain of the TCR molecule is SEQ ID NO 2.

In a preferred embodiment of the invention, the TCR molecules of the invention are single chain TCR molecules consisting of part or all of the α chain and/or part or all of the β chain. Single chain TCR molecules are described in Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-. From the literature, those skilled in the art are readily able to construct single chain TCR molecules comprising the CDRs regions of the invention. In particular, the single chain TCR molecule comprises V α, V β and C β, preferably linked in order from N-terminus to C-terminus.

The alpha chain variable domain amino acid sequence of the single chain TCR molecule comprises the CDR1(SEQ ID NO: 5), CDR2(SEQ ID NO: 6) and CDR3(SEQ ID NO:7) of the alpha chain described above. Preferably, the single chain TCR molecule comprises the alpha chain variable domain amino acid sequence SEQ ID NO 1. More preferably, the α chain variable domain amino acid sequence of the single chain TCR molecule is SEQ ID NO 1. The amino acid sequence of the beta chain variable domain of the single chain TCR molecule comprises the CDR1(SEQ ID NO:8), CDR2(SEQ ID NO: 9) and CDR3(SEQ ID NO:10) of the above-described beta chain. Preferably, the single chain TCR molecule comprises the beta chain variable domain amino acid sequence SEQ ID NO 2. More preferably, the amino acid sequence of the β chain variable domain of the single chain TCR molecule is SEQ ID NO 2.

In a preferred embodiment of the invention, the constant domain of the TCR molecules of the invention is a human constant domain. The human constant domain amino acid sequences are known to those skilled in the art or can be obtained by consulting published databases of relevant books or IMGT (international immunogenetic information system). For example, the constant domain sequence of the α chain of the TCR molecules of the invention can be "TRAC 01", and the constant domain sequence of the β chain of the TCR molecules can be "TRBC 1 01" or "TRBC 2 01". Arg at position 53 of the amino acid sequence given in TRAC 01 of IMGT, here denoted: TRAC × 01 Arg53 of exon 1, and so on.

Preferably, the first and second electrodes are formed of a metal,

the amino acid sequence of the TCR molecule alpha chain is SEQ ID NO 17

MGDSVTQTEGLLNVPEGLPVSINCTYQTIYSNPFLFWYRQDPGKSPRLLLKSFTDNKRTEHQRFHATLHKSDSSFHLQIERIQPNDSGTYFCAFDTNAYKVIFGKGTHLHVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDT；

And/or the amino acid sequence of the beta chain is SEQ ID NO 18

MGDMKITQTPRYLIVKTGENVTLECGQDMSHETMYWYRQDPGQGLQLIYISYDVDSNSEGDIPKRYRVSRKKREHFSLRIDSVKTSDSALYLCASSSTNTEVFFGPGTRLTVVEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD。

Naturally occurring TCRs are membrane proteins that are stabilized by their transmembrane regions. Like immunoglobulins (antibodies) as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, where soluble TCR molecules are required. Soluble TCR molecules do not include their transmembrane regions. Soluble TCRs have a wide range of uses, not only for studying the interaction of TCRs with pmhcs, but also as diagnostic tools for detecting infections or as markers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic or immunostimulatory compounds) to cells presenting a specific antigen, and in addition, soluble TCRs can be conjugated to other molecules (e.g., anti-CD3 antibodies) to redirect T cells to target them to cells presenting a particular antigen. Soluble TCRs specific for the MAGE-a3 antigen short peptide were also obtained by the present invention.

To obtain a soluble TCR, in one aspect, the inventive TCR may be one in which an artificial disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a disulfide bond is formed by substituting Thr48 of exon 1 of TRAC × 01 and a cysteine residue of Ser57 of exon 1 of TRBC1 × 01 or TRBC2 × 01. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 50, or up to 30, or up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include a cysteine residue for the purpose of deleting the native disulphide bond, or by mutating the cysteine residue forming the native disulphide bond to another amino acid.

As described above, the TCRs of the invention may comprise artificial disulfide bonds introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native disulfide bond present in the TCR.

To obtain a soluble TCR, on the other hand, the inventive TCR also comprises a TCR having a mutation in its hydrophobic core region, preferably a mutation that enables an improved stability of the inventive soluble TCR, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or

positions

3,5,7 of the reciprocal amino acid position of the short peptide of the alpha chain J gene (TRAJ), and/or

positions

2,4,6 of the reciprocal amino acid position of the short peptide of the beta chain J gene (TRBJ), wherein the position numbering of the amino acid sequence is according to the position numbering listed in the International immunogenetic information System (IMGT). The above-mentioned international system of immunogenetics information is known to the skilled person and the position numbering of the amino acid residues of the different TCRs in IMGT can be derived from this database.

The TCR with the mutated hydrophobic core region of the invention can be a stable soluble single chain TCR formed by connecting the variable domains of the alpha and beta chains of the TCR by a flexible peptide chain. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains.

For the purposes of the present invention, the inventive TCRs are moieties having at least one TCR α and/or TCR β chain variable domain. They typically comprise both a TCR α chain variable domain and a TCR β chain variable domain. They may be α β heterodimers or single chain forms or any other form that is stable. In adoptive immunotherapy, the entire long chain (containing both cytoplasmic and transmembrane domains) of an α β heterodimeric TCR can be transfected. The TCRs of the invention are useful as targeting agents for delivering therapeutic agents to antigen presenting cells or in combination with other molecules to produce bifunctional polypeptides for targeting effector cells, where the TCRs are preferably in soluble form.

For stability, it is disclosed in the prior art that the introduction of an artificial interchain disulfide bond between the α and β chain constant domains of a TCR enables soluble and stable TCR molecules to be obtained, as described in patent document PCT/CN 2015/093806. Thus, the inventive TCR may be one in which an artificial interchain disulfide bond is introduced between residues of the constant domains of its alpha and beta chains. Cysteine residues form an artificial interchain disulfide bond between the alpha and beta chain constant domains of the TCR. Cysteine residues may be substituted for other amino acid residues at appropriate positions in native TCRs to form artificial interchain disulfide bonds. For example, a substitution of Thr48 for exon 1 of TRAC × 01 and a substitution of Ser57 for exon 1 of TRBC1 × 01 or TRBC2 × 01 form disulfide bonds. Other sites for introducing cysteine residues to form disulfide bonds may also be: thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser77 of TRBC2 × 01 exon 1; tyr10 and TRBC1 x 01 of exon 1 of TRAC x 01 or Ser17 of exon 1 of TRBC2 x 01; thr45 and TRBC1 × 01 of TRAC × 01 exon 1 or Asp59 of TRBC2 × 01 exon 1; ser15 and TRBC1 × 01 of TRAC × 01 exon 1 or Glu15 of TRBC2 × 01 exon 1; arg53 and TRBC1 × 01 of TRAC × 01 exon 1 or Ser54 of TRBC2 × 01 exon 1; pro89 and TRBC1 and 01 of exon 1 of TRAC 01 or Ala19 of exon 1 of TRBC2 and 01; or Tyr10 and TRBC1 and 01 of TRAC 01 exon 1 or Glu20 of TRBC2 and 01 exon 1. I.e., a cysteine residue, in place of any of the above-described alpha and beta chain constant domains. The TCR constant domains of the invention may be truncated at one or more of their C-termini by up to 15, or up to 10, or up to 8 or fewer amino acids, so as not to include cysteine residues for the purpose of deleting the native interchain disulphide bond, or by mutating the cysteine residues forming the native interchain disulphide bond to another amino acid.

