Imported: 10 Mar '17 | Published: 27 Nov '08
USPTO - Utility Patents
The present invention provides isolated T cell receptors (TCRs) having the property of specifically binding to the VYGFVRACL-HLA-A24 peptide-MHC. Such TCRs are useful, either alone or associated with a therapeutic agent, for targeting cancerous cells presenting the complex.
The present invention relates to isolated T cell receptors (TCRs) having the property of specifically binding to VYGFVRACL-HLA-A24.
The VYGFVRACL peptide is derived from the catalytic sub-unit of the Telomerase protein. (See Meyerson et al., (1997) Cell 90: 785-795 and Nakamura et al., (1997) Science 277: 955-9) These studies describe the near-simultaneous discovery of the DNA and deduced amino acid sequence of the Telomerase catalytic subunit from database sequences. Both studies note that Telomerase catalytic sub-unit activity is associated with human cancer. The Class I HLA molecules of these cancerous cells present peptides from this protein, including VYGFVRACL. This peptide is presented in the context of HLA-A24 (Arai et al., (2001) Blood 97 (9): 2903-2907, and Tajima et al., Int. J. Cancer (2004) 110: 403-412). Therefore, the VYGFVRACL-HLA-A24 complex provides a cancer marker that the TCRs of the invention can target, for example for the purpose of delivering cytotoxic agents to the cancer cells.
This invention makes available for the first time isolated T cell receptors (TCRs) having the property of specifically binding to VYGFVRACL-HLA-A24. Such TCRs are useful, either alone or associated with a therapeutic agent for targeting cancer cells presenting that complex.
The present invention provides isolated TCRs having the property of specifically binding to VYGFVRACL-HLA-A24. Preferably, the VYGFVRACL (SEQ ID NO: 1) peptide is presented in the context of HLA-A*2402.
The TCRs of the having the property of specifically binding to VYGFVRACL-HLA-A24, are referred to herein as VYG-A24 TCRs. Parental VYG-A24 TCRs are defined as those which comprise the alpha chain variable region shown in FIG. 1a (SEQ ID NO: 2) and the beta chain variable region shown in FIG. 1b (SEQ ID NO: 3). For example, a disulfide-linked soluble version of the parental VYG-A24 TCR consists of the TCR alpha chain shown in FIG. 5a (SEQ ID NO: 15) and the TCR beta chain shown in FIG. 5b (SEQ ID NO: 16).
One embodiment of the invention is provided by non-natural TCRs having the property of specifically binding to VYGFVRACL-HLA-A24. That is such TCRs of the invention consist of sequences not found in nature.
Another embodiment provides a TCR of the invention CHARACTERISED IN THAT said TCR has a KD for the said VYGFVRACL-HLA-A*02402 complex of 5 M or less. Example 4 herein provides details of a Biacore-based method suitable for determining the KD for the interaction between soluble TCRs and pMHC molecules.
A further aspect is provided by an isolated TCR of the invention comprising the Complimentarity Determining Regions (CDRs) present in FIG. 10b (SEQ ID NO 20) and/or FIG. 3b (SEQ ID No: 12). The CDRs of these TCR chains are underlined in the corresponding figures.
The parental VYG-A24 TCR specific for VYGFVRACL-HLA-A24 complex has the following Valpha chain and Vbeta chain gene usage:
VYG-A24 TCRs can be used as a template from which other TCRs of the invention can be produced. Thus the invention includes in one embodiment TCRs which are mutated relative to the parental VYG-A24 TCR chain variable region (see FIG. 1a and SEQ ID No: 2) and/or chain variable region (see FIG. 1b and SEQ ID NO: 3) in at least one complementarity determining region (CDR) and/or variable domain framework region thereof. In a related embodiment the invention also encompasses TCRs which are mutated relative to the VYG-A24 TCR chain variable region (see FIG. 10a and SEQ ID No: 19) and in at least one complementarity determining region (CDR) and/or variable domain framework region thereof.
It is also contemplated that other hypervariable regions in the variable domains of the TCRs of the invention, such as the hypervariable 4 (HV4) regions, may be mutated to produce a high affinity mutant TCR which retains the property of specifically binding to VYGFVRACL-HLA-A24.
Phage display provides one means by which libraries of TCR variants can be generated. Methods suitable for the phage display and subsequent screening of libraries of TCR variants each containing a non-native disulfide interchain bond are detailed in (Li et al., (2005) Nature Biotech 23 (3): 349-354) and WO 2004/04404.
Native TCRs exist in heterodimeric or forms. However, recombinant TCRs consisting of a single TCR or TCR chain have previously been shown to bind to peptide MHC molecules. Furthermore, recombinant TCRs consisting of or homodimers have previously been shown to bind to peptide MHC molecules. Therefore, other embodiments of the invention are provided by TCR or TCR homodimers.
In one embodiment TCRs of the invention comprise both an chain variable region and an TCR chain variable region.
