WO2008037943A1

WO2008037943A1 - Cells transformed with nucleic acid encoding ny-eso t cell receptors

Info

Publication number: WO2008037943A1
Application number: PCT/GB2006/003649
Authority: WO
Inventors: Bent Karsten Jakobsen; Jonathan Michael Boulter; Yi Li; Peter Eamon Molloy; Steven Mark Dunn
Original assignee: Medigene Limited
Priority date: 2006-09-29
Filing date: 2006-09-29
Publication date: 2008-04-03

Abstract

The present invention provides cells transformed with expressible nucleic acids encoding a TCR specific for the SLLMWITQC-HLA-A*0201 complex, said nucleic acids consisting of: (i) a sequence comprising bases 15 to 836 of SEQ ID Nos: 1, 3, 5, 7, or 9, and (ii) a sequence comprising bases 16 to 948 of SEQ ID Nos: 11, 13, 15, 17, 19, or 21 or; (a) a sequence comprising bases 15 to 836 of SEQ ID Nos: 23, 25, 27, 29 or 31 and (b) a sequence comprising bases 16 to 948 of SEQ ID Nos: 33, 35, 37, 39, 41, or 43. Such cells are useful for targeting NY-ESO+ cancer cells presenting the SLLMWITQC-HLA-A*0201 complex.

Description

Cells transformed with nucleic acid encoding NY-ESO T cell receptors

The present invention relates to a cell transformed with expressible nucleic acids encoding a TCR specific for the SLLMWITQC-HLA-A*0201 complex, said nucleic acids consisting of: (i) a sequence comprising bases 15 to 836 of SEQ ID Nos: 1, 3, 5, 7, or 9, and (ii) a sequence comprising bases 16 to 948 of SEQ ID Nos: 11, 13, 15, 17, 19, or 21 or; (a) a sequence comprising bases 15 to 836 of SEQ ID Nos: 23, 25, 27, 29 or 31 and (b) a sequence comprising bases 16 to 948 of SEQ ID Nos: 33, 35, 37, 39, 41, or 43.

Background to the Invention

WO 2005/113595 and a recent paper (Li et al, (2005) Nature Biotech 23 (3) 349-354) disclose TCRs which have a K₀ for the SLLMWITQC-HLA-A*0201 complex of less than or equal to 1 μM and/or has an off-rate (k_off) for the SLLMWITQC-HLA-A*0201 complex of 1x10^"3 S^'W slower. For many purposes it is desirable that the nucleic acid or nucleic acids encoding such TCRs, and the wild-type TCR from which they were derived, are adapted for high level expression in cells whether for in-vitro production or in-vivo use. Such adaptations are known to those skilled in the art and the sequences provided herein are so adapted.

The SLLMWITQC peptide is derived from the NY-ESO-I protein that is expressed by a range of tumours (Chen et al, (1997) PNAS USA 94 1914-1918). The Class I HLA molecules of these cancerous cells present peptides from this protein, including SLLMWITQC. Therefore, the SLLMWITQC-HLA-A2 complex provides a cancer marker that TCRs can target, for example T cells expressing said TCRs can be used to target and directly kill, or aid in the destruction of, cancer cells expressing the SLLMWITQC-HLA-A* 0201 complex. Brief Description of the Invention

This invention makes available for the first time a cell transformed with expressible nucleic acids encoding a TCR specific for the SLLMWITQC-HLA-A*0201 complex, said nucleic acids consisting of: (i) a sequence comprising bases 15 to 836 of SEQ ID Nos: 1, 3, 5, 7, or 9, and (ii) a sequence comprising bases 16 to 948 of SEQ ID Nos: 11, 13, 15, 17, 19, or 21 or; (a) a sequence comprising bases 15 to 836 of SEQ ID Nos: 23, 25, 27, 29 or 31 and (b) a sequence comprising bases 16 to 948 of SEQ ID Nos: 33, 35, 37, 39, 41, or 43. These cells will be of particular use in adoptive T cell therapy.

Detailed Description of the Invention

The present invention provides a cell transformed with expressible nucleic acids encoding a TCR specific for the SLLMWITQC-HLA-A*0201 complex, said nucleic acids consisting of: (i) a sequence comprising bases 15 to 836 of SEQ ID Nos: 1, 3, 5, 7, or 9, and (ii) a sequence comprising bases 16 to 948 of SEQ ID Nos: 11, 13, 15, 17, 19, or 21 or; (a) a sequence comprising bases 15 to 836 of SEQ ID Nos: 23, 25, 27, 29 or 31 and (b) a sequence comprising bases 16 to 948 of SEQ ID Nos: 33, 35, 37, 39, 41, or 43.