As described above, the TCRs of the invention may comprise an artificial interchain disulfide bond introduced between residues of the constant domains of their alpha and beta chains. It should be noted that the TCRs of the invention may each contain both TRAC constant domain sequences and TRBC1 or TRBC2 constant domain sequences, with or without the artificial disulfide bonds introduced as described above between the constant domains. The TRAC constant domain sequence and TRBC1 or TRBC2 constant domain sequences of the TCR may be linked by the native interchain disulfide bonds present in the TCR.

In addition, for stability, patent document PCT/CN2016/077680 also discloses that the introduction of an artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR can significantly improve the stability of the TCR. Thus, the high affinity TCRs of the invention may also contain an artificial interchain disulfide bond between the α chain variable region and the β chain constant region. Specifically, the cysteine residues that form the artificial interchain disulfide bond between the α chain variable region and the β chain constant region of the TCR are substituted for: amino acid 46 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 47 of TRAV and amino acid 61 of exon 1 of TRBC1 x 01 or TRBC2 x 01; amino acid 46 of TRAV and amino acid 61 of TRBC1 x 01 or TRBC2 x 01 exon 1; or amino acid 47 of TRAV and amino acid 60 of exon 1 of TRBC1 x 01 or TRBC2 x 01. Preferably, such a TCR may comprise (i) all or part of a TCR α chain, excluding its transmembrane domain, and (ii) all or part of a TCR β chain, excluding its transmembrane domain, wherein (i) and (ii) both comprise the variable domain and at least part of the constant domain of the TCR chain, the α chain forming a heterodimer with the β chain. More preferably, such a TCR may comprise the a chain variable domain and the β chain variable domain and all or part of the β chain constant domain, excluding the transmembrane domain, but which does not comprise the a chain constant domain, the a chain variable domain of the TCR forming a heterodimer with the β chain.

For stability, on the other hand, the inventive TCRs also include TCRs having mutations in their hydrophobic core region, preferably mutations that improve the stability of the inventive TCRs, as described in the patent publication WO 2014/206304. Such TCRs may be mutated at the following variable domain hydrophobic core positions: (alpha and/or beta chain) variable region amino acid positions 11, 13, 19, 21, 53, 76, 89, 91, 94, and/or

positions

More specifically, the TCR with the mutated hydrophobic core region of the invention can be a high stability single chain TCR with a flexible peptide chain connecting the variable domains of the α and β chains of the TCR. The CDR regions of the variable region of the TCR determine the affinity with the short peptide-HLA complex, and the mutation of the hydrophobic core can stabilize the TCR without affecting the affinity with the short peptide-HLA complex. It should be noted that the flexible peptide chain of the present invention can be any peptide chain suitable for linking the TCR α and β chain variable domains. The template chain for screening high affinity TCR constructed in example 1 of the present invention is the above-described high stability single chain TCR comprising the hydrophobic core mutation. The affinity between the TCR and the KVAELVHFL-HLA-a 0201 complex can be more conveniently assessed using TCRs with higher stability.

The TCRs of the invention may also be provided in the form of multivalent complexes. Multivalent TCR complexes of the invention comprise polymers formed by association of two, three, four or more TCRs of the invention, such as might be produced as a tetramer using the tetrameric domain of p53, or a complex formed by association of a plurality of TCRs of the invention with another molecule. The TCR complexes of the invention can be used to track or target cells presenting a particular antigen in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes having such applications.

The TCRs of the invention may be used alone or in covalent or other association, preferably covalently, with a conjugate. The conjugates include a detectable label (for diagnostic purposes, where the TCR is used to detect the presence of cells presenting the MAGE-A3:112-120(KVAELVHFL): HLA-A0201 complex), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that may be associated or conjugated with the TCRs of the invention include, but are not limited to: 1. radionuclides (Koppe et al, 2005, Cancer metastasis reviews (Cancer metastasis) 24, 539); 2. biotoxicity (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and Immunotherapy 51, 565); 3. cytokines such as IL-2 and the like (Gillies et al, 1992, Proc. Natl. Acad. Sci. USA (PNAS)89, 1428; Card et al, 2004, Cancer Immunology and Immunotherapy)53, 345; Halin et al, 2003, Cancer Research 63, 3202); 4. antibody Fc fragment (Mosquera et al, 2005, Journal Of Immunology 174, 4381); 5. antibody scFv fragments (Zhu et al, 1995, International Journal of Cancer 62,319); 6. gold nanoparticles/nanorods (Lapotko et al, 2005, Cancer letters 239, 36; Huang et al, 2006, Journal of the American Chemical Society 128, 2115); 7. viral particles (Peng et al, 2004, Gene therapy 11, 1234); 8. liposomes (Mamot et al, 2005, Cancer research 65, 11631); 9. nano magnetic particles; 10. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 11. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

Antibodies or fragments thereof that bind to the TCRs of the invention include anti-T cell or NK-cell determining antibodies, such as anti-CD3 or anti-CD 28 or anti-CD 16 antibodies, whose binding to the TCR directs effector cells to better target cells. A preferred embodiment is the binding of a TCR of the invention to an anti-CD3 antibody or a functional fragment or variant of said anti-CD3 antibody. Specifically, the fusion molecule of the TCR and the anti-CD3 single-chain antibody comprises TCR alpha chain variable domain amino acid sequence SEQ ID NO 1 and TCR beta chain variable domain amino acid sequence SEQ ID NO 2.

It should be understood that the amino acid names herein are expressed in terms of international single-letter or three-letter english letters, and the single-letter english letter and three-letter english letters of the amino acid names correspond to the following relationships: ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).

In the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Addition of one or several amino acids at the C-terminus and/or N-terminus will not generally alter the structure and function of the protein. Thus, the TCR of the invention also includes TCRs in which up to 5, preferably up to 3, more preferably up to 2, most preferably 1 amino acid (especially outside the CDR regions) of the TCR of the invention has been replaced by amino acids of similar or analogous nature, and still retain its functionality.

The invention also includes TCRs that are slightly modified from the TCRs of the invention. Modified (generally without altering primary structure) forms include: chemically derivatized forms of the inventive TCR, such as acetylation or carboxylation. Modifications also include glycosylation, such as those that result from glycosylation modifications made during synthesis and processing or during further processing steps of the inventive TCR. Such modification may be accomplished by exposing the TCR to an enzyme that effects glycosylation, such as mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are TCRs that have been modified to improve their resistance to proteolysis or to optimize solubility.

Binding affinity (equilibrium constant K to dissociation) can be determined by any suitable method_DInversely proportional) and binding half-life (denoted T)_1/2). It will be appreciated that doubling the affinity of the TCR will result in K_DAnd (4) halving. T is_1/2Calculated as In2 divided by dissociation rate (K)_off). Thus, T_1/2Doubling can result in K_offAnd (4) halving. Preferably, the binding affinity or binding half-life of a given TCR is measured several times, e.g. 3 times or more, using the same assay protocol, and the results are averaged. In a preferred embodiment, these assays are performed using the surface plasmon resonance (BIAcore) method in the examples herein.

Nucleic acid molecules

The invention also relates to nucleic acid molecules encoding the inventive TCRs. The nucleic acid molecules of the invention may be in the form of DNA or in the form of RNA. The DNA may be the coding strand or the non-coding strand. For example, a nucleic acid sequence encoding a TCR of the present invention may be identical to or a degenerate variant of a nucleic acid sequence as set out in the figures of the present invention. By way of illustration of the meaning of "degenerate variant", as used herein, is meant a nucleic acid sequence which encodes a protein sequence having SEQ ID NO. 1, but differs from the sequence of SEQ ID NO. 3.

A second aspect of the invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the invention or a portion thereof, which may be one or more CDRs, variable domains of the alpha and/or beta chains, and the alpha and/or beta chains.

