# Patent

## Title
Cells expressing modified T cell receptor

## Abstract

This invention provides a cell presenting at least one T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, said TCR comprising an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs.

## Background
RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
This application is a division of U.S. application Ser. No. 11/597,252 filed Apr. 28, 2009, now U.S. Pat. No. 8,361,794, which is a National Stage application of co-pending PCT application PCT/GB2005/002570 filed Jun. 29, 2005, which was published in English under PCT Article 21(2) on Jan. 5, 2006, and which claims the benefit of Great Britain patent applications Serial No. GB 0414499.4 filed Jun. 29, 2004; Serial No. 0421831.9 filed Oct. 1, 2004; and Serial No. 0511123.2 filed Jun. 1, 2005. These applications are incorporated herein by reference in their entireties.


REFERENCE TO A SEQUENCE LISTING
This application incorporates by reference the contents of a 80.6 kb text file created on Oct. 29, 2009 and named “SN11597252_sequencelisting.txt, which is the sequence listing for this application.
FIELD OF THE INVENTION
The present invention relates to cells, particularly T cells, expressing modified T cell receptors (TCRs), their preparation, and their use in therapy.
BACKGROUND TO THE INVENTION
Native TCRs
As is described in, for example, WO 99/60120 TCRs mediate the recognition of specific Major Histocompatibility Complex (MHC)-peptide complexes by T cells and, as such, are essential to the functioning of the cellular arm of the immune system.
Antibodies and TCRs are the only two types of molecules which recognise antigens in a specific manner, and thus the TCR is the only receptor for particular peptide antigens presented in MHC, the alien peptide often being the only sign of an abnormality within a cell. T cell recognition occurs when a T-cell and an antigen presenting cell (APC) are in direct physical contact, and is initiated by ligation of antigen-specific TCRs with pMHC complexes.
The native TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist in αβ and γδ forms, which are structurally similar but have quite distinct anatomical locations and probably functions. The MHC class I and class II ligands are also immunoglobulin superfamily proteins but are specialised for antigen presentation, with a highly polymorphic peptide binding site which enables them to present a diverse array of short peptide fragments at the APC cell surface.
Two further classes of proteins are known to be capable of functioning as TCR ligands. (1) CD1 antigens are MHC class I-related molecules whose genes are located on a different chromosome from the classical MHC class I and class II antigens. CD1 molecules are capable of presenting peptide and non-peptide (eg lipid, glycolipid) moieties to T cells in a manner analogous to conventional class I and class II-MHC-pep complexes. See, for example (Barclay et al, (1997) The Leucocyte Antigen Factsbook 2nd Edition, Academic Press) and (Bauer (1997) Eur J Immunol 27 (6) 1366-1373)) (2) Bacterial superantigens are soluble toxins which are capable of binding both class II MHC molecules and a subset of TCRs. (Fraser (1989) Nature 339 221-223) Many superantigens exhibit specificity for one or two Vbeta segments, whereas others exhibit more promiscuous binding. In any event, superantigens are capable of eliciting an enhanced immune response by virtue of their ability to stimulate subsets of T cells in a polyclonal fashion.
The extracellular portion of native heterodimeric αβ and γδ TCRs consist of two polypeptides each of which has a membrane-proximal extracellular constant domain, and a membrane-distal variable region. Each of the extracellular constant domain and variable region includes an intra-chain disulfide bond. The variable regions contain the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies. CDR3 of αβ TCRs interact with the peptide presented by MHC, and CDRs 1 and 2 of αβ TCRs interact with the peptide and the MHC. The diversity of TCR sequences is generated via somatic rearrangement of linked variable (V), diversity (D), joining (J), and constant genes, the genes products thereof making up the variable region.
Functional α and γ chain polypeptides are formed by rearranged V-J-C domains, whereas β and δ chains consist of V-D-J-C domains. (See FIG. 1) Each functional TCR possessing one of the possible variants of each domain. (See FIGS. 7 and 8 for the DNA sequences of all known TCR C and V domains from TCR α and β chains respectively, also see (LeFranc et al, (2001) The T cell receptor Factsbook, Academic Press) for a complete listing of the DNA and amino acid sequences of all known TCR domains) A further level of diversity is introduced to αβ TCRs by the in-vivo recombination of shortened TCR domains. The extracellular constant domain has a membrane proximal motif and an immunoglobulin motif. There are single α and δ chain constant domains, known as TRAC and TRDC respectively. The β chain constant domain is composed of one of two different β constant domains, known as TRBC1 and TRBC2 (IMGT nomenclature). There are four amino acid changes between these β constant domains, three of which are in exon 1 of TRBC1 and TRBC2: N4K5->K4N5 and F37->Y, the final amino acid change between the two TCR β chain constant regions being in exon 3 of TRBC1 and TRBC2: V1->E. (IMGT numbering, differences TRBC1->TRBC2) The constant γ domain is composed of one of either TRGC1, TRGC2(2×) or TRGC2(3×). The two TRGC2 constant domains differ only in the number of copies of the amino acids encoded by exon 2 of this gene that are present. TCR constant domains include a transmembrane sequence, the amino acids of which anchor the TCR chains into the cell surface membrane. There are 46 different TRAY domains and 56 TRBV domains. 52 different functional genes encode the TRAJ domains, whereas 12-13 functional genes encode the TRBJ domains. 2 different functional genes encode the TRBD domains.
The extent of each of the TCR extracellular constant domains, bounded by the transmembrane sequences, is somewhat variable. However, a person skilled in the art can readily determine the position of the domain boundaries using a reference such as The T Cell Receptor Facts Book, Lefranc & Lefranc, Publ. Academic Press 2001.
Immunotherapy
Immunotherapy involves enhancing the immune response of a patient to cancerous or infected cells. Active immunotherapy is carried out by stimulation of the endogenous immune system of tumour bearing patients. Passive, or adoptive, immunotherapy involves the transfer of immune competent cells into the patient. (Paul (2002) Curr Gene Therapy 2 91-100) There are three broad approaches to adoptive immunotherapy which have been applied in the clinic for the treatment of metastatic diseases; lymphokine-activated killer (LAK) cells, auto-lymphocyto therapy (ALT) and tumour-infiltrating lymphocutes (TIL). (Paul (2002) Curr Gene Therapy 2 91-100).
A recent proposed variation of T cell adoptive therapy is the use of gene therapy techniques to introduce TCRs specific for known cancer-specific MHC-peptide complexes into the T cells of cancer patients. For example, (WO 01/55366) discloses retrovirus-based methods for transfecting, preferably, T cells with heterologous TCRs. This document states that these transfected cells could be used for either the cell surface display of TCR variants as a means of identifying high affinity TCRs or for immunotherapy. Methods for the molecular cloning of cDNA of a human p53-specific, HLA restricted murine TCR and the transfer of this cDNA to human T cells are described in published US patent application no. 20020064521. This document states that the expression of this murine TCR results in the recognition of endogenously processed human p53 expressed in tumour cells pulsed with the p53-derived peptide 149-157 presented by HLA A*0201 and claims the use of the murine TCR in anti-cancer adoptive immunotherapy. However, the concentration of peptide pulsing required achieving half maximal T cell stimulation of the transfected T cells was approximately 250 times that required by T cells expressing solely the murine TCR. As the authors noted “The difference in level of peptide sensitivity is what might be expected of a transfectant line that contained multiple different TCR heterodimers as a result of independent association of all four expressed hu and mu TCR chains.”
There are also a number of recent papers relating to T cell adoptive therapy. In one study (Rosenberg (1988) N Engl J Med 319 (25) 1676-80) lymphocytes from melanomas were expanded in vitro and these tumor-infiltrating lymphocytes, in combination with IL-2 were used to treat 20 patients with metastatic melanoma by means of adoptive transfer. The authors note that objective regression of the cancer was observed in 9 of 15 patients (60 percent) who had not previously been treated with interleukin-2 and in 2 of 5 patients (40 percent) in whom previous therapy with interleukin-2 had failed. Regression of cancer occurred in the lungs, liver, bone, skin, and subcutaneous sites and lasted from 2 to more than 13 months. A further study describes the administration of an expanded population of Melan-A specific cytotoxic T cells to eight patients with refractory malignant melanoma. These T cells were administered by i.v. infusion at fortnightly intervals, accompanied by s.c. administration of IL-2. The T cell infusions were well tolerated with clinical responses noted as one partial, one mixed with shrinkage of one metastatic deposit and one no change (12 months) among the eight patients. (Meidenbauer (2003) J Immunol 170 2161-2169) As noted in this study, recent advances regarding the in vitro stimulation T cells for the generation of cell populations suitable for T cell adoptive therapy have made this approach more practical. See, for example (Oelke (2000) Clin Cancer Res 6 1997-2005) and (Szmania (2001) Blood 98 505-12).
SUMMARY OF THE INVENTION
The present invention relates to cells presenting at least one T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, said TCR comprising an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs. Such T cells are expected to be particularly suited for use in T cell adoptive immunotherapy.