Another embodiment of the invention provides a cell transformed with expressible nucleic acids encoding a TCR specific for the SLLMWITQC-HLA-A*0201 complex, wherein the said nucleic acids are selected from the nucleic acid pairs listed in the following table:

The nucleic acids corresponding to bases 15 to 836 of the DNA encoding TCR alpha chains are written in italics in Figures Ia, 2a, 3a, 4a, 5a, 12a, 13a, 14a, 15a, and 16a. (SEQ ID Nos: 1, 3, 5, 7, 9, 23, 25, 27, 29, and 31 respectively) These bases correspond to the Open Reading Frames (ORFs) within these DNA sequences.

The nucleic acids corresponding to bases 16 to 948 of the DNA encoding TCR beta chains are written in italics in Figures 6a, 7a, 8a, 9a, 10a, 11a, 17a, 18a, 19a, 20a, 21a and 22a. (SEQ ID Nos: 11, 13, 15, 17, 19, 21, 33, 35, 37, 39, 41 and 43 respectively) These bases correspond to the Open Reading Frames (ORFs) within these DNA sequences.

The ORFs of SEQ ID NOs: 23, 25, 27, 29 and 31 and of SEQ ID Nos: 33, 35, 37, 39, 41, and 43 encode modified variants of the full length TCR chains encoded by the ORFs of SEQ ID Nos: 1, 3, 5, 7, and 9 and modified variants of the full length TCR chains encoded by the ORFs of SEQ ID Nos: 11, 13, 15, 17, 19, and 21 respectively. These modified nucleic acids contain mutations which result in the following substitutions within the TCR chains thereby encoded:

The substitution of a cysteine amino acid residue for a native threonine amino acid residue within the constant domains of the TCR alpha chains encoded by SEQ ID Nos: 23, 25, 27, 29 and 31 and the substitution of a cysteine amino acid residue for a native serine amino acid residue within the constant domains of the TCR beta chains encoded by SEQ ID 33, 35, 37, 39, 41, and 43. More precisely, the introduced cysteine amino acid residue in the TCR alpha chains replaces the threonine amino acid residue occurring at residue 48 of exon 1 of TRAC*01, and the introduced cysteine in the TCR beta chains replaces the serine amino acid occurring at residue 57 of exon 1 of both TRBC*01 and TRBC*02. (TCR residue numbering follows the IMGT format as described in LeFranc et al. , (2001) The T Cell Receptor Fact shook, Academic Press) As will be obvious to those skilled in the art any DNA sequences corresponding to the two known cysteine-encoding codons can be used to introduce these cysteine residues.

The substitution of a serine amino acid residue for a native cysteine amino acid residue within the constant domains of the TCR alpha chains encoded by SEQ ID Nos: 23, 25, 27, 29 and 31. More precisely, the introduced serine amino acid residue in the TCR alpha chain replaces the alpha chain cysteine amino acid residue involved in the formation of the native disulfide interchain bond in TCRs occurring at residue 4 of exon 2 of TRAC*01 (TCR residue numbering follows the IMGT format as described in LeFranc et al, (2001) The T Cell Receptor Factsbook, Academic Press) As will be obvious to those skilled in the art any DNA sequences corresponding to any of the six known serine-encoding codons can be used to introduce these serine residues

The substitution of a serine amino acid residue for a native cysteine amino acid residue within the constant domains of the TCR beta chains encoded by SEQ ID Nos: 33, 35, 37, 39, 41, and 43. More precisely, the introduced serine amino acid residue in the TCR alpha chain replaces the beta chain cysteine amino acid residue involved in the formation of the native disulfide interchain bond in TCRs occurring at residue 2 of exon 2 of TRBC* 01 or TRBC* 02 (TCR residue numbering follows the IMGT format as described in LeFranc et al, (2001) The TCeIl Receptor Factsbook, Academic Press) As will be obvious to those skilled in the art any DNA sequences corresponding to any of the six known serine-encoding codons can be used to introduce these serine residues