The nucleotide sequence encoding the α chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:

CDR1α：ACCATTTATTCCAATCCCTTC，SEQ ID NO:11

CDR2α：TCATTTACTGATAATAAACGA，SEQ ID NO:12

CDR3α：GCCTTTGACACCAATGCTTATAAGGTGATC，SEQ ID NO:13

the nucleotide sequence encoding the β chain CDR regions of the TCR molecules of the first aspect of the invention is as follows:

CDR1β：ATGTCCCATGAGACC，SEQ ID NO:14

CDR2β：AGCTATGACGTGGATAGC，SEQ ID NO:15

CDR3β：GCAAGTTCAAGCACCAACACAGAGGTGTTC，SEQ ID NO:16

thus, the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR α chain of the invention comprises SEQ ID NO 19 and/or the nucleotide sequence of a nucleic acid molecule of the invention encoding a TCR β chain of the invention comprises SEQ ID NO 20.

Preferably, the nucleotide sequence encoding the TCR α chain is SEQ ID NO:19

GGCGACTCTGTCACCCAGACAGAGGGACTGCTGAACGTGCCTGAAGGCCTGCCAGTCAGCATCAACTGTACTTACCAGACCATTTATTCCAATCCCTTCCTGTTTTGGTACAGACAGGACCCCGGAAAGAGTCCTAGGCTGCTGCTGAAGTCATTTACTGATAATAAACGAACCGAGCACCAGCGGTTCCATGCTACCCTGCACAAATCTGACAGCTCCTTTCACCTGCAGATCGAACGGATTCAGCCAAACGATAGCGGCACTTACTTCTGCGCCTTTGACACCAATGCTTATAAGGTGATCTTCGGCAAAGGGACACACCTGCATGTCCTGCCCTACATTCAGAACCCAGATCCCGCCGTGTATCAGCTGAGGGACTCAAAGTCTAGTGATAAAAGCGTGTGCCTGTTCACCGACTTTGATTCTCAGACAAATGTCTCCCAGTCTAAGGACAGTGATGTGTATATCACTGACAAATGTGTCCTGGATATGCGCAGCATGGACTTTAAGAGTAACTCAGCCGTGGCTTGGAGTAATAAATCAGACTTCGCATGCGCCAACGCTTTTAACAATTCAATCATTCCTGAGGATACATTCTTTCCTAGCCCAGAATCAAGCTGTGACGTGAAGCTGGTCGAGAAATCTTTCGAAACTGATACCAACCTGAATTTTCAGAACCTGAGCGTGATCGGCTTCCGGATTCTGCTGCTGAAGGTCGCCGGGTTCAATCTGCTGATGACCCTGAGACTGTGGTCCTCT。

Preferably, the nucleotide sequence encoding the TCR β chain is SEQ ID NO:20

GATATGAAGATCACACAGACTCCTAGGTACCTGATTGTGAAAACAGGGGAGAACGTCACTCTGGAATGCGGACAGGACATGTCCCATGAGACCATGTACTGGTATCGACAGGACCCCGGACAGGGACTGCAGCTGATCTACATTAGCTATGACGTGGATAGCAATTCCGAGGGCGATATCCCCAAGAGGTACCGCGTGTCCAGAAAGAAAAGGGAACACTTCAGCCTGCGGATTGATTCCGTGAAAACCTCTGACAGTGCTCTGTATCTGTGTGCAAGTTCAAGCACCAACACAGAGGTGTTCTTTGGCCCAGGGACAAGACTGACTGTGGTCGAAGACCTGAAGAATGTGTTCCCCCCTGAGGTGGCTGTCTTTGAACCTTCTGAGGCAGAAATCAGTCATACCCAGAAAGCAACACTGGTGTGCCTGGCCACAGGGTTCTACCCAGATCATGTGGAGCTGTCCTGGTGGGTCAACGGCAAGGAAGTGCACTCTGGGGTCTGTACTGACCCACAGCCCCTGAAAGAGCAGCCCGCCCTGAATGATAGTAGATACGCTCTGTCCTCTCGACTGCGAGTGTCCGCAACCTTCTGGCAGGACCCTCGGAACCACTTCAGATGCCAGGTGCAGTTTTATGGCCTGTCTGAGAATGATGAATGGACACAGGACCGCGCTAAGCCCGTGACTCAGATTGTCAGCGCAGAGGCCTGGGGGCGAGCAGATTGTGGATTTACATCAGAAAGCTATCAGCAGGGGGTGCTGAGCGCCACTATCCTGTACGAGATTCTGCTGGGAAAGGCTACCCTGTATGCAGTGCTGGTCAGCGCCCTGGTGCTGATGGCTATGGTCAAGAGGAAAGACTCCCGCGGCTAA。

The nucleotide sequence of the nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not comprise an intron. Preferably, the nucleotide sequence of the nucleic acid molecule of the invention does not comprise an intron but is capable of encoding a polypeptide of the invention, e.g. the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR alpha chain variable domain of the invention comprises SEQ ID No. 3 and/or the nucleotide sequence of the nucleic acid molecule of the invention encoding a TCR beta chain variable domain of the invention comprises SEQ ID No. 3. More preferably, the nucleotide sequence of the nucleic acid molecule of the invention comprises SEQ ID NO 3 and/or SEQ ID NO 4.

Preferably, the nucleotide sequence encoding the variable domain of the TCR α chain is SEQ ID NO:3

GGCGACTCTGTCACCCAGACAGAGGGACTGCTGAACGTGCCTGAAGGCCTGCCAGTCAGCATCAACTGTACTTACCAGACCATTTATTCCAATCCCTTCCTGTTTTGGTACAGACAGGACCCCGGAAAGAGTCCTAGGCTGCTGCTGAAGTCATTTACTGATAATAAACGAACCGAGCACCAGCGGTTCCATGCTACCCTGCACAAATCTGACAGCTCCTTTCACCTGCAGATCGAACGGATTCAGCCAAACGATAGCGGCACTTACTTCTGCGCCTTTGACACCAATGCTTATAAGGTGATCTTCGGCAAAGGGACACACCTGCATGTCCTGCCC。

Preferably, the nucleotide sequence encoding the variable domain of the TCR β chain is SEQ ID NO:4

GATATGAAGATCACACAGACTCCTAGGTACCTGATTGTGAAAACAGGGGAGAACGTCACTCTGGAATGCGGACAGGACATGTCCCATGAGACCATGTACTGGTATCGACAGGACCCCGGACAGGGACTGCAGCTGATCTACATTAGCTATGACGTGGATAGCAATTCCGAGGGCGATATCCCCAAGAGGTACCGCGTGTCCAGAAAGAAAAGGGAACACTTCAGCCTGCGGATTGATTCCGTGAAAACCTCTGACAGTGCTCTGTATCTGTGTGCAAGTTCAAGCACCAACACAGAGGTGTTCTTTGGCCCAGGGACAAGACTGACTGTGGTC。

The nucleotide sequence may be codon optimized. Different cells differ in the utilization of specific codons, and the expression level can be increased by changing the codons in the sequence according to the type of the cell. Codon usage tables for mammalian cells as well as for various other organisms are well known to those skilled in the art.

The full-length sequence of the nucleic acid molecule of the present invention or a fragment thereof can be obtained by, but not limited to, PCR amplification, recombination, or artificial synthesis. At present, DNA sequences encoding the TCRs of the invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. The DNA may be the coding strand or the non-coding strand.

Carrier

The invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, i.e. constructs capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages and animal and plant viruses.

Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.