BRIEF DESCRIPTION OF THE DRAWINGS
The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.
FIG. 1 illustrates the domains that comprise TCR α and β chains: wherein “S” denotes the signal peptide, “V” denotes the variable domain, “J” denotes the joining domain, D denotes the diversity domain, and “C” denotes the constant domain which contains the transmembrane sequence;
FIG. 2 illustrates the structure of a cell surface TCR containing a non-native interchain disulfide bond;
FIGS. 3a and 3b show respectively the nucleic acid sequences of the a (SEQ ID NO:34) and β (SEQ ID NO:35) chains of a soluble A6 TCR, mutated so as to introduce a cysteine codon. The shading indicates the introduced cysteine codons;
FIG. 4a shows the amino acid sequence (SEQ ID NO:36) encoded by the DNA sequence of FIG. 3a, including the T48→C mutation (underlined) used to produce the novel disulfide inter-chain bond, and FIG. 4b shows the amino acid sequence (SEQ ID NO:37) encoded by the DNA sequence of FIG. 3b, including the S57→C mutation (underlined) used to produce the novel disulfide inter-chain bond;
FIG. 5 graphically illustrates the PCR reactions required to produce a DNA sequence encoding a full-length disulfide-linked A6 Tax TCR using DNA encoding soluble disulfide-linked A6 Tax TCR and wild-type A6 Tax TCR as templates;
FIG. 6a shows the nucleic acid (SEQ ID NO:38) and protein sequences (SEQ ID NO:39) of the membrane anchored α chain of A6 TCR, mutated so as to introduce a new cysteine codon and mutate the Cys involved in forming the native inter-chain disulfide bridge to Ser. The first shading indicates the introduced cysteine codon; the underlined Ser codon indicates the position of the Cys->Ser mutation disrupting the capacity to form the native inter-chain disulfide link.
FIG. 6b shows nucleic acid (SEQ ID NO:40) and protein sequences (SEQ ID NO:41) of the membrane anchored β chain of A6 TCR, using the native constant domain, TRBC2 (nomenclature according to the IMGT format as described in (LeFranc et al, (2001) The T cell receptor Factsbook, Academic Press), mutated so as to introduce a new cysteine codon and mutate the Cys involved in forming the native inter-chain disulfide bridge to Ser. The first shading indicates the introduced cysteine codon; the underlined Ser codon indicates the position of the Cys->Ser mutation disrupting the capacity to form the native inter-chain di-sulfide link.
FIGS. 7a-7h detail the DNA sequence of all known TCR α chain constant and variable domains (SEQ ID NOS: 41-89.
FIGS. 8a-8j detail the DNA sequence of all known TCR β chain constant and variable domains SEQ ID NOS:90-145.
FIGS. 9a and 9b show respectively the DNA sequences of the α (SEQ ID NO:146) and β (SEQ ID NO:147) chains of a soluble AH-1.23 TCR, mutated so as to introduce a novel cysteine codon (indicated by shading).
FIGS. 10a and 10b show respectively the AH-1.23 TCR α (SEQ ID NO:148) and P (SEQ ID NO:149) chain extracellular amino acid sequences produced from the DNA sequences of FIGS. 9a and 9b. 



DETAILED DESCRIPTION OF THE INVENTION
This invention provides a cell presenting at least one T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, said TCR comprising an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs.
As noted above native TCRs exist in αβ and γδ forms, the present invention encompasses cells presenting either of these TCR forms, wherein said TCR comprises an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs.
Another embodiment provides a cell presenting at least one αβ T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, said TCR comprising a disulfide bond between α and β extracellular constant domain residues which is not present in native TCRs.
The presence of the novel cysteine residues (creating the novel disulfide bond) in the transfected heterodimeric TCR (dTCR) chains favour the production of the desired transfected TCRs over TCRs comprising a native TCR chain associated with a transfected TCR chain. Without wishing to be bound by theory, this result is interpreted as being due to the two transfected TCR chains preferentially self-associating due to the formation of the novel inter-chain disulfide bond between the introduced cysteine residues. The formation of any TCR comprising a mismatched pair of TCR chains (one native and one from the transfected TCR) may be further inhibited by ensuring the transfected chains lack the cysteine residue involved in the formation of the native inter-chain disulfide bond. Cells expressed such transfected TCR chains therefore provide a preferred embodiment of the invention.
Use of a single chain TCR (scTCR) in accordance with the invention also avoids formation of mismatched pairs.
The αβ TCRs which comprise a disulfide bond between α and β extracellular constant domain residues which is not present in native TCRs presented by the cells of the invention are targeting moieties. The TCRs of the invention target TCR ligands such as peptide-MHC or CD1-antigen complexes. As such, it would be desirable if the affinity of these TCR could be altered. For example it may be desirable if these TCR had a higher affinity and/or a slower off-rate for the TCR ligands than native TCRs specific for that ligand. The inventors co-pending application WO 2004/044004 details methods of producing and testing TCRs having a higher affinity and/or a slower off-rate for the TCR ligand than native TCRs specific for that ligand.
The TCR functionality of cells transfected to express and present the membrane anchored scTCRs and dTCRs may be tested by confirming that transfected cells bind to the relevant TCR ligand (pMHC complex, CD1-antigen complex, superantigen or superantigen/pMHC complex)—if it binds, then the requirement is met. The binding of the transfected cells to a TCR ligand can be detected by a number of methods. These include attaching a detectable label to the TCR ligand. For example, where the method uses pMHC tetramers, the pMHC may include a fluorescent label. Example 6 herein provides a detailed description of the methods required to analyse the binding of cells transfected to express disulfide-linked TCRs to MHC-peptide complexes. This method is equally applicable to the study of TCR/CD1 interactions. In order to apply this method to the study of TCR/CD1 interactions soluble forms of CD1 are required, the production of which are described in (Bauer (1997) Eur J Immunol 27 (6) 1366-1373).
The Cell Membrane Anchored dTCR
In the case of a dTCR, the TCR □ and □ chains may each comprise a transmembrane sequence, fused at its N terminus to an extracellular constant domain sequence, in turn fused at its N terminus to a variable region sequence. Furthermore, at least the said sequences of the TCR α and β chains other than the complementarity determining regions of the variable region, may correspond to human TCR α and β sequences.
The Cell Membrane Anchored scTCR
In the case of an scTCR, the scTCR comprises

    
    
        (i) a first segment constituted by an α chain variable region sequence fused to the N terminus of an α chain extracellular constant domain sequence, and a second segment constituted by a β chain variable region sequence fused to the N terminus of a sequence β chain extracellular constant and transmembrane sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment, or
        (ii) a first segment constituted by a TCR β chain variable region sequence fused to the N terminus of a β chain extracellular constant domain sequence, and a second segment constituted by an α chain variable region sequence fused to the N terminus of a sequence α chain extracellular constant and transmembrane sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
    
    