The substitution of an alanine amino acid residue for a native cysteine amino acid residue within the constant domains of the TCR beta chains encoded by SEQ ID Nos: 33, 35, 37, 39, 41 and 43. More precisely, the introduced serine amino acid residue in the TCR beta chain replaces the "unpaired" cysteine amino acid residue occurring at residue 75 of exon 1 of TRBC*01 or TRBC*02 (TCR residue numbering follows the IMGT format as described in LeFranc et al, (2001) The T Cell Receptor Factsbook, Academic Press) As will be obvious to those skilled in the art any DNA sequences corresponding to any of the four known alanine-encoding codons can be used to introduce these alanine residues

The substitution of an aspartic acid amino acid residue for a native asparagine amino acid residue within the constant domains of the TCR beta chains encoded by SEQ ID Nos: 33, 35, 37, 39, 41, or 43. More precisely, the introduced aspartic acid amino acid residue in the TCR beta chain replaces the asparagine amino acid residue occurring at residue 89 of exon 1 of TRBC*01 or TRBC*02 (TCR residue numbering follows the IMGT format as described in LeFranc et al, (2001) The T Cell Receptor Factsbook, Academic Press) As will be obvious to those skilled in the art either of the DNA sequences corresponding to the two known aspartic acid-encoding codons can be used to introduce these aspartic acid residues

WO 03/020763 contains detailed information relating to the introduction of cysteine residues into various locations of TCR constant domains in order to create non-native interchain disulfide bonds. The cell of the invention is transformed with the nucleic acids such that the latter are expressible in the cell. This will normal involve incorporating the nucleic acids into suitable expression vectors, of which many are known. The technology of recombinant DNA expression is well understood and described in many laboratory manuals and textbooks. (See, for example, Sambrook and Russell (2001) Molecular Cloning, a Laboratory Manual 3^rd edition, ISBN 0-87969-576-5)

Although the nucleic acids of the invention are defined uniquely by their sequence information, they are intended to benefit from one or more of the following known general design considerations:

Relative abundance of transfer RNA (tRNA) - It is known that the cells of different species possess varying amounts of the tRNA molecules which each recognise the different codons which can be used to encode a given amino acid residue. Therefore, in general, it is preferable to use the codon recognised by the most abundance of these tRNA molecules. However, it may be advisable to use the selection of different codons encoding a given amino acid residues if these residue are encoded with high frequency within a sub-sequence of the nucleic acids, in order to prevent localised depletion of the most abundant tRNA species which may otherwise occur.

Removal of inverted repeat motifs - Such sequences introduce strong secondary structures, such as "hair-pins", into nucleic acids which may result in a slowing down of the translation process and a reduction of the level of expression.

Avoidance of other unwanted motifs — For example, the removal of inappropriate splice sites or polyadenylation signals, and undesirable restriction enzyme recognition sequences.

Introduction of desired motifs - For example, translation initiation consensus signals ("Kozak" signals) 5' of the ORF, and/or a strong translation termination codon, such as TAA immediately 3' of the ORF. Optimisation of nucleic acid GC content - The overall ratio of CG: AT bases in a nucleic acid can also influence the rate of transcription and/or translation of a nucleic acid encoding a given polypeptide.

Note however, it is not necessarily preferable to induce the fastest possible rate of transcription and/or translation. Overly high rates of either of these processes may result in the production of inappropriately folded and therefore inactive polypeptides.

In another embodiment the cells of the invention are human cells. In yet another embodiment the cells of the invention are human T cells or human haematopoietic cells.

Example 1 herein details a suitable method for transfecting human cells with DNA encoding any SLLMWITQC-HLA- A* 0201 -specific TCR that has been adapted for high level expression in human cells.

Further embodiments are provided by a pharmaceutical composition comprising a plurality of cells of the invention, together with a pharmaceutically acceptable carrier.

The invention also provides a method of treatment of cancer comprising administering to a subject suffering such cancer disease an effective amount of a plurality of cells of the invention. In a related embodiment the invention provides for the use of a plurality of cells of the invention in the preparation of a composition for the treatment of cancer.

Therapeutic or imaging cells in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier. This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

The pharmaceutical composition may be adapted for administration by any appropriate route, for example parenteral, transdermal or via inhalation, preferably a parenteral (including subcutaneous, intramuscular, or, most preferably intravenous) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.

Dosages of the cells and compositions of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

Preferred features of each aspect of the invention are as for each of the other aspects mutatis mutandis. The prior art documents mentioned herein are incorporated to the fullest extent permitted by law.