Preferably, the vector can transfer the nucleotide of the invention into a cell, for example a T cell, such that the cell expresses a TCR specific for the MAGE-a3 antigen. Ideally, the vector should be capable of sustained high level expression in T cells.

Cells

The invention also relates to genetically engineered host cells that have been engineered with the vectors or coding sequences of the invention. The host cell comprises a vector of the invention or has integrated into its chromosome a nucleic acid molecule of the invention. The host cell is selected from: prokaryotic and eukaryotic cells, such as E.coli, yeast cells, CHO cells, and the like.

In addition, the invention also includes isolated cells, particularly T cells, that express the TCRs of the invention. The T cell may be derived from a T cell isolated from a subject, or may be part of a mixed population of cells isolated from a subject, such as a population of Peripheral Blood Lymphocytes (PBLs). For example, the cells may be isolated from Peripheral Blood Mononuclear Cells (PBMC), which may be CD4⁺Helper T cell or CD8⁺Cytotoxic T cells. The cell may be in CD4⁺Helper T cell/CD 8⁺A mixed population of cytotoxic T cells. Generally, the cells can be activated with an antibody (e.g., an anti-CD3 or anti-CD 28 antibody) to render them more amenable to transfection, e.g., transfection with a vector comprising a nucleotide sequence encoding a TCR molecule of the invention.

Alternatively, the cell of the invention may also be or be derived from a stem cell, such as a Hematopoietic Stem Cell (HSC). Gene transfer to HSCs does not result in TCR expression on the cell surface, since the CD3 molecule is not expressed on the stem cell surface. However, when stem cells differentiate into lymphoid precursors (lymphoid precursors) that migrate to the thymus, expression of the CD3 molecule will initiate expression of the introduced TCR molecule on the surface of the thymocytes.

The invention also includes isolated cells, particularly T cells, expressing a TCR of the invention. There are many methods suitable for T cell transfection using DNA or RNA encoding the high affinity TCRs of the invention (e.g., Robbins et al, (2008) J.Immunol.180: 6116-. T cells expressing the high affinity TCRs of the invention may be used for adoptive immunotherapy. Those skilled in the art will be able to recognize many suitable methods for adoptive therapy (e.g., Rosenberg et al, (2008) Nat Rev Cancer8 (4): 299-308).

Pharmaceutical composition

The invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention.

The TCR of the invention, the TCR complex or the TCR-transfected T cell of the invention may be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier. The TCRs, multivalent TCR complexes or cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier. The pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It may be provided in unit dosage form, typically in a sealed container, and may be provided as part of a kit. Such kits (but not necessarily) include instructions for use. It may comprise a plurality of said unit dosage forms.

In addition, the TCRs of the invention may be used alone, or in combination or coupling with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent. The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

Pharmaceutically acceptable carriers in therapeutic compositions can comprise liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.

Generally, the therapeutic compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; solid forms suitable for constitution with a solution or suspension, or liquid carrier, before injection, may also be prepared.

Once formulated, the compositions of the present invention may be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated may be an animal; especially a human.

When the pharmaceutical composition of the present invention is used for practical treatment, various dosage forms of the pharmaceutical composition may be used depending on the use case. Preferably, injections, oral agents and the like are exemplified.

These pharmaceutical compositions may be formulated by mixing, dilution or dissolution according to a conventional method, and occasionally, suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents (isotonicities), preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizing agents are added, and the formulation process may be carried out in a conventional manner according to the dosage form.

The pharmaceutical compositions of the present invention may also be administered in the form of sustained release formulations. For example, the inventive TCR may be incorporated into a pellet or microcapsule carried by a slow release polymer, which pellet or microcapsule is then surgically implanted into the tissue to be treated. As examples of the sustained-release polymer, ethylene-vinyl acetate copolymer, polyhydroxymethacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer and the like can be exemplified, and biodegradable polymers such as lactic acid polymer and lactic acid-glycolic acid copolymer can be preferably exemplified.

When the pharmaceutical composition of the present invention is used for practical treatment, the TCR or TCR complex of the present invention or the cells presenting the TCR of the present invention as an active ingredient can be determined reasonably according to the body weight, age, sex, degree of symptoms of each patient to be treated, and finally the reasonable amount is decided by a physician.

Method of treatment

The invention also provides a method of treating a disease comprising administering to a subject in need thereof an amount of a TCR of the invention, or a TCR complex of the invention, or a cell presenting a TCR of the invention, or a pharmaceutical composition of the invention.

The invention also relates to a method of treating and/or preventing a disease associated with the human MAGE-A3 antigen in a subject, comprising the step of adoptively transferring MAGE-A3 specific T cells to the subject. The MAGE-A3-specific T cells recognize a complex of MAGE-A3:112-120(KVAELVHFL) and HLA-A0201.

The MAGE-A3 specific T cells of the invention can be used to treat any MAGE-A3 related disease presenting the human MAGE-A3 antigen short peptide KVAELVHFL and HLA-A0201 complex. Including but not limited to non-small cell lung cancer, breast cancer, melanoma, squamous cell carcinoma, liver cancer.

Treatment may be effected by isolating T cells from patients or volunteers suffering from a disease associated with the MAGE-A3 antigen and introducing the TCR of the invention into such T cells, followed by reinfusion of these genetically engineered cells into the patient. Accordingly, the present invention provides a method of treating a disease associated with MAGE-a3 comprising infusing into a patient isolated T cells expressing a TCR of the invention, preferably the T cells are derived from the patient themselves. Generally, this involves (1) isolating T cells from the patient, (2) transducing T cells in vitro with a nucleic acid molecule of the invention or a nucleic acid molecule capable of encoding a TCR molecule of the invention, and (3) infusing the genetically modified T cells into the patient. The number of cells isolated, transfected and transfused can be determined by a physician.

The main advantages of the invention include:

(1) the humanized TCR of the invention shows better affinity, and the binding dissociation constant is 2.3 times of that of the murine TCR before modification.

(2) The present invention is derived from the TCR in mice and thus solves the problem of finding a limited source of TCR.

(3) The humanized TCR prepared by the invention not only shows better affinity, but also maintains the specificity of the humanized TCR.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.

General procedure

Method for transforming humanized TCR and verifying function thereof

(1) Finding out a human TCR sequence with the highest homology with the mouse TCR sequence through sequence comparison;

(2) grafting the CDR region of the mouse TCR to the human TCR framework;

(3) grafting the CDR region of the mouse TCR and introducing a site for maintaining the conformational stability after CDR grafting;

(4) performing in-vitro renaturation expression on the humanized TCR subjected to CDR transplantation, measuring the antigen recognition capacity and affinity of the humanized TCR, and comparing the antigen recognition capacity and affinity with the murine TCR;

(5) transfer of murine and humanized TCRs into CD3, respectively⁺T cells, and the expression ability, the ability to recognize short peptides, the ability to release factors, and the ability to kill tumor cells are measured for comparison.

Example 1

The detailed method or steps for the transformation of the humanized TCR and the experimental verification of the cell function are as follows:

TCR engineering

1. Sequence alignment

The CDR regions of the murine TCR were grafted onto the human TCR backbone by sequence alignment and the sequence of the stability site was introduced to give SRm1g13t (see FIG. 1).

1.1 sequence alignment of the V regions of the α and β chains of murine TCRs with the TRAV and TRBV database sequences in the human TCR library on IMGT to find the most similar sequences, e.g., TRAV18 x 01 and TRBV28 x 01 (FIGS. 1-A and 1-B).