Again, in the scTCR embodiment, the said sequences of the TCR α and β chains other than the complementarity determining regions of the variable region, correspond to human TCR α and β sequences.
Linker in the Membrane Anchored scTCR Polypeptide
For the cell presented scTCRs of the present invention, a linker sequence links the first and second TCR segments, to form a single polypeptide strand. The linker sequence may, for example, have the formula -P-AA-P- wherein P is proline and AA represents an amino acid sequence wherein the amino acids are glycine and serine.
For the cell presented scTCRs of the present invention to bind to a TCR ligand, such as MHC-peptide or CD1-antigen complexes, the first and second segments must be paired so that the variable region sequences thereof are orientated for such binding. Hence the linker should have sufficient length to span the distance between the C terminus of the first segment and the N terminus of the second segment, or vice versa. On the other hand excessive linker length should preferably be avoided, in case the end of the linker at the N-terminal variable region sequence blocks or reduces bonding of the scTCR to the target ligand.
For example, in the case where the constant region extracellular sequences present in the first segment correspond to the constant regions of the α and β chains of a native TCR truncated at their C termini such that the cysteine residue that forms the native interchain disulfide bond of the TCR is excluded, and the linker sequence links the C terminus of the first segment to the N terminus of the second segment, the linker may consist of from 26 to 41, for example 29, 30, 31 or 32 amino acids, and a particular linker has the formula -PGGG-(SGGGG)5-P- wherein P is proline, G is glycine and S is serine (SEQ ID NO:1).
The Cell Membrane Anchored scTCR and dTCR
As mentioned above, preferred embodiments the dTCR or scTCR α and β chain sequences correspond to human TCR α and β sequences, with the exception of the complementarity determining regions (CDRs) of the variable regions which may or may not correspond to human CDR sequences. However, correspondence between such sequences need not be 1:1 on an amino acid level. N- or C-truncation, and/or amino acid deletion and/or substitution relative to corresponding human TCR sequences is acceptable, provided the overall result is a cell membrane anchored TCR comprising mutual orientation of the α and β variable region sequences is as in native αβ T cell receptors respectively. In particular, because the constant domain extracellular sequences are not directly involved in contacts with the ligand to which the cell membrane anchored scTCR or dTCR binds, they may be shorter than, or may contain substitutions or deletions relative to, extracellular constant domain sequences of native TCRs.
Included in the scope of this invention are cells presenting membrane anchored TCRs comprising amino acids encoded by any appropriate combination of the nucleic acid sequences corresponding to those disclosed in FIGS. 7 and 8. As is known to those skilled in the art, TCRs can also be produced by combination of amino acid sequences encoded by truncated variants of the sequences disclosed in FIGS. 7 & 8. Such TCRs form an additional embodiment of the present invention. Also included within the scope of this invention are membrane anchored TCRs encoded by any variants of these nucleic acid molecules.
Usually, cells according to the invention will present a plurality of the said scTCR or dTCR (the exogenous TCRs). Each of the plurality of the said scTCRs or dTCRs is preferably identical, but if the cell is a T-cell, it may also present some native (endogenous) TCRs, residually encoded by the T cell chromosomes.
Another preferred embodiment provides T cells having the said membrane anchored scTCR or dTCR, or a plurality thereof. In a further preferred embodiment these T cells are cytotoxic T cells.
Another preferred embodiment provides cells that reduces the cellular or pro-inflammatory arms of an auto-immune response having the said membrane anchored scTCR or dTCR, or a plurality thereof. Examples of such cells, include, but are not limited to macrophages, γδ T cells, Th3 T cells, Tr1 T cells, NK T cells, macrophages and regulatory T cells. In a further preferred embodiment these cells are regulatory T cells.
Regulatory T cells are characterised by the cell-surface expression of CD4 and CD25. (Bluestone and Tang Proc Natl Acad Sci USA. 2004 101 Suppl 2: 14622-6.) provides a review of regulatory T cells.
In a further embodiment of the invention the cells present scTCR or dTCR which contains a covalent disulfide bond linking a residue of the immunoglobulin region of the constant domain of the α chain to a residue of the immunoglobulin region of the constant domain of the β chain.
A further embodiment of the invention provides a cell presenting scTCR or dTCR wherein in the said TCR an interchain disulfide bond in native TCR is not present.
A further embodiment of the invention provides a cell presenting scTCR or dTCR wherein in the said TCR cysteine residues which form the native interchain disulfide bond are substituted to another residue.
A further embodiment of the invention provides a cell presenting scTCR or dTCR wherein in the said TCR cysteine residues which form the native interchain disulfide bond are substituted to serine or alanine.
A further embodiment of the invention provides a cell presenting scTCR or dTCR wherein in the said TCR an unpaired cysteine residue present in native TCR β chain is not present.
Inter-Chain Disulfide Bond
A principal characterising feature of the cell membrane anchored scTCRs and dTCRs of the present invention, is a disulfide bond between the constant region extracellular sequences of the dTCR polypeptide pair or first and second segments of the scTCR polypeptide. That bond may correspond to the native inter-chain disulfide bond present in native dimeric αβ TCRs, or may have no counterpart in native TCRs, being between cysteines specifically incorporated into the constant region extracellular sequences of dTCR polypeptide pair or first and second segments of the scTCR polypeptide. In some cases, both a native and a non-native disulfide bond may be desirable.
The position of the disulfide bond is subject to the requirement that the variable region sequences of dTCR polypeptide pair or first and second segments of the scTCR polypeptide are mutually orientated substantially as in native αβ T cell receptors.
The disulfide bond may be formed by mutating non-cysteine residues on the first and second segments to cysteine, and causing the bond to be formed between the mutated residues. Residues whose respective β carbons are approximately 6 Å (0.6 nm) or less, and preferably in the range 3.5 Å (0.35 nm) to 5.9 Å (0.59 nm) apart in the native TCR are preferred, such that a disulfide bond can be formed between cysteine residues introduced in place of the native residues. It is preferred if the disulfide bond is between residues in the constant immunoglobulin region, although it could be between residues of the membrane proximal region. Preferred sites where cysteines can be introduced to form the disulfide bond are the following residues in exon 1 of TRAC*01 for the TCR α chain and TRBC1*01 or TRBC2*01 for the TCR β chain:















TCR α chain
TCR β chain
Native β carbon separation (nm)




















Thr 48
Ser 57
0.473



Thr 45
Ser 77
0.533



Tyr 10
Ser 17
0.359



Thr 45
Asp 59
0.560



Ser 15
Glu 15
0.59










Now that the residues in human TCRs which can be mutated into cysteine residues to form a new interchain disulfide bond in cell membrane bound dTCRs or scTCRs according to the invention have been identified, those of skill in the art will be able to mutate TCRs of other species in the same way to produce a dTCR or scTCR of that species for cell membrane bound expression. In humans, the skilled person merely needs to look for the following motifs in the respective TCR chains to identify the residue to be mutated (the shaded residue is the residue for mutation to a cysteine).













αChain Thr 48:






 


αChain Thr 45:






 


αChain Tyr 10:






 


αChain Ser 15:






 


β Chain Ser 57:






 


β Chain Ser 77:






 


β Chain Ser 17:






 


β Chain Asp 59:






 


β Chain Glu 15:













In other species, the TCR chains may not have a region which has 100% identity to the above motifs. However, those of skill in the art will be able to use the above motifs to identify the equivalent part of the TCR α or β chain and hence the residue to be mutated to cysteine. Alignment techniques may be used in this respect. For example, ClustalW, available on the European Bioinformatics Institute website at www.ebi.ac.uk/index.html can be used to compare the motifs above to a particular TCR chain sequence in order to locate the relevant part of the TCR sequence for mutation.
The present invention includes within its scope cell membrane bound αβ scTCRs and dTCRs, as well as those of other mammals, including, but not limited to, mouse, rat, pig, goat and sheep. As mentioned above, those of skill in the art will be able to determine sites equivalent to the above-described human sites at which cysteine residues can be introduced to form an inter-chain disulfide bond. For example, the following shows the amino acid sequences of the mouse Cα and Cβ soluble domains, together with motifs showing the murine residues equivalent to the human residues mentioned above that can be mutated to cysteines to form a TCR interchain disulfide bond (where the relevant residues are shaded):












Mouse Cα soluble domain:


(SEQ ID NO: 11)


PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTV


 


LDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVP


 


Mouse Cβ soluble domain:


(SEQ ID NO: 12)


EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGR


 


EVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLS


 


EEDKWPEGSPKPVTQNISAEAWGRAD


 


Murine equivalent of human α Chain Thr 48:


(SEQ ID NO: 13)


 








 


Murine equivalent of human α Chain Thr 45:


(SEQ ID NO: 14)


 








 


Murine equivalent of human α Chain Tyr 10:


(SEQ ID NO: 15)


 








 


Murine equivalent of human α Chain Ser 15:


(SEQ ID NO: 16)


 








 


Murine equivalent of human β Chain Ser 57:


(SEQ ID NO: 17)


 








 


Murine equivalent of human β Chain Ser 77:


(SEQ ID NO: 18)


 








 


Murine equivalent of human β Chain Ser 17:


(SEQ ID NO: 19)


 








 


Murine equivalent of human β Chain Asp 59:


(SEQ ID NO: 20)


 








 


Murine equivalent of human β Chain Glu 15:


(SEQ ID NO: 21)


 















A nucleic acid molecule or molecules comprising a sequence or sequences encoding a membrane anchored scTCR or dTCR are also provided, as are vectors comprising said nucleic acid molecules. Included in the scope of this invention are nucleic acid sequences encoding membrane anchored TCR comprising any appropriate combination of nucleic acid sequence corresponding to those disclosed in FIGS. 7 and 8. As is known to those skilled in the art, TCRs can also be produced that comprise combinations of amino acids encoded by truncated variants of the nucleic sequences disclosed in FIGS. 7 and 8, such nucleic acid sequences form an additional embodiment of the present invention. Also included within the scope of this invention are variants of these nucleic acid molecules that encode membrane anchored TCRs.
The nucleic acid or nucleic acids encoding TCRs of the invention may be provided in a form which has been adapted for expression in a prokaryote or eukaryote host cell. Suitable host cells include, but are not limited to, bacterial, yeast, mammalian or insect cells. For example, the host cell may be a human T cell or a human haematopoietic stem cell.
Such adapted nucleic acid or nucleic acids is/are mutated to reflect the codon preference of the host cell in to which it is introduced. The mutations introduced are silent mutations which do not affect the amino acid sequence of the polypeptide or polypeptides thereby encoded. GeneArt (Regensburg, Germany) offer a suitable nucleic acid optimisation service (GeneOptimizerTM). WO 2004/059556, owned by GeneArt, provides further details of the optimisation process. Nucleic acid complementary to any such adapted nucleic acid sequence or a RNA sequence corresponding thereto also forms part of this invention. Furthermore, as will be obvious to those skilled in the art such nucleic acid or nucleic acids encoding TCRs of the invention may also comprise non-coding (intron) sequences.
As will be obvious to those skilled in the art such full-length TCR chain DNA sequences encode for the following sequences:

    
    
        A leader sequence and the extracellular, transmembrane, and cytoplasmic TCR sequences.
    
    


A method for obtaining a cell expressing a membrane anchored scTCR or dTCR is also provided, said method comprises incubating a host cell harbouring a vector encoding the membrane anchored scTCR or dTCR under conditions causing expression of the scTCR or dTCR.
Preparation of Cells Expressing TCRs Comprising a Non-Native Disulfide Interchain Bond
Another embodiment provides a method for the preparation of cells of the invention said method comprising:

    
    
        (a) isolation of a population of cells, preferably a population of T cells
        (b) in vitro transfection of said population of cells with an expression vector encoding a TCR of the invention specific for a target cell,
        (c) optional in vitro growth of the transfected cells.
    
    


In a preferred embodiment the population of cells is isolated from a patient to be treated by a method of directing said cells to a population of target cells.
The following provides details of the isolation, transformation and optional in-vitro growth of T cells.
Isolation of T cells
T cells are found in both the bloodstream and lymphatic system. Generally, in order to obtain a suitable sample of T cells a venous blood sample is first obtained. In a preferred embodiment of the invention this blood sample is obtained from the patient requiring treatment.
The skilled person will be able to prepare a suitable sample of T cells for use in the present invention. For example, the sample may be whole blood, or a sample prepared from blood including, but not limited to, peripheral blood leucocytes (PBLs) or peripheral blood mononuclear cells (PBMC).
The T cells in the blood sample obtained are then be isolated by fluorescent activated cell sorting (FACS). Briefly, this involves the addition of florescent labels which specifically bind to T cell-specific ‘marker’ proteins and sorting the cells into populations based on the presence or absence of these labels. These fluorescent labels typically comprise an antibody, or fragment thereof, to which is attached a fluorescent moiety such as phycoerythicin (PE). The choice of label, or labels, used will determine the cell types present in the sorted populations:














Label Used
Cell Population isolated









Anti CD3 fluorescent label
All (cytotoxic and helper) T cells



Anti CD8 fluorescent label
CD8+ (cyto-toxic) T cells



Anti CD4 fluorescent label
CD4+ (helper) T cells



Anti CD4 and anti CD25
Regulatory T cells



fluorescent label










In Vitro Transfection T Cells with a Vector Encoding a TCR Specific for the Target Cell