Example

Reference is made in the following Example to the accompanying drawings in which:

Figure Ia provides a DNA sequence adapted for high level expression in human cells encoding a full-length WT 1G4 TCR alpha chain. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure Ib provides the amino acid sequence of a full-length WT 1G4 TCR alpha chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORP) in the DNA sequence of Figure Ia.

Figure 2a provides a DNA sequence adapted for high level expression in human cells encoding a full-length c5 high affinity 1G4 TCR alpha chain. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 2b provides the amino acid sequence of a full-length c5 high affinity 1G4 TCR adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 2a.

Figure 3a provides a DNA sequence adapted for high level expression in human cells encoding a full-length clO high affinity 1G4 TCR alpha chain. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 3 b provides the amino acid sequence of a full-length clO high affinity 1G4 TCR adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 3 a.

Figure 4a provides a DNA sequence adapted for high level expression in human cells encoding a full-length cl2 high affinity 1G4 TCR alpha chain. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 4b provides the amino acid sequence of a full-length cl2 high affinity 1G4 TCR alpha chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 4a.

Figure 5a provides a DNA sequence adapted for high level expression in human cells encoding a full-length c58 high affinity 1G4 TCR alpha chain. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 5b provides the amino acid sequence of a full-length c58 high affinity 1G4 TCR alpha chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 5 a.

Figure 6a provides a DNA sequence adapted for high level expression in human cells encoding a full-length WT 1G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 6b provides the amino acid sequence of a full-length WT 1G4 TCR beta chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 6a. Figure 7a provides a DNA sequence adapted for high level expression in human cells encoding a full-length cl high affinity IG4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 7b provides the amino acid sequence of a full-length cl high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 7a.

Figure 8a provides a DNA sequence adapted for high level expression in human cells encoding a full-length c2 high affinity IG4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 8b provides the amino acid sequence of a full-length c2 high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 8a.

Figure 9a provides a DNA sequence adapted for high level expression in human cells encoding a full-length c59 high affinity IG4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 16-948) Figure 9b provides the amino acid sequence of a full-length c59 high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 9a.

Figure 10a provides a DNA sequence adapted for high level expression in human cells encoding a full-length c61 high affinity IG4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 10b provides the amino acid sequence of a full-length c61 high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 10a.

Figure 11a provides a DNA sequence adapted for high level expression in human cells encoding a full-length cl 00 high affinity IG4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure lib provides the amino acid sequence of a full-length clOO high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 11a. Figure 12a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length WT 1 G4 TCR alpha chain. This sequence has been mutated to contain an introduced cysteine codon and to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine- encoding codon. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 12b provides the amino acid sequence of a modified full-length WT 1G4 TCR alpha chain adapted for high level expression in human cells. This sequence has been mutated to contain an introduced cysteine residue and to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 12a. The mutated TCR residues are highlighted.

Figure 13a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length c5 high affinity 1G4 TCR alpha chain. This sequence has been mutated to contain an introduced cysteine codon and to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon. This DNA sequence also contains Xlτol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 13b provides the amino acid sequence of a modified full-length c5 high affinityl G4 TCR adapted for high level expression in human cells. This sequence has been mutated to contain an introduced cysteine residue and to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 13 a. The mutated TCR residues are highlighted.

Figure 14a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length clO high affinity 1G4 TCR alpha chain. This sequence has been mutated to contain an introduced cysteine codon and to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 14b provides the amino acid sequence of a modified full-length clO high affinity 1G4 TCR adapted for high level expression in human cells. This sequence has been mutated to contain an introduced cysteine residue and to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 14a. The mutated TCR residues are highlighted.

Figure 15a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length cl2 high affinity 1G4 TCR alpha chain. This sequence has been mutated to contain an introduced cysteine codon and to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 15b provides the amino acid sequence of a modified full-length cl2 high affinityl G4 TCR alpha chain adapted for high level expression in human cells. This sequence has been mutated to contain an introduced cysteine residue and to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 15 a. The mutated TCR residues are highlighted.

Figure 16a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length c58 high affinity 1G4 TCR alpha chain. This sequence has been mutated to contain an introduced cysteine codon and to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon. This DNA sequence also contains Xhol and Mini restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 16b provides the amino acid sequence of a modified full-length c58 high affinity 1G4 TCR alpha chain adapted for high level expression in human cells. This sequence has been mutated to contain an introduced cysteine residue and to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 16a. The mutated TCR residues are highlighted.