1.2 grafting the CDR regions (CDR1, CDR2, CDR3) of the α, β chain V regions of the murine TCR directly into the corresponding framework regions of TRAV18 x 01 and TRBV28 x 01 to form the corresponding V regions of the humanized TCR α, β chains which are not optimized for stability, plus the corresponding C regions to form the humanized TCR α, β chains which are not optimized for stability (which can be named SRm1CLa and SRm1 CLb). Meanwhile, stability optimization is carried out on the basis of SRm1CLa and SRm1CLb, stability sites for stabilizing humanized TCR conformation are analyzed through computer simulation, so that humanized TCR alpha and beta chains with optimized stability (SRm1a g13t and SRm1b g13t) are formed, corresponding C regions are added to the V regions (figure 1) to form humanized TCR alpha and beta chains with optimized stability, and then in vitro renaturation and affinity determination are also needed.

2. In vitro renaturation:

2.1 Synthesis of the α, β chain sequence of the TCR (the C region used in vitro renaturation is the human C region), cloning it into pet28a vector, transferring it into BL23(DE3) competence, picking the positive clones and sequencing.

2.2 inoculating the clone with correct sequence, detecting the pre-expression of the inclusion body, and observing whether the protein is successfully expressed and soluble under induction.

2.3 inoculating the bacterial liquid into LB culture medium containing antibiotic Kan, inducing for 4h by 0.5M IPTG, centrifuging to collect bacteria, and purifying inclusion bodies.

2.4 denaturation of the inclusion bodies of purified alpha, beta chains with 6M guanidine hydrochloride and 15uM at 37 ℃ for 40min, followed by addition of 50mg/ml SRm1a g13t and 30mg/ml SRm1b g13t refolding buffers (comprising 100mM Tris-HCl,0.4M L-argine, 5M Urea,2mM EDTA,6.5mM cysteamine and 1.87mM cystamine, pH adjusted to 8.1) to H₂Dialyzed in a freezer overnight in O and then twice with 10mM Tris-HCl.

2.5 renaturation and dialysis of the sample using anion exchange column (QHP) gradient elution, the eluent is 10mMTris-HCl, pH 8.1/1M NaCl, the elution sample in SDS-PAGE on the target protein identification, the target protein with 10KD ultra filtration tube to 500 ul.

2.6 then passing through a molecular sieve, eluting with PBS, identifying the target protein of the eluted sample on SDS-PAGE, concentrating the target protein by using an ultrafiltration tube with 10KD, and quantitatively storing the target protein by using BCA.

3. Affinity identification

3.1 affinity assay with Biacore T200, Streptavidin (SA) -coated CM5 chip to capture biotinylated pHLA (prepared in advance) molecules, TCR with good renaturation at different concentrations for binding dissociation test, and Biacore T200 analysis software 1: 1 simulation, calculating the affinity.

Second, TCR cell function experiment verification

1. Construction of electrotransport vectors for mRNA preparation

1.1 the α, β chain (where the C region sequence needs to use the murine C region, because the murine C region is not found to be immunogenic and can solve the problem of endogenous TCR mismatching and improve the expression of the transferred TCR) sequence of the TCR is constructed on the RNA expression vector pGEM-4Z by sequence optimization, and plasmids are extracted to prepare mRNA.

1.2 mRNA is prepared by using the mMESSAGE mMECHANINE kit, and is identified by running glue.

2. Electrotransfer CD3⁺Functional assay of T cells

2.1 isolation of CD3⁺T cells were cultured using magnetic beads (magnetic beads of Human T-activation anti-CD 3/28) in a volume of 1: 1, cultured in 12-well plates for 48-72h in RPMI-1640+50U/ml IL-2+ 10% FBS.

2.2 stimulated CD3⁺T cells were then electroporated using a Lonza's electrotransfer apparatus and cultured overnight.

2.3 the next day, staining with corresponding anti-mouse beta chain (anti-mouse beta chain) and anti-CD3 antibody, detecting the positive rate of the transferred TCR by running flow, staining with anti-CD8 antibody and pHLA tetramer, and observing the relationship between affinity and staining tetramer according to the positive rate of tetramer.

2.4 after the positive rate is detected, the specificity and the non-specificity of the TCR are firstly verified, IFN-gamma Elispot is used for verification, and the corresponding specific cell is HLA-A0201⁺/MAGE-A3⁺T2 was loaded with the specific peptides MAGE-A3:112-120(KVAELVHFL), NCI-H1299-A2, U266B1, the corresponding nonspecific cells were HLA-A0201⁺/MAGE-A3^-Cell: t2 and HLA-A0201^-/MAGE-A3⁺Cell: NCI-H1299. 2X 10³Positive expression of TCR-transferred CD3⁺ T cellsAnd 1X 10⁴The target cells were co-incubated at 37 ℃ for 24h and then read using an Elispot reader.

2.5 killing validation 7500 Positive expression of TCR-transferred CD3⁺T cells and 1500 target cells (U266B1, NCI-H1299, NCI-H1299-A2) were CO-incubated in a 96-well round bottom plate at 5% CO₂At 37 ℃. After 24h, the supernatant was centrifuged and the killing efficiency was calculated by the equation by detecting Lactate Dehydrogenase (LDH) in the supernatant.

Calculating the formula: killing rate ═ experimental well-effector cell spontaneous well-target cell spontaneous well)/(target cell maximal lysis well-target cell spontaneous well)

2.6 carrying out the experimental verification of the capability of the T cells to release the factors: 1X 10⁵Positive expression of TCR-transferred CD3⁺T cells and 2X 10⁵Target cells in 48 wells at 5% CO₂Cultured overnight at 37 ℃ in RPMI-1640+ 10% FBS. Golgi blocking agent (Brefeldin A) is added 6h before detection, and after 6h, anti-IFN-gamma, anti-IL-2 and anti-TNF alpha antibodies are co-dyed simultaneously and respectively by dyeing anti-CD3 antibodies, so that the capacities of TCR endowing T cells with release factors are compared by analyzing the positive rates of intracellular IFN-gamma, IL-2 and TNF alpha in CD3 positive cells.

Results and discussion of engineering of humanized TCR

Alpha, beta chain inclusion body purification of SRm1 and SRm1g13t

After codon optimization, gene synthesis is carried out, a required sequence (shown in figure 2-A) is cloned to a pET28a vector through enzyme digestion, and then is transferred into a BL21(DE3) strain, and expression is induced through IPTG. The α, β chain inclusion body proteins of SRm1 and SRm1g13t were purified using BugBuster, and identified by running SDS-PAGE after dilution with 6M urea fold-ratio, and the purity was estimated approximately by comparing the ratio of inclusion body protein to hetero-protein. As shown in FIGS. 2-B and 2-C, the inclusion bodies of alpha and beta chains of purified SRm1 and SRm1g13t were more than 90% pure and matched in size to the expected 22kDa alpha chain and 28kDa beta chain.

In vitro renaturation purification of SRm1 and SRm1g13t TCR and its ligand pHLA

Through in vitro renaturation after deformation of alpha and beta chain inclusion body proteins of SRm1 and SRm1g13t, a target sample is collected through a primary anion exchange column, and then concentrated to 500 mu l, and then the target sample passes through a molecular sieve and is eluted by PBS (phosphate buffer solution), as shown in a figure 3-A and a figure 3-B, the main elution peaks of SRm1 and SRm1g13t TCR are obvious and single. Meanwhile, in order to determine the affinity of SRm1 and SRm1g13t TCR, a ligand pHLA molecule needs to be prepared. HLA-A0201 and beta 2m inclusion body protein is denatured and then is subjected to in vitro renaturation with synthetic antigen peptide MAGE-A3:112-120(KVAELVHFL), biotin labeling is added through a BirA biotin ligase substrate peptide fused with the C terminal of the HLA-A0201 gene, and the target protein is purified and eluted through a final molecular sieve, wherein the sample elution peak is obvious as shown in figure 3-C, and finally, the biotinylation efficiency is about 95% through the biotinylation analysis of figure 3-D.