There are many techniques suitable for the transfection of mammalian cells, such as human T cells, that are known to those skilled in the art. Textbooks including the following provide experimental examples that describe the methods involved: Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1992; Glover DNA Cloning, I and II, Oxford Press, Oxford, 1985; B. D. Hames & S. J. Higgins Nucleic Acid Hybridization 1984; J. H. Miller and M. P. Calos, Gene Transfer Vectors For Mammalian Cells, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1987).
As stated above two recent patent applications are directed to the transfection of T cells with TCRs. (WO 01/55366 and US 20020064521) The methods disclosed in these applications are also applicable to the transfection of T cells with the TCRs of this invention that comprise a disulfide bond between residues not present in native TCRs. Briefly, WO 01/55366 discloses a retro-viral method for the introduction of TCRs with defined specificity into T cells. The application describes methods for the production of a retro-viral vector containing the α and β chains of a high affinity murine TCR specific for a nucleoprotein peptide (ASNENMDAM) (SEQ ID NO:22) presented by the murine class I MHC H-2Db. This vector was then replicated in a human embryonic kidney cell line and the retroviral supernatant was collected to provide the material required for T cell transfection. US 20020064521 describes methods for the molecular cloning of cDNA of a human p53-specific, HLA restricted murine TCR and the transfer of this cDNA to human T cells. α and β chain TCR cDNAs were subcloned separately into a mammalian expression vector. This vector was then transferred into Jurkat cells using standard liposome transfection procedures. Surface expression Of the transfected TCR was then confirmed by flow cytometry.
In Vitro Growth of the Transfected T Cells
Once the T cells required for adoptive therapy have been transfected with the required TCR they can optionally be cultured in vitro to provide an expanded population of T cells using standard techniques.
One preferred method for the expansion of transfected T cells of the invention relies on the use of magnetic beads coated with the specific TCR ligand recognised by the introduced TCR, and a combination of anti CD28 and anti-CD3. Briefly, the use of these beads allows the selective expansion of T cells possessing functional transfected TCRs. The beads are commercially available in an anti-biotin coated form (Miltenyi Biotec, Bisley UK) which can then be coated with the biotinylated ligands of choice. (Example 9 herein details the required methodology)
Once the T cells have been prepared using the above methods they can be administered to patients together with a pharmaceutically acceptable carrier.
Administration of the Transfected Cells to the Patient
The invention provides a method of directing cells to a population of target cells in a patient, said method comprising administering to a patient a plurality of cells expressing a surface anchored TCR, wherein said TCR comprises a disulfide interchain bond between extracellular constant domain residues which is not present in native TCRs and wherein the TCR presented by such cells is specific for a TCR ligand on the population of target cells.
The invention also provides a method of directing a T cell response to a target cell phenotype in a patient, said method comprising administering to a patient a plurality of T cells expressing a surface anchored TCR, wherein said TCR comprises a disulfide interchain bond between extracellular constant domain residues which is not present in native TCRs and wherein the TCR presented by such T cells is specific for a TCR ligand on the target cell type.
In another embodiment of the invention the TCR ligand on the target cell type is a peptide-MHC complex or a CD1-antigen complex.
In a further embodiment of the invention the administered cells are not cytotoxic T cells.
In a further embodiment of the invention the target cell is a cancer cell or infected cell and the administered cells are cytotoxic T cells.
In a further embodiment of the invention the TCR ligand is unique to one tissue-type or to cells characteristic of one organ of the body.
In another embodiment of the invention the target cell is a target for auto-reactive T cells in autoimmune disease, organ rejection or Graft Versus Host Disease (GVHD). In a specific embodiment the target cells is an islet cell.
Examples of suitable MHC-peptide targets for the TCR according to the invention include, but are not limited to, viral epitopes such as HTLV-1 epitopes (e.g. the Tax peptide restricted by HLA-A2; HTLV-1 is associated with leukaemia), HIV epitopes, EBV epitopes, CMV epitopes; insulin and/or IGRP-derived diabetes epitopes; melanoma epitopes (e.g. MAGE-1 HLA-A1 restricted epitope) and other cancer-specific epitopes (e.g. the renal cell carcinoma associated antigen G250 restricted by HLA-A2). Further disease-associated pMHC targets, suitable for use in the present invention, are listed in the HLA Factsbook (Barclay (Ed) Academic Press), and many others are being identified.
In a further embodiment of the invention the population of T cells is isolated from a patient to be treated.
T cells expressing the transfected TCRs can be administered to the patients by a number of routes. For example, i.v. infusion at regular intervals, optionally accompanied by the administration of a cytokine such as IL-2.
A further embodiment of the invention provides an infusible or injectable pharmaceutical composition comprising a plurality of cells expressing a surface anchored TCR, said TCR comprises a disulfide bond between α and β extracellular constant domain residues which is not present in native TCRs together with a pharmaceutically acceptable carrier.
Such pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection formulations which may contain suspending agents, anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient.
Dosages of the cells 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. For example, a effective dosage may vary between 105 to 1010 cells/kg body weight. The practice of therapeutic administration by infusion is described in a number of papers. See, for example (Rosenberg 1988 New Eng. J Med 319 1676-1680). The dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be reduced, in accordance with normal clinical practice.
Additional Aspects
The invention provides a method of treatment of cancer, GVHD, infection, organ rejection, or auto-immune disease comprising administering a plurality of cells presenting at least one αβ T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, said TCR comprising a disulfide interchain bond between extracellular constant domain residues which is not present in native TCRs. A specific embodiment is provided wherein the auto-immune disease is a disease selected from Rheumatoid Arthritis, Diabetes, Multiple Sclerosis or Reactive Arthritis
Another aspect of the invention is provided by the use of a cell presenting at least one αβ T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, said TCR comprising a disulfide interchain bond between extracellular constant domain residues which is not present in native TCRs in the preparation of a medicament for treatment of cancer, GVHD, infection, organ rejection, or auto-immune disease.
Cancers which may benefit the methods of the present invention include: leukaemia, head, neck, lung, breast, colon, cervical, liver, pancreatic, ovarian and testicular.
Auto-immune diseases which may benefit the methods of the following invention include:
Acute disseminated encephalomyelitis
Adrenal insufficiency
Allergic angiitis and granulomatosis
Amylodosis
Ankylosing spondylitis
Asthma
Autoimmune Addison's disease
Autoimmune alopecia
Autoimmune chronic active hepatitis
Autoimmune haemolytic anaemia
Autoimmune Neutrogena
Autoimmune thrombocytopenic purpura
Behçet's disease
Cerebellar degeneration
Chronic active hepatitis
Chronic inflammatory demyelinating polyradiculoneuropathy Chronic neuropathy with monoclonal gammopathy
Classic polyarteritis nodosa
Congenital adrenal hyperplasia
Cryopathies
Dermatitis herpetiformis
Diabetes
Eaton-Lambert myasthenic syndrome
Encephalomyelitis
Epidermolysis bullosa acquisita
Erythema nodosa
Gluten-sensitive enteropathy
Goodpasture's syndrome
Guillain-Barre syndrome
Hashimoto's thyroiditis
Hyperthyroidism
Idiopathic hemachromatosis
Idiopathic membranous glomerulonephritis
Isolated vasculitis of the central nervous system
Kawasaki's disease
Minimal change renal disease
Miscellaneous vasculitides
Mixed connective tissue disease
Multifocal motor neuropathy with conduction block
Multiple sclerosis
Myasthenia gravis
Opsoclonus-myoclonus syndrome
Pemphigoid
Pemphigus
pernicious anaemia Polymyositis/dermatomyositis
Post-infective arthritides
Primary biliary sclerosis
Psoriasis
Reactive arthritides
Reiter's disease
Retinopathy
Rheumatoid arthritis
Sclerosing cholangitis
Sjögren's syndrome
Stiff-man syndrome
Subacute thyroiditis
Systemic lupus erythematosis
Systemic necrotizing vasculitides
Systemic sclerosis (scleroderma)
Takayasu's arteritis
Temporal arteritis
Thromboangiitis obliterans
Type I and type II autoimmune polyglandular syndrome
Ulcerative colitis
Uveitis
Wegener's granulomatosis
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.
The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.
EXAMPLES
The following example describes the preparation of the DNA sequences of FIGS. 3a and 3b. This example is usable for the preparation of the coding sequences of any given αβ TCR including a non-native disulfide interchain bond.
Example 1
Design of Primers and Mutagenesis of A6 Tax TCR α and β Chains to Introduce the Cysteine Residues Required for the Formation of a Novel Inter-Chain Disulfide Bond
For mutating A6 Tax threonine 48 of exon 1 in TRAC*01 to cysteine, the following primers were designed (mutation shown in lower case):









(SEQ ID NO: 23)



5′-C ACA GAC AAA tgT GTG CTA GAC AT



 



(SEQ ID NO: 24)



5′-AT GTC TAG CAC Aca TTT GTC TGT G






For mutating A6 Tax serine 57 of exon 1 in TRBC1*01 or TRBC2*01 to cysteine, the following primers were designed (mutation shown in lower case):









(SEQ ID NO: 25)



5′-C AGT GGG GTC tGC ACA GAC CC



 



(SEQ ID NO: 26)



5′-GG GTC TGT GCa GAC CCC ACT G






PCR Mutagenesis:
Expression plasmids containing the genes for the A6 Tax TCR α or β chain were mutated using the α-chain primers or the β-chain primers respectively, as follows. 100 ng of plasmid was mixed with 5 μl 10 mM dNTP, 25 μl 10×Pfu-buffer (Stratagene), 10 units Pfu polymerase (Stratagene) and the final volume was adjusted to 240 μl with H2O. 48 μl of this mix was supplemented with primers diluted to give a final concentration of 0.2 μM in 50 μl final reaction volume. After an initial denaturation step of 30 seconds at 95° C., the reaction mixture was subjected to 15 rounds of denaturation (95° C., 30 sec.), annealing (55° C., 60 sec.), and elongation (73° C., 8 min.) in a Hybaid PCR express PCR machine. The product was then digested for 5 hours at 37° C. with 10 units of DpnI restriction enzyme (New England Biolabs). 10 μl of the digested reaction was transformed into competent E. coli XL1-Blue bacteria and grown for 18 hours at 37° C. A single colony was picked and grown over night in 5 ml TYP+ampicillin (16 g/l Bacto-Tryptone, 16 g/l Yeast Extract, 5 g/l NaCl, 2.5 g/l K2HPO4, 100 mg/l Ampicillin). Plasmid DNA was purified on a Qiagen mini-prep column according to the manufacturer's instructions and the sequence was verified by automated sequencing. The respective mutated nucleic acid and amino acid sequences are shown in FIGS. 3a and 4a for the α chain and FIGS. 3b and 4b for the β chain.
The following example describes the extension of the DNA of FIGS. 3a and 3b to add sequences coding for the remainder of the constant domains of the A6 TCR. Again, this example is usable for the extension of the constant domain encoding sequences of a corresponding soluble variant of any given αβ TCR.
Example 2
Design of A6 Tax TCR α and β Chain Nucleic Acid Sequences Required to Produce a Cell Surface Membrane Anchored A6 Tax TCR Including Cysteine Residues Required for the Formation of a Novel Inter-Chain Disulfide Bond
The constructs encoding the α and β chains of a soluble disulfide-linked A6 Tax TCR molecule prepared as described in example 1 are used, along with cDNA from human peripheral blood mononuclear cells (PBMCs), in the production of constructs encoding the α and β chains of a membrane anchored A6 Tax TCR including cysteine residues required for the formation of a novel inter-chain disulfide bond. (Refer to FIG. 5 for a diagrammatic representation of the method involved).
TCR α chain DNA corresponding to Fragment 1 (See FIG. 5a) is amplified from cDNA encoding wild-type TRAY 12-2 TCR by PCR using the following primer pair specific for the TRAY 12-2 TCR signal peptide (Fwd primer) and the TRAY 12-2 TCR variable domain (Rev primer):









5′ Fwd α primer:



(SEQ ID NO: 27)



5′- ATG ATG AAA TCC TTG AGA GTTTT -3′



 