Figure 17a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length WT 1G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. This sequence has been mutated so as to; contain an introduced cysteine codon, to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon, to replace the codon encoding the "unpaired" cysteine in native TCR beta chains with an alanine-encoding codon and to replace a native asparagine-encoding codon with an aspartic acid-encoding codon. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 17b provides the amino acid sequence of a modified full-length WT 1G4 TCR beta chain adapted for high level expression in human cells. This sequence has been mutated so as to; contain an introduced cysteine residue, to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue, to replace the "unpaired" cysteine in native TCR beta chains with an alanine residue and to replace a native asparagine residue with an aspartic acid residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 17a. The mutated TCR residues are highlighted.

Figure 18a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length cl high affinity 1G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. This sequence has been mutated so as to; contain an introduced cysteine codon, to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon, to replace the codon encoding the "unpaired" cysteine in native TCR beta chains with an alanine-encoding codon and to replace a native asparagine-encoding codon with an aspartic acid-encoding codon. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 16-948) Figure 18b provides the amino acid sequence of a modified full-length cl high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This sequence has been mutated so as to; contain an introduced cysteine residue, to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue, to replace the "unpaired" cysteine in native TCR beta chains with an alanine residue and to replace a native asparagine residue with an aspartic acid residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 18a. The mutated TCR residues are highlighted.

Figure 19a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length c2 high affinity 1G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. This sequence has been mutated so as to; contain an introduced cysteine codon, to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon, to replace the codon encoding the "unpaired" cysteine in native TCR beta chains with an alanine-encoding codon and to replace a native asparagine-encoding codon with an aspartic acid-encoding codon. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 19b provides the amino acid sequence of a modified full-length c2 high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This sequence has been mutated so as to; contain an introduced cysteine residue, to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue, to replace the "unpaired" cysteine in native TCR beta chains with an alanine residue and to replace a native asparagine residue with an aspartic acid residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 19a. The mutated TCR residues are highlighted. Figure 20a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length c59 high affinity 1G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. This sequence has been mutated so as to; contain an introduced cysteine codon, to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon, to replace the codon encoding the "unpaired" cysteine in native TCR beta chains with an alanine-encoding codon and to replace a native asparagine-encoding codon with an aspartic acid-encoding codon. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 20b provides the amino acid sequence of a modified full-length c59 high affinity 1 G4 TCR beta chain adapted for high level expression in human cells. This sequence has been mutated so as to; contain an introduced cysteine residue, to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue, to replace the "unpaired" cysteine in native TCR beta chains with an alanine residue and to replace a native asparagine residue with an aspartic acid residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 20a.

Figure 21a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length c61 high affinity 1G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. This sequence has been mutated so as to; contain an introduced cysteine codon, to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon, to replace the codon encoding the "unpaired" cysteine in native TCR beta chains with an alanine-encoding codon and to replace a native asparagine-encoding codon with an aspartic acid-encoding codon. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 21b provides the amino acid sequence of a modified full-length c61 high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This sequence has been mutated so as to; contain an introduced cysteine residue, to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue, to replace the "unpaired" cysteine in native TCR beta chains with an alanine residue and to replace a native asparagine residue with an aspartic acid residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORF) in the DNA sequence of Figure 21a. The mutated TCR residues are highlighted.

Figure 22a provides a DNA sequence adapted for high level expression in human cells encoding a modified full-length cl 00 high affinity 1 G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. This sequence has been mutated so as to; contain an introduced cysteine codon, to replace the codon encoding the cysteine involved in forming the native TCR interchain disulfide bond with a serine-encoding codon, to replace the codon encoding the "unpaired" cysteine in native TCR beta chains with an alanine-encoding codon and to replace a native asparagine-encoding codon with an aspartic acid-encoding codon. The restriction enzyme recognition sequences are underlined, the mutated TCR bases are highlighted and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Figure 22b provides the amino acid sequence of a modified full-length clOO high affinity 1G4 TCR beta chain adapted for high level expression in human cells. This sequence has been mutated so as to; contain an introduced cysteine residue, to replace the cysteine involved in forming the native TCR interchain disulfide bond with a serine residue, to replace the "unpaired" cysteine in native TCR beta chains with an alanine residue and to replace a native asparagine residue with an aspartic acid residue. This is the amino acid sequence of the polypeptide that is encoded by the open reading frame (ORP) in the DNA sequence of Figure 22a. The mutated TCR residues are highlighted.