SRm1 and SRm1g13t TCR affinity assays

To analyze the ability of SRm1 and SRm1g13t TCRs to recognize and bind pHLA (MAGE-A3), Biacore was used to assay the affinity of the TCRs. Both SRm1 and SRm1g13t TCRs showed the ability to recognize binding to pHLA (MAGE-A3), where K of SRm1_D587.2. mu.M. However, for humanized antibodies, the humanized antibody is often exposed to a phenomenon of reduced affinity following humanization, whereas for humanized TCR, SRm1g13t K_D238.3. mu.M was reached, showing higher affinity. It is clear that the half-lives of SRm1 and SRm1g13t are almost 1.8s and 1.9s, respectively, and that the dissociation constants are also almost 3.8X 10, respectively^-1s^-1And 3.6X 10^-1s^-1. However, the binding constant of SRm1g13t was 1.5X 10 higher³M^-1s^-1Whereas SRm1 is only 6.4X 10²M^-1s^-1See fig. 4-a and 4-B.

Positive rates of determination after transfection of CD3+ T cells with SRm1 and SRm1g13T TCR

In order to examine whether SRm1g13T TCR could be expressed on the surface of T cells after humanization, RNA expression vectors for SRm1 and SRm1g13T TCR were first designed, and as shown in FIG. 5-A, the α, β chains of SRm1 and SRm1g13T TCR were constructed on pGEM-4Z vectors, respectively. Then, the RNA of the alpha and beta chains of SRm1 and SRm1g13t TCR was electrotransferred into CD3 by electrotransfer⁺T cells expressingBy using anti-mouse TCR beta chain (anti-mouse TCR beta chain) antibody, it can be found that both SRm1 and SRm1g13t TCR can be in CD3⁺Expressed on T cells and the positive rates were comparable, 94.2% and 91.8%, respectively. Meanwhile, by detecting the binding of pHLA (MAGE-A3) tetramer, it can be found that SRm1g13t has 7% more pHLA (MAGE-A3) tetramer binding than SRm1 binding, which is more than 3% of SRm1, and this also indicates that the affinity affects the binding ability of pHLA (MAGE-A3) tetramer, as shown in FIG. 5-B and FIG. 5-C.

5. CD3 transfected with SRm1 and SRm1g13t TCR⁺Specific assay for T cells

To verify CD3 transfected with SRm1 and SRm1g13t TCR⁺Recognition of cell specificity by T cells CD3 transfected with SRm1 and SRm1g13T TCR⁺T cells were incubated with T2-loaded with specific short peptides, NCI-H1299-A2, U266B1, T2 and NCI-H1299, respectively, to detect IFN-. gamma.release. As shown in FIG. 6-A, for specific cell T2 loaded with specific short peptide, NCI-H1299-A2, U266B1, CD3 transfected with SRm1g13T TCR⁺T cells showed better IFN-. gamma.releasing ability, while NCI-H1299, non-specific cells, CD3 transfected with SRm1 and SRm1g13T TCR⁺None of the T cells recognized, suggesting the specificity of SRm1 and SRm1g13T TCR for HLA-A0201. Also for MAGE-A3 negative cells, as shown in FIG. 6-B, CD3 transfected with SRm1 and SRm1g13t TCR was found⁺Neither T cells recognized T2 cells, indicating that the TCR after humanization still retains better specificity.

6. CD3 transfected with SRm1 and SRm1g13t TCR⁺Comparison of killing Capacity of T cells

To verify CD3 transfected with SRm1 and SRm1g13t TCR⁺The ability of both T cells to kill tumor cells would be to transfect CD3 of SRm1 and SRm1g13T TCR⁺Validation of LDH assay after incubation of T cells with NCI-H1299-A2, U266B1 and NCI-H1299, respectively, for 24H, as shown in FIG. 7, it was found that CD3 transfected with SRm1 and SRm1g13T TCR⁺Both T cells have comparable ability to kill tumor cells and show nonspecific killing for NCI-H1299.

7. CD3 transfected with SRm1 and SRm1g13t TCR⁺Ability of T cells to release factor

To verify that SRm1 and SRm1g13t TCR were transfected with CD3⁺The ability of T cells to release different factors, CD3 transfected with SRm1 and SRm1g13T TCR⁺T cells were incubated overnight with NCI-H1299-A2 and CD3 transfected with different TCRs was compared by intracellular staining⁺The ability of T cells to release IFN-. gamma.IL-2, TNF. alpha. As shown in FIG. 8, CD3 transfected with SRm1g13t TCR⁺T cells showed a greater capacity to release IFN-. gamma.IL-2 than SRm1, almost twice that of SRm1, whereas for TNF. alpha, SRm1 and SRm1g13T TCR conferred CD3⁺T cells have comparable ability to release the factor. These results indicate that SRm1g13t TCR conferred CD3 over SRm1⁺T cells have a greater ability to release factors.

Discussion verification of immunogenicity with respect to engineered TCR

1. Non-clinical immunogenicity assessment

Immunogenicity was evaluated in non-clinical studies primarily to investigate the magnitude of immunogenicity of the tested TCRs and the possible impact of immunogenicity on safety assessments. In non-clinical toxicology testing, detection of antibodies raised by a humanized TCR may reflect to some extent the magnitude of immunogenicity of the humanized TCR. However, the immune response observed in animal experiments does not necessarily represent the same immune response of the humanized TCR in human, and particularly, the immunogenicity data of animals is of little significance to some humanized TCRs with few intergeneric cross reactions or intergeneric immune response mechanisms. Therefore, animals, such as monkeys, having a toxicological effect consistent with that of humans should be selected. In the absence of related species, it is contemplated to use a related transgenic animal expressing a human receptor. Detection of antibody responses is critical to the validation of toxicity tests because drug-induced antibody responses can affect pharmacokinetics, pharmacodynamics, and biological activities, and thus the data obtained from toxicity tests does not truly reflect the exposure level and activity of the tested drug.

2. Clinical study and evaluation of immunogenicity after marketing

In the immunogenicity assay, there is no clear data showing that the antibody response elicited by the humanized TCR may affect pharmacokinetics, efficacy and toxicity, as part of the evaluation of clinical safety of the humanized TCR. However, given the unpredictability of the incidence and frequency of immunogenicity, humanized TCRs should be marketed at intervals during the year when drug-induced antibody changes are detected.

3. Content of immunogenicity evaluation

3.1 antibody Titers and characteristics of antibodies

Before and after the back transfusion of T cells transfected with humanized TCR, blood samples are respectively taken and an immunological detection method is adopted to detect whether antibodies are generated or not and analyze antibody subtype, concentration and relative affinity.

3.2 neutralizing Activity of antibodies

Humanized TCRs produce antibodies that do not necessarily have neutralizing activity if the antibody is found to have neutralizing activity in vitro and it is observed that the return transfusion can be stopped by a reasonable majority of the experimental animal pharmacological or toxicological effects in the antibody produced.