5′ Rev α primer:



(SEQ ID NO: 28)



5′- GTA AGT GCA GTT GAGAGAGG -3′






TCR β chain DNA corresponding to Fragment 1 (See FIG. 5a) is amplified from cDNA encoding wild-type TRBV 6-5 TCR by PCR using the following primer pair specific for the TRBV 6-5 TCR signal peptide (Fwd primer) and the TRBV 6-5 TCR variable domain (Rev primer):









5′ Fwd β primer:



(SEQ ID NO: 29)



5′- ATG AGC ATC GGC CTC CTG T -3′



 



5′ Rev β primer:



(SEQ ID NO: 30)



5′- TT CAT ATC CTGGGC ACA CTG -3′






The above primers are designed to produce a PCR product that incorporates an overlap with the DNA encoding the variable region of the soluble disulfide-linked A6 Tax TCR produced in example 1.
TCR α chain DNA corresponding to Fragment 2 (See FIG. 5b) is amplified from cDNA from PBMC using the following primer pair specific for the 3′ end of TRAC, this primer pair also introduces a Cys to Ser mutation disrupting the formation of the native inter-chain disulfide bond:







3′ Fwd α primer:


(SEQ ID NO: 31)


5′- TC CCC AGC CCA GAA AGT TCC TCT GAT GTC 


 


AAG CTG GTC GAG AAA AG -3′


 


3′ Rev α primer:


(SEQ ID NO: 32)


5′- TTA GCT GGA CCA CAG CCG CAG -3′






TCR β chain DNA corresponding to Fragment 2 (See FIG. 5b) is amplified from cDNA from PBMC using the following primer pair specific for the 3′ end of TRBC2, this primer pair also introduces a Cys to Ser mutation disrupting the formation of the native inter-chain disulfide bond:







3′ Fwd β primer:


(SEQ ID NO: 33)


5′- CC GAG GCC TGG GGT AGA GCA GAC TCT GGC


 


TTC ACC TCC GAG TCT TAC C -3′


 


3′ Rev β primer:


(SEQ ID NO: 34)


5′- TTA GCC TCT GGA ATC CTT TCT C- 3′






These primers are designed to produce a PCR product that incorporates an overlap with the DNA encoding the constant region of the soluble disulfide-linked A6 Tax TCR produced in example 1.
Final PCRs are required to assemble the entire genes for the two TCR-chains. For the alpha chain fragments 1 and 2 are mixed with the plasmid coding for the soluble alpha-chain and the full length coding region is amplified using the 5′ Fwd a primer and the 3′ Rev a primer with suitable restriction site sequences added to the primers as flanking sequences to facilitate sub-cloning in the required vector (for example, the retroviral pLXSN vector, BD Clontech, UK). The fragment is sub-cloned into the expression vector and sequenced.
For the beta chain fragments 1 and 2 are mixed with the plasmid coding for the soluble beta-chain and the full length coding region is amplified using the 5′ Fwd β primer and the 3′ Rev β primer with suitable restriction site sequences added to the primer as flanking sequences to facilitate sub-cloning into the required vector (for example, the retroviral pLXSN vector, BD Clontech, UK). The fragment is sub-cloned into the expression vector and sequenced.
FIGS. 6a and 6b show the nucleic acid and protein sequences of the membrane anchored α and β chain of A6 TCR respectively, mutated so as to introduce a new cysteine codon and mutate the cysteine residues involved in forming the native inter-chain disulfide bridge to Ser.
The above PCR reactions are all carried out using the following methodology.
For a 100 μl reaction mix:

    
    
        1. 18 MO quality H2O to 100
        2. 50 pmol Forward Primer
        3. 50 pmol Reverse Primer
        4. 2 μl 10 mM dNTP (10 mM each of dATP, dTTP, dCTP, dGTP).
        5. 10 μl 10× Buffer (Pfu buffer for cloning purposes and Taq buffer for diagnostic PCR).
        6. 5 units of enzyme (Pfu DNA Polymerase or Taq polymerase according to the particular application).
    
    


PCR program:

    
    
        1. A denaturation step where the sample is heated to 94° for 10 minutes.
        2. A number of cycles (20-40) including
        
            a denaturation step 1 minute @94°
            an annealing step 1 minute @45-60° (use the gradient block in PCR-1 if you need to establish the optimal annealing temperature).
            an elongation step 5-10 minutes @72-73°.
        
        
        3. A final elongation step 10 minutes @72-73° to ensure that all products are full length
        4. followed by a soak step at 4°.
    
    


The following example describes the preparation of the DNA sequences of FIGS. 9a and 9b. This example is usable for the preparation of the coding sequences of any given αβ TCR which includes a non-native disulfide interchain bond.
Example 3
Production of DNA Encoding a Soluble AH-1.23 TCR Comprising a Non-Native Disulfide Inter-Chain Bond
Synthetic genes encoding the TCR α and TCR β chains of a soluble AH1.23 TCR can be manufactured to order. There are a number of companies which carry out this service such as GeneArt (Germany).
FIGS. 9a and 9b show respectively the DNA sequences of the α and β chains of a soluble AH-1.23 TCR, mutated so as to introduce a novel cysteine codon (indicated by shading).
FIGS. 10a and 10b show respectively the AH-1.23 TCR α and β chain extracellular amino acid sequences produced from the DNA sequences of FIGS. 9a and 9b 
The DNA sequences shown in FIGS. 9a and 9b can then be sub-cloned into the required vector containing the DNA sequences of FIGS. 3a and 3b respectively in such a way as to replace the DNA encoding the corresponding extracellular portions of the A6 TCR.
The following example describes a means of preparing DNA sequences encoding full-length TCRs containing a non-native disulfide interchain bond for use in the current invention. Preferably, said DNA sequences will comprise restriction enzyme recognition site to facilitate ligation of the sequences into the vector of choice. This example is usable for the production of any αβ or γδ TCR.
Example 4
Production of Nucleic Acids Encoding Alternative TCR α and β Chains of Membrane Anchored TCRs Including Cysteine Residues Required for the Formation of a Novel Inter-Chain Disulfide Bond
To incorporate DNA encoding an alternative TCR into the vector(s) of choice synthetic genes encoding the required full-length TCR α and TCR β chains, altered in order to encode the required introduced cysteine residues in the constant domains thereof, can be manufactured to order. There are a number of companies which carry out this service such as GeneArt (Germany). Such DNA sequences can be produced which incorporate restriction enzyme recognition sequences to facilitate ligation of the DNA produced into the vector of choice.
For transfection of the desired cells with the expression vectors prepared according to example 6, selection of the appropriate vector is required:
Example 5
Vector Choice for the Transfection of T Cells with DNA Encoding TCRs Containing Cysteine Residues Required for the Formation of a Novel Inter-Chain Disulfide Bond
As will be obvious to those skilled in the art the primary difference between transient and stable transfection methods is the choice of vector. The following table provides a summary of a number of vectors suitable for the transient transfection and/or stable transfection of T cells with DNA encoding TCRs containing cysteine residues required for the formation of a novel inter-chain disulfide bond:
















Random
Stable Site-
Stable


Vector and
Transient
Stable
Specific
Episomal


Supplier
Expression
Integration
Integration
Maintenance







pCI
✓
 ✓*
x
x


(Promega)


pCIneo
✓
 ✓*
x
x


(Promega)


pREP4
✓
x
x
✓


(Invitrogen)


pCEP4
✓
x
x
✓


(Invitrogen)


pcDNA5/FRT
✓
(✓)
✓
x


(Invitrogen)


FRT
✓
✓
✓
x


Retroviral


Vectors


Standard
✓
✓
x
x


Retroviral


Vectors





✓* Suitable when in combination with an appropriate vector (e.g. pCIneo with pCI).


(✓) Capable of random integration, but designed for site-specific integration into specialised recipient cells.