Figure 23 a provides a DNA sequence encoding a full-length WT 1G4 TCR alpha chain. This DNA sequence also contains Xhol and MIuI restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 15-839)

Figure 23b provides a DNA sequence encoding a full-length WT 1G4 TCR beta chain. This DNA sequence also contains BamHI and Notl restriction enzyme recognition sequences to aid ligation of the DNA sequence into an expression vector. The restriction enzyme recognition sequences are underlined and the bases within the open reading frame (ORF) are in italics. (Bases 16-948)

Example 1 - Comparison of TCR expression levels on Jurkat cells transfected with codon-optimised and non-codon optimised DNA encoding 1G4 TCRs

4 x 10⁶ Jurkat cells grown in RMPI containing 10% heat-inactivated fetal calf serum medium cells are washed in serum-free medium and transfected with either:

a) 5μg of endotoxin-free plasmid pCIneo containing the non-codon optimised sequence encoding wild-type 1G4 full-length TCRα chain plus 5μg of endotoxin-free plasmid pCI containing the non-codon optimised sequence encoding wild-type 1 G4 full-length TCRβ chain (The ORFs of these sequences are provided in Figures 23 a (SEQ ID NO: 45) and 23b (SEQ ID NO: 46) respectively.

b) 5μg of endotoxin-free plasmid pCIneo (Promega) containing an ORF codon- optimised encoding wild-type 1 G4 full-length TCRα chain plus 5 μg of endotoxin-free plasmid pCI (Promega) containing an ORF codon- encoding wild-type 1G4 full-length TCRβ chain The ORFs of these sequences are provided in Figure Ia (SEQ ID NO: 1) and Figure 6a (SEQ ID NO: 11) respectively.

Transfection is achieved by electroporation using 0.4cm cuvettes using conditions of 0.27 kV and 975 μF in a BioRad Genepulser apparatus.

Cells are placed in 6ml of RPMI containing 20% heat-inactivated fetal calf serum at 37 ⁰C for 72 hours.

Cells are stained in a volume of 1 OOμl PBS using 1 μl (0.54μg) of PE-labelled streptavidin p/HLA-A2 tetramer (peptide was either the cognate NY-ESO peptide SLLMWITQC or an irrelevant peptide as a control).

After 20 minutes at room temperature the cells are washed once in 5ml RPMI and re- suspended in 800μl RPMI and analysed on a FC500 Beckman Coulter instrument.

Results

An increase in FACs staining data using the cognate SLLMWITQC-HLA-A2 tetramers obtained for Jurkat cells transfected with the codon optimised wild-type 1 G4 TCR compared to that obtained using cells transfected with the corresponding non- codon optimised DNA would demonstrate that a higher level of TCR surface expression was achieved using the codon optimised DNA compared to that achieved using the corresponding non-codon optimised DNA.

Claims

1. A cell transformed with expressible nucleic acids encoding a TCR specific for the SLLMWITQC-HLA-A* 0201 complex, said nucleic acids consisting of:

(i) a sequence comprising bases 15 to 836 of SEQ ID Nos: 1, 3, 5, 7, or 9, and (ii) a sequence comprising bases 16 to 948 of SEQ ID Nos: 11, 13, 15, 17, 19, or 21 or;

(a) a sequence comprising bases 15 to 836 of SEQ ID Nos: 23, 25, 27, 29 or 31 and (b) a sequence comprising bases 16 to 948 of SEQ ID Nos: 33, 35, 37, 39, 41, or 43.

2. A cell as claimed in claim 1 wherein the said nucleic acids are selected from the nucleic acid pairs listed in the following table:

3. A cell as claimed in claim 1 wherein the said nucleic acids are selected from the nucleic acid pairs listed in the following table:

4. A cell as claimed in any preceding claim which is a human cell

5. A cell as claimed in any preceding claim which is a human T cell or a human haematopoietic cell.

6. A pharmaceutical composition comprising a plurality of cells as claimed in any preceding claim, together with a pharmaceutically acceptable carrier.

7. A method of treatment of cancer comprising administering to a subject suffering such cancer an effective amount of a plurality of cells as claimed in any preceding claim.

8. The use of a plurality of cells as claimed in any preceding claim in the preparation of a composition for the treatment of cancer.