3.3 immune Complex deposition

The antibody and the antigen are combined to generate immune complexes, the immune complexes are deposited on organs to cause case change, the analysis of the cause of formation reaction becomes difficult, whether tissues are always deposited or not can be detected by a cotton-padded clothes histochemical method, and whether the immune complexes are deposited to cause tissue lesion or not is analyzed by combining blood biochemical indexes and urine examination results which indicate organ function numbers.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Guangzhou biomedical and health research institute of Chinese academy of sciences

<120> MAGE-A3 humanized T cell receptor

<130> P2017-1923

<160> 27

<170> PatentIn version 3.5

<210> 1

<211> 112

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

Gly Asp Ser Val Thr Gln Thr Glu Gly Leu Leu Asn Val Pro Glu Gly

1 5 10 15

Leu Pro Val Ser Ile Asn Cys Thr Tyr Gln Thr Ile Tyr Ser Asn Pro

20 25 30

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

35 40 45

Leu Lys Ser Phe Thr Asp Asn Lys Arg Thr Glu His Gln Arg Phe His

50 55 60

Ala Thr Leu His Lys Ser Asp Ser Ser Phe His Leu Gln Ile Glu Arg

65 70 75 80

Ile Gln Pro Asn Asp Ser Gly Thr Tyr Phe Cys Ala Phe Asp Thr Asn

85 90 95

Ala Tyr Lys Val Ile Phe Gly Lys Gly Thr His Leu His Val Leu Pro

100 105 110

<210> 2

<211> 111

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 2

Asp Met Lys Ile Thr Gln Thr Pro Arg Tyr Leu Ile Val Lys Thr Gly

1 5 10 15

Glu Asn Val Thr Leu Glu Cys Gly Gln Asp Met Ser His Glu Thr Met

20 25 30

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

35 40 45

Ser Tyr Asp Val Asp Ser Asn Ser Glu Gly Asp Ile Pro Lys Arg Tyr

50 55 60

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

65 70 75 80

Val Lys Thr Ser Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Ser Thr

85 90 95

Asn Thr Glu Val Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Val

100 105 110

<210> 3

<211> 336

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ggcgactctg tcacccagac agagggactg ctgaacgtgc ctgaaggcct gccagtcagc 60