Ligation of the DNA sequences encoding a TCR containing a non-native disulfide interchain bond, prepared for example, as described in examples 1 and 2, or examples 3 or 4, into the desired vector or vectors is required. These vectors may be one of those listed in example 5. This example is usable for the ligation of the coding sequences of any given αβ or γδ TCR including a non-native disulfide interchain bond into the vector(s) of choice:
Example 6
Ligation of DNA Sequences Encoding a TCR Containing a Non-Native Disulfide Interchain Bond into the Desired Vector
In order to facilitate the insertion of DNA encoding the TCR chains in the desired orientation into the vector or vectors of choice the vector(s) and DNA encoding the TCR chains should each have the same pair of different complementary ends. To achieve this the desired recipient vector or vectors, and the DNA sequences encoding the TCR chains are digested with the same appropriate pair of differing restriction enzymes. The cut DNA chains and the cut vector or vectors are then ligated using the Rapid DNA Ligation kit (Roche) following the manufacturers instructions.
Example 7 describes a general procedure for the isolation of T cell sub-populations for transformation to produce cells in accordance with the invention.
Example 7
Isolation of T Cell Sub Populations
PBMCs are isolated from venous blood samples using Leucosep® tubes (Greiner Bio-one, Germany) following the manufacturer's instruction. The isolated PBMCs are washed and used immediately. Freshly isolated PBMCs are washed twice in 10% autologous human serum/RPMI (Gibco BRL). Finally, the cells are re-suspended in RPMI medium.
T cell sub-populations are isolated from PBMCs by FACS using the relevant combination of antibodies in the table below for the T cell sub-population required and the following procedure:














Label Used
Cell Population isolated









Anti CD3 fluorescent label
All (cytotoxic and helper) T cells



Anti CD8 fluorescent label
CD8+ (cyto-toxic) T cells



Anti CD4 fluorescent label
CD4+ (helper) T cells



Anti CD4 and anti CD25
Regulatory T cells



fluorescent label










Under sterile conditions, the relevant fluorescently-labeled antibodies (0.01 mg/ml final concentration) are incubated with PBMCs (1×107/ml) for 30 mins at 37° C., 5% CO2. Cells are then washed using medium (37° C.), centrifuged for 10 mins at 250×g and the supernatant discarded. The pellet is re-suspended and the cells are then bulk-sorted by FACS. The selected T cells are collected in either medium containing 10% autologous serum (for in-vitro culturing), or in the appropriate infusion medium, such as Hank's balanced buffer solution (Sigma, UK) with 10% autologous human serum albumin for immediate therapeutic use.
Alternatively, the required T cell sub-population may be isolated using magnetic beads coated with the same antibodies and antibody combinations described above.
Minimacs beads, produced by Miltenyi Biotech, are suitable for use in the isolation of T sub-populations and the manufacturer provides instructions for their use. This method “positively” selects and isolates the desired T cell sub-population. It is also possible to “negatively” select the desired T cell sub-population. This is achieved by coating the beads with a range of antibodies that will bind to all but the required T cell population in PBMCs.
Example 8 describes one method, usable in accordance with the invention, of modifying isolated cells for expression of TCRs containing a non-native disulfide interchain bond.
Example 8
Retro-Viral Transduction of T Cell with TCRs Containing Introduced Cysteine Residues Capable of Forming a Non-Native Disulfide Interchain Bond
Primary T cells or T cell lines/clones are transduced with an appropriate retroviral vector, (e.g. the pLXSN retrovirus (BD ClonTech, UK)) following a T cell transduction methodology based on that described in (Clay (1999) J Immunol 163 507-513 and Bunnel (1995) PNAS USA 92 7739)
Production of Retroviral Supernatant
Briefly, in order to produce retroviral supernatant, the PG13 retrovirus producer cell line is transduced with the retroviral vector (pLXSN, BD Clontech, UK) produced in example 2 that contains DNA encoding the α and β chains of a membrane anchored A6 Tax TCR including cysteine residues required for the formation of a novel inter-chain disulfide bond. High titre clones are then isolated using standard techniques familiar to those skilled in the art. (See, for example (Miller (1991) J Virol 65 2220) A high titre clone is then grown to 80% confluence and the supernatant is then harvested.
Transduction of T Cells with Retroviral Supernatant
T cells are then re-suspended at 1×106 cells/ml in microtitre well plates in retroviral supernatant containing 8 μg/ml polybrene and 600 IU/ml IL-2. The plates are then centrifuged at 1000×g for 90 mins and incubated overnight at 37° C. in a humidified 5% CO2 incubator. This transduction procedure is repeated after 2 days. The transduction procedure described in (Clay (1999) J. Immunol. 163 507-513) is then followed, thereby providing transfected T cells ready for in-vitro testing.
Example 9 describes a general method for enriching and enlarging a population of T cells in accordance with the invention. This method is not TCR specific.
Example 9
In-Vitro Growth of Transfected T Cells
After the transfection of T cells to express modified TCRs as described in Example 8 these T cells can, if necessary, be grown in-vitro to produce an enriched and enlarged populations of cells for in-vitro evaluation or therapeutic use using the following method.
Anti-biotin coated Clinimacs beads (Miltenyi Biotec, Germany) are coated with biotinylated anti-CD28 and anti-CD3 antibodies. 500,000 T cells and 500,000 autologous irradiated (33 Gy) APCs pulsed with the appropriate peptide (Tax peptide), are added to RPMI 1640 buffer containing 10-50 U/ml IL-2 and 10% autologous serum. 5×106/ml anti-CD28 and anti-CD3 antibody coated Clinibeads are then added to the cells.
The cells are then incubated under sterile conditions at 37° C., 5% CO2 for 7 days. During this incubation period the buffer is replaced every 3 days. The cells can be re-stimulated the following week with the same ratio of beads to T-cells and fresh peptide-pulsed APCs. Once the required total number of transfected T cells has been reached the T cells are then re-suspended in the appropriate buffer for in-vitro evaluation or therapeutic use.
Example 10 describes one method of testing for successful cell surface expression of the desired TCRs on the chosen modified cell. This method is generally applicable, and not restricted to any particular cell surface TCR.
Example 10
Fluorescence Activated Cell Sorting (FACS)-Based Assay to Demonstrate Specific Binding of Cognate Peptide-MHC Complexes to T Cells Transfected to Express an A6 Tax TCR Incorporating Cysteine Residues Required for the Formation of a Novel Inter-Chain Disulfide Bond
Preparation of T Cell Samples for Staining
The transfected T cells are re-suspended in FACS staining buffer (2% FCS/PBS, at 37° C.) and counted. The cells are aliquoted into FACS tubes and pre-incubated at 37° C. for 5-10 minutes prior to staining.
Staining of T cells with HLA-A2 Tax Tetramers to Assess TCR-pMHC Binding
In order to stain the transfected T cells HLA-A2 Tax monomers are prepared using the methods described in WO 99/60120, and tetramerised using Phycoerythrin (PE)-labelled streptadivin via the methods described in (O'Callaghan (1999) Anal Biochem 266 9-15)
The following fluorescently labelled molecules are also used in the FACS assay as controls:
FITC-labelled isotype controls
PE-labelled “irrelevant” peptide-HLA-A2 tetramers
PE-labelled HLA-A2 Tax tetramer (48 μg) is incubated with 1×106 transfected T cells and 5 μg anti-CD8-FITC labelled antibody (or 5 μg anti-CD4-FITC labelled antibody) for 20 mins at 37° C. Cells are then washed using FACS buffer (37° C.), centrifuged for 10 mins at 250×g and the supernatant discarded.
After the wash, transfected T cells are re-suspended in 0.5 ml PBS. The T cell populations present in the samples are then analysed by flow cytometry.
Any T cells that are double-labelled by both the PE-HLA-A2 Tax tetramers and the αCD8-FITC labels (or anti-CD4-FITC labelled antibody) are CD8+ T cells (or CD4+ T cells) expressing the transfected A6 Tax TCR.
The above HLA tetramer FACs staining method can be adapted to assess the expression level of any exogenous TCR on the surface of T cells by using tetramers of the cognate peptide-HLA for the desired exogenous TCR.
Staining of Transfected T Cells with Antibodies to Assess Exogenous TCR Expression
As will be obvious to those skilled in the art there are other binding agent that can be utilised in such FACS methods, or any other suitable detection methods, for the assessment of exogenous TCR. The following table provides a summary of some antibodies suitable for this purpose:











Antibody Specificity
Usage







Specific TCR variable domain
Assessment of exogenous TCR expression


(e.g. anti-Vβ30)
on T cells posessing an endogenous TCR



of differing V domain usage


Pan TCR
Assessment of exogenous TCR expression



on TCR- cells


CD3
Assessment of exogenous TCR expression



on CD3− TCR- cells. The presence of the



exogenous TCR should “rescue” cell



surface CD3 presentation









Example 11 describes one method of testing for successful cell surface expression of functional exogenous TCRs on the surface of a CTL. This method is specific for such CTL cells. However, the method is not limited to a specific TCR.
Example 11
Europium-Release Method for Assessing the Ability of CTL ‘Killer’ T Cells Transfected to Express the Membrane-Anchored A6 Tax TCR to Specifically Lyse Target Cells
The following assay is used to assess the ability of CTLs transfected to express the membrane-anchored A6 Tax TCR to specifically lyse HLA-A*0201+ target cells.
The following mixtures are prepared for the assays:

    
    
        Experimental wells: 50 μl of Transfected CTLs, 50 μl of media, 50 μl targets cells pulsed with the cognate HLA-A2 peptide.
        Negative control wells: 50 μl of Transfected CTLs (effector cells), 50 μl of media, 50 μl targets cells pulsed with an irrelevant HLA-A2 peptide.
        Background wells: APC Target cells are spun down after dilution to final concentration and the 50 μl of supernatant added to 100 μl media.
        Spontaneous release wells: Target cells alone (no effector cells)+100 μl media
        Maximum release wells: spontaneous release wells +15 μl of 10% Triton (Sigma T-9284)
    
    


Briefly, the above mixtures of effector and target cells are incubated for 2 to 4 hours and the Europium release assay is then carried out following the instructions supplied with the Delfia EuTDA Cytotoxicity Kit (Perkin Elmer).
Example 12 describes one method of testing for successful cell surface expression of functional exogenous TCRs on the surface of regulatory T cells or CTLs. The method is not limited to a specific TCR.
Example 12
Thymidine Incorporation Assay for Assessing the Ability of T Cells Transfected to Express the Membrane-Anchored AH1.23 TCR to Specifically Alter T Cell Proliferation
5×106 PMBCs are pulsed with 1 μM of the cognate peptide for the AH1.23 TCR and then cultured in RPMI 1640 medium at 37° C., 5% CO2 for 14 days. A control group of 5×106 PMBCs cultured at 37° C., 5% CO2 for 14 days without peptide pulsing. Both cultures are fed with 40 units/ml recombinant human IL-2 every 3 days.
The following are then added to 1×105 cells in a 96 well plate both the cultures prepared above:

    
    
        1×105 fresh autologous irradiated (33Gy) PBMCs, and a range (0 cells, 5×104, 1×105, 2×105, 5×105) of T cells transfected with the AH1.23 TCR using the methods described in the previous examples. These cultures are then incubated in RPMI 1640 medium for 3 days at 37° C., 5% CO2.
        1.85 MBq/ml of H3 Thymidine is then added to these cultures and they are incubated for a further 8 hours at 37° C., 5% CO2. The cells are harvested using a cell-harvester, and the level of thymidine incorporation into the cells is measured using a TopCount scintillation counter.
    
    


A reduction in thymidine incorporation into the previously peptide-pulsed PBMCs, compared to that seen in the non-pulsed PMBCs indicates that the transfected Regulatory T cells are causing a pMHC-specific down-regulation of cell proliferation.
An increase in thymidine incorporation into the previously peptide-pulsed PBMCs, compared to that seen in the non-pulsed PMBCs indicates that the transfected CTLs are causing a pMHC-specific up-regulation of cell proliferation.
Example 13 describes the treatment of patients with cells in accordance with the invention. This treatment method can be used for T cells transfected with any exogenous TCR.
Example 13
Infusion into Patients of T Cells Transfected to Express TCRs Including Cysteine Residues Required for the Formation of a Novel Inter-Chain Disulfide Bond
In order to infuse the transfected T cells expressing TCRs including cysteine residues required for the formation of a novel inter-chain disulfide bond into patients the following methodology, as described in (Hague (2002) Lancet 360 436-442), is used. Briefly, the transfected T cells are washed in Hank's balanced buffer solution (Sigma, UK) with 10% autologous human serum albumin and then re-suspended in 20 ml of the same buffer solution. The transfected T cells are then slowly infused into the patient requiring treatment at a dose of 106 cells per kg bodyweight over a 15 minute period. The patient's vital signs are regularly checked over the next 4 hours to detect any toxic effects.
These infusions are then repeated periodically, and the condition of the patient assessed by the most appropriate method. For example, in the case of a patient receiving the transfected TCRs as a means of treating a tumour these could include one or more of the following palpation, radiography, CT scanning or biopsy. The dosage and frequency of the infusions is varied if required. Finally the outcome of the treatment at 6 months after the final infusion is also recorded in accordance with WHO criteria.

## Claims

1. A method for obtaining modified mammalian cell, wherein the modified mammalian cell comprising a membrane presenting at least one modified T cell receptor (TCR) anchored to the membrane by a transmembrane sequence, the modified TCR comprising an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs, which method comprises incubating mammalian cell containing an expression vector encoding the modified TCR under conditions whereby the modified TCR is expressed, thereby obtaining the modified mammalian cell.

2. A method of directing immune cells to a population of target cells in a patient, said method comprising administering to the patient a plurality of immune cells comprising a membrane presenting at least one modified TCR anchored to the membrane by a transmembrane sequence, wherein the modified TCR comprises an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs wherein the modified TCR presented by such cells is specific for a TCR ligand presented on the target cells thereby directing the cells to the population of target cells in the patient.

3. A method of directing a T cell response to a target cell in a patient, said method comprising administering to the patient a plurality of T cells comprising a membrane presenting at least one modified TCR anchored to the membrane by a transmembrane sequence, wherein the modified TCR comprises an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs, wherein the modified TCR presented by such T cells is specific for a TCR ligand presented on the target cell, thereby directing the T cell response to the target cell in the patient.

4. The method according to claim 2 wherein the TCR ligand presented on the target cells is a peptide-MHC complex or a CD1-antigen complex.

5. The method according to claim 2 wherein the administered cells are not cytotoxic T cells.

6. The method according to claim 2 wherein the target cell is a cancer cell or infected cell and the administered cells are cytotoxic T cells.

7. The method according to claim 2 wherein the TCR ligand is unique to one tissue type or to cells characteristic of one organ of the body.

8. The method according to claim 7 wherein the target cell is a target for autoreactive T cells in autoimmune disease, organ rejection or graft versus host disease (GVHD).

9. The method according to claim 8 wherein the target cell is an islet cell.

10. A method for the preparation of cells comprising a membrane presenting a plurality of modified (TCRs) anchored to the membrane by a transmembrane sequence, wherein the modified TCRs comprise an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs, said method comprising: (a) isolation of a population of cells (b) in vitro transfection of said population of cells with an expression vector encoding the modified TCRs, said TCRs being specific for a target cell, and optionally (c) in vitro growth of the transfected cells.

11. The method according to claim 10 wherein the cells are T cells.

12. The method according to claim 10 wherein the population of cells is isolated from a patient who is to be treated by directing immune cells comprising a membrane presenting at least one modified TCR anchored to the membrane by a transmembrane sequence, wherein the modified TCR comprises an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs wherein the modified TCR presented by such cells is specific for a TCR ligand on the population of target cells to a population of target cells in the patient.

13. A method of treatment of cancer, GVHD, infection, organ rejection, or autoimmune disease comprising administering a plurality of immune cells comprising a membrane presenting at least one modified TCR anchored to the membrane by a transmembrane sequence, wherein the modified TCR comprises an interchain disulfide bond between extracellular constant domain residues which is not present in native TCRs.

14. The method according to claim 13 wherein the auto-immune disease is rheumatoid arthritis, diabetes, multiple sclerosis or reactive arthritis.

15. The method according to claim 3 wherein the TCR ligand on the target cell type is a peptide-MHC complex or a CD1-antigen complex.

16. The method according to claim 3 wherein the administered cells are not cytotoxic T cells.

17. The method according to claim 3 wherein the target cell is a cancer cell or infected cell and the administered cells are cytotoxic T cells.

18. The method according to claim 3 wherein the TCR ligand is unique to one tissue type or to cells characteristic of one organ of the body.

19. The method according to claim 18 wherein the target cell is a target for autoreactive T cells in autoimmune disease, organ rejection or (GVHD).

20. The method according to claim 19 wherein the target cell is an islet cell.