atcaactgta cttaccagac catttattcc aatcccttcc tgttttggta cagacaggac 120

cccggaaaga gtcctaggct gctgctgaag tcatttactg ataataaacg aaccgagcac 180

cagcggttcc atgctaccct gcacaaatct gacagctcct ttcacctgca gatcgaacgg 240

attcagccaa acgatagcgg cacttacttc tgcgcctttg acaccaatgc ttataaggtg 300

atcttcggca aagggacaca cctgcatgtc ctgccc 336

<210> 4

<211> 333

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

gatatgaaga tcacacagac tcctaggtac ctgattgtga aaacagggga gaacgtcact 60

ctggaatgcg gacaggacat gtcccatgag accatgtact ggtatcgaca ggaccccgga 120

cagggactgc agctgatcta cattagctat gacgtggata gcaattccga gggcgatatc 180

cccaagaggt accgcgtgtc cagaaagaaa agggaacact tcagcctgcg gattgattcc 240

gtgaaaacct ctgacagtgc tctgtatctg tgtgcaagtt caagcaccaa cacagaggtg 300

ttctttggcc cagggacaag actgactgtg gtc 333

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 5

Thr Ile Tyr Ser Asn Pro Phe

1 5

<210> 6

<211> 7

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 6

Ser Phe Thr Asp Asn Lys Arg

1 5

<210> 7

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 7

Ala Phe Asp Thr Asn Ala Tyr Lys Val Ile

1 5 10

<210> 8

<211> 5

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 8

Met Ser His Glu Thr

1 5

<210> 9

<211> 6

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 9

Ser Tyr Asp Val Asp Ser

1 5

<210> 10

<211> 10

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 10

Ala Ser Ser Ser Thr Asn Thr Glu Val Phe

1 5 10

<210> 11

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 11

accatttatt ccaatccctt c 21

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 12

tcatttactg ataataaacg a 21

<210> 13

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 13

gcctttgaca ccaatgctta taaggtgatc 30

<210> 14

<211> 15

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 14

atgtcccatg agacc 15

<210> 15

<211> 18

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 15

agctatgacg tggatagc 18

<210> 16

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 16

gcaagttcaa gcaccaacac agaggtgttc 30

<210> 17

<211> 199

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 17

Met Gly Asp Ser Val Thr Gln Thr Glu Gly Leu Leu Asn Val Pro Glu

1 5 10 15

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

20 25 30

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

35 40 45

Leu Leu Lys Ser Phe Thr Asp Asn Lys Arg Thr Glu His Gln Arg Phe

50 55 60

His Ala Thr Leu His Lys Ser Asp Ser Ser Phe His Leu Gln Ile Glu

65 70 75 80

Arg Ile Gln Pro Asn Asp Ser Gly Thr Tyr Phe Cys Ala Phe Asp Thr

85 90 95

Asn Ala Tyr Lys Val Ile Phe Gly Lys Gly Thr His Leu His Val Leu

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

180 185 190

Ser Ile Ile Pro Glu Asp Thr

195

<210> 18

<211> 243

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 18

Met Gly Asp Met Lys Ile Thr Gln Thr Pro Arg Tyr Leu Ile Val Lys

1 5 10 15

Thr Gly Glu Asn Val Thr Leu Glu Cys Gly Gln Asp Met Ser His Glu

20 25 30

Thr Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Gln Leu Ile

35 40 45

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

50 55 60

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

65 70 75 80

Asp Ser Val Lys Thr Ser Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser

85 90 95

Ser Thr Asn Thr Glu Val Phe Phe Gly Pro Gly Thr Arg Leu Thr Val

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Arg Ala Asp

<210> 19

<211> 759

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

ggcgactctg tcacccagac agagggactg ctgaacgtgc ctgaaggcct gccagtcagc 60

atcaactgta cttaccagac catttattcc aatcccttcc tgttttggta cagacaggac 120

cccggaaaga gtcctaggct gctgctgaag tcatttactg ataataaacg aaccgagcac 180

cagcggttcc atgctaccct gcacaaatct gacagctcct ttcacctgca gatcgaacgg 240

attcagccaa acgatagcgg cacttacttc tgcgcctttg acaccaatgc ttataaggtg 300

atcttcggca aagggacaca cctgcatgtc ctgccctaca ttcagaaccc agatcccgcc 360

gtgtatcagc tgagggactc aaagtctagt gataaaagcg tgtgcctgtt caccgacttt 420

gattctcaga caaatgtctc ccagtctaag gacagtgatg tgtatatcac tgacaaatgt 480

gtcctggata tgcgcagcat ggactttaag agtaactcag ccgtggcttg gagtaataaa 540

tcagacttcg catgcgccaa cgcttttaac aattcaatca ttcctgagga tacattcttt 600

cctagcccag aatcaagctg tgacgtgaag ctggtcgaga aatctttcga aactgatacc 660

aacctgaatt ttcagaacct gagcgtgatc ggcttccgga ttctgctgct gaaggtcgcc 720

gggttcaatc tgctgatgac cctgagactg tggtcctct 759

<210> 20

<211> 873

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

gatatgaaga tcacacagac tcctaggtac ctgattgtga aaacagggga gaacgtcact 60

ctggaatgcg gacaggacat gtcccatgag accatgtact ggtatcgaca ggaccccgga 120

cagggactgc agctgatcta cattagctat gacgtggata gcaattccga gggcgatatc 180

cccaagaggt accgcgtgtc cagaaagaaa agggaacact tcagcctgcg gattgattcc 240

gtgaaaacct ctgacagtgc tctgtatctg tgtgcaagtt caagcaccaa cacagaggtg 300

ttctttggcc cagggacaag actgactgtg gtcgaagacc tgaagaatgt gttcccccct 360

gaggtggctg tctttgaacc ttctgaggca gaaatcagtc atacccagaa agcaacactg 420

gtgtgcctgg ccacagggtt ctacccagat catgtggagc tgtcctggtg ggtcaacggc 480

aaggaagtgc actctggggt ctgtactgac ccacagcccc tgaaagagca gcccgccctg 540

aatgatagta gatacgctct gtcctctcga ctgcgagtgt ccgcaacctt ctggcaggac 600

cctcggaacc acttcagatg ccaggtgcag ttttatggcc tgtctgagaa tgatgaatgg 660

acacaggacc gcgctaagcc cgtgactcag attgtcagcg cagaggcctg ggggcgagca 720

gattgtggat ttacatcaga aagctatcag cagggggtgc tgagcgccac tatcctgtac 780

gagattctgc tgggaaaggc taccctgtat gcagtgctgg tcagcgccct ggtgctgatg 840

gctatggtca agaggaaaga ctcccgcggc taa 873

<210> 21

<211> 26

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 21

Gly Asp Ser Val Thr Gln Thr Glu Gly Leu Leu Asn Val Pro Glu Gly

1 5 10 15

Leu Pro Val Ser Ile Asn Cys Thr Tyr Gln

20 25

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 22

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

1 5 10 15

Lys

<210> 23

<211> 34

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 23

Thr Glu His Gln Arg Phe His Ala Thr Leu His Lys Ser Asp Ser Ser

1 5 10 15

Phe His Leu Gln Ile Glu Arg Ile Gln Pro Asn Asp Ser Gly Thr Tyr

20 25 30

Phe Cys

<210> 24

<211> 26

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 24

Asp Met Lys Ile Thr Gln Thr Pro Arg Tyr Leu Ile Val Lys Thr Gly

1 5 10 15

Glu Asn Val Thr Leu Glu Cys Gly Gln Asp

20 25

<210> 25

<211> 17

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 25

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

1 5 10 15

Ile

<210> 26

<211> 37

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 26

Asn Ser Glu Gly Asp Ile Pro Lys Arg Tyr Arg Val Ser Arg Lys Lys

1 5 10 15

Arg Glu His Phe Ser Leu Arg Ile Asp Ser Val Lys Thr Ser Asp Ser

20 25 30

Ala Leu Tyr Leu Cys

35

<210> 27

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 27

Lys Val Ala Glu Leu Val His Phe Leu

1 5

Claims

1. A humanized T Cell Receptor (TCR) having binding activity to the human antigen pHLA (MAGE-A3) and comprising a TCR a chain variable domain and a TCR β chain variable domain,

the variable domain of the TCR alpha chain comprises 3 CDR regions, the amino acid sequence of the 3 CDR regions of the variable domain of the TCR alpha chain is as follows,

CDR1α：TIYSNPF，SEQ ID NO:5；

CDR2α：SFTDNKR，SEQ ID NO:6；

CDR3α：AFDTNAYKVI，SEQ ID NO:7；

and, the variable domain of the TCR beta chain comprises 3 CDR regions, the amino acid sequence of the 3 CDR regions of the variable domain of the TCR beta chain is as follows,

CDR1β：MSHET，SEQ ID NO:8；

CDR2β：SYDVDS，SEQ ID NO:9；

CDR3β：ASSSTNTEVF，SEQ ID NO:10。

2. the humanized T cell receptor of claim 1, wherein the humanized TCR binds the human antigen MAGE-A3 with a dissociation constant that is 1.5-3 times the dissociation constant for binding of the murine TCR to the human antigen MAGE-A3.

3. The humanized T cell receptor of claim 1, wherein the framework region of the TCR α chain variable domain comprises 3 FR regions, the amino acid sequences of the 3 FR regions being as follows:

FR1α：GDSVTQTEGLLNVPEGLPVSINCTYQ，SEQ ID NO: 21；

FR2α：LFWYRQDPGKSPRLLLK，SEQ ID NO: 22；

FR3α：TEHQRFHATLHKSDSSFHLQIERIQPNDSGTYFC，SEQ ID NO: 23；

and, the framework region of the variable domain of the TCR β chain comprises 3 FR regions, the amino acid sequences of the 3 FR regions are as follows:

FR1β：DMKITQTPRYLIVKTGENVTLECGQD，SEQ ID NO: 24；

FR2β：MYWYRQDPGQGLQLIYI，SEQ ID NO: 25；

FR3β： NSEGDIPKRYRVSRKKREHFSLRIDSVKTSDSALYLC，SEQ ID NO: 26。

4. the humanized T cell receptor of claim 3, wherein the FR region of the framework region of the TCR α chain variable domain is derived from TRAV18 x 01.

5. The humanized T cell receptor of claim 3, wherein the FR region of the framework region of the TCR β chain variable domain has a sequence derived from TRBV28 x 01.

6. The humanized T cell receptor of claim 1, having a TCR α chain variable domain as set forth in SEQ ID No. 1 and a TCR β chain variable domain as set forth in SEQ ID No. 2.

7. The humanized T cell receptor of claim 1, wherein the TCR is soluble.

8. The humanized T cell receptor of claim 7, wherein the soluble TCR is an α β heterodimer comprising both TCR α chain constant regions and TCR β chain constant regions being human.

9. The humanized T cell receptor of claim 7, wherein the soluble TCR is an α β heterodimer comprising the FR region of TCR α chain TRAV18 x 01 and the FR region of TCR β chain TRBV28 x 01.

10. The humanized T cell receptor of claim 7, wherein the soluble TCR is an α β heterodimer comprising a TCR α chain constant region TRAC 01 and a TCR β chain constant region TRBC 101 or TRBC2 01.

11. The humanized T cell receptor of claim 1, wherein the α chain amino acid sequence of the TCR is SEQ ID NO:17 and the beta chain amino acid sequence of the TCR are SEQ ID NO 18.

12. The humanized T cell receptor of claim 1, wherein the TCR is formed by a peptide linker sequence linking an alpha chain variable domain to a beta chain variable domain.

13. The humanized T cell receptor of claim 1, wherein the C-or N-terminus of the α chain and/or β chain of the TCR is conjugated to a conjugate.

14. A multivalent TCR complex comprising at least two TCR molecules, and wherein at least one TCR molecule is a TCR as claimed in any one of claims 1 to 13.

15. A nucleic acid molecule comprising a nucleic acid sequence encoding a TCR as claimed in any one of claims 1 to 13.

16. The nucleic acid molecule of claim 15, comprising the nucleotide sequence encoding the TCR α chain variable domain of SEQ ID NO: 3.

17. The nucleic acid molecule of claim 15, wherein said nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 4.

18. the nucleic acid molecule of claim 15, wherein the nucleic acid molecule comprises the nucleotide sequence encoding a TCR α chain of SEQ ID NO:19 and a nucleic acid sequence comprising the nucleotide sequence encoding a TCR β chain of SEQ ID NO: 20.

19. a vector comprising the nucleic acid molecule of claim 15.

20. A host cell comprising the vector of claim 19 or a nucleic acid molecule of claim 15 integrated into the chromosome.

21. An isolated cell expressing a TCR as claimed in any one of claims 1 to 13.

22. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a TCR according to any one of claims 1 to 13 or a TCR complex according to claim 14 or a cell according to claim 21.

23. Use of a T cell receptor according to any one of claims 1 to 13, a TCR complex as claimed in claim 14 or a cell according to claim 21 for the manufacture of a medicament for the treatment of a tumour or an autoimmune disease.

24. A method of preparing a T cell receptor according to any one of claims 1 to 13, comprising the steps of:

(i) culturing the host cell of claim 20 so as to express the T cell receptor of any one of claims 1-13;

(ii) isolating or purifying said T cell receptor.