Unless stated to the contrary, the TCR amino acid sequences herein are generally provided including an N-terminal methionine (Met or M) residue. As will be known to those skilled in the art this residue may be removed during the production of recombinant proteins. As will also be obvious to those skilled in the art, it may be possible to truncate the sequences provided at the C-terminus and/or N-terminus thereof, by 1, 2, 3, 4, 5 or more residues, without substantially affecting the pMHC binding characteristics of the TCR, all such trivial variants are encompassed by the present invention.
As used herein the term isolated TCRs means TCRs in a format other than one found in nature, for example a soluble TCR or a TCR presented on a cell which was non-naturally transfected with the genetic material encoding said TCR.
As used herein the term variable region is understood to encompass all amino acids of a given TCR which are not included within the constant domain as encoded by the TRAC gene for TCR chains and either the TRBC1 or TRBC2 for TCR chains. (T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8)
Preferred embodiments provide a TCR of the invention comprising:
the alpha chain variable region shown in FIG. 1a (SEQ ID NO: 2) and the beta chain variable region shown in FIG. 1b (SEQ ID NO: 3), or phenotypically silent variants thereof. For example, a TCR comprising the amino acid sequences of SEQ ID NO: 15 (FIG. 5a) and SEQ ID NO: 16 (FIG. 5b). This TCR alpha chain is that of a known TCR specific for the ILAKFLHWL-HLA-A*0201 complex. The DNA and amino acid sequence of this TCR chain alpha was first published in WO 2005/116075.
Other preferred embodiments provide a TCR of the invention comprising:
the alpha chain variable region shown in FIG. 10a (SEQ ID NO: 19) and the beta chain variable region shown in FIG. 1b (SEQ ID NO: 3), or phenotypically silent variants thereof. For example, a TCR comprising the amino acid sequences of SEQ ID NO: 20 (FIG. 10b) and SEQ ID NO: 16 (FIG. 5b).
In a related embodiment such TCRs of the invention may further comprise the truncated alpha chain constant region amino acid sequence shown in FIG. 6a (SEQ ID NO: 4) and one of the truncated beta chain amino acid constant region sequences shown in FIGS. 6b and 6c (SEQ ID NOs: 5 and 6) or phenotypically silent variants thereof.
As used herein the term phenotypically silent variants is understood to refer to those TCRs which retain the property of binding to the Telomerase-derived VYGFVRACL peptide presented in the context of HLA-A24. For example, as is known to those skilled in the art, it may be possible to produce TCRs that incorporate minor changes in the constant domains and/or variable regions thereof compared to those detailed above without altering the affinity and/or off-rate for the interaction with the VYGFVRACL-HLA-A24 complex. Such trivial variants are included in the scope of this invention. Those TCRs in which one or more conservative substitutions have been made also form part of this invention.
In one broad aspect, the TCRs of the invention are in the form of either single chain TCRs (scTCRs) or dimeric TCRs (dTCRs), including but not limited to, those described in WO 04/033685 and WO 03/020763 respectively.
A suitable scTCR form comprises a first segment constituted by an amino acid sequence corresponding to a TCR chain variable region, a second segment constituted by an amino acid sequence corresponding to a TCR chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
Alternatively the first segment may be constituted by an amino acid sequence corresponding to a TCR chain variable region; the second segment may be constituted by an amino acid sequence corresponding to a TCR chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR chain constant domain extracellular sequence
The above scTCRs may further comprise a disulfide bond between the first and second chains, said disulfide bond being one which has no equivalent in native T cell receptors, and wherein the length of the linker sequence and the position of the disulfide bond being such that the variable region sequences of the first and second segments are mutually orientated substantially as in native T cell receptors.
More specifically the first segment may be constituted by an amino acid sequence corresponding to a TCR , chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR chain constant domain extracellular sequence, the second segment may be constituted by an amino acid sequence corresponding to a TCR chain variable region fused to the N terminus of an amino acid sequence corresponding to TCR chain constant domain extracellular sequence, and a disulfide bond may be provided between the first and second chains, said disulfide bond being one which has no equivalent in native T cell receptors.
In the above scTCR forms, the linker sequence may link the C terminus of the first segment to the N terminus of the second segment, and may have the formula -PGGG-(SGGGG)n-P- wherein n is 5 or 6 and P is proline, G is glycine and S is serine:
A suitable dTCR form of the TCRs of the present invention comprises a first polypeptide wherein a sequence corresponding to a TCR chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR chain constant domain extracellular sequence, and a second polypeptide wherein a sequence corresponding to a TCR chain variable region sequence fused to the N terminus a sequence corresponding to a TCR chain constant domain extracellular sequence, the first and second polypeptides being linked by a disulfide bond which has no equivalent in native T cell receptors.
The first polypeptide may comprise a TCR chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR chain constant domain extracellular sequence, and a second polypeptide wherein a sequence corresponding to a TCR chain variable region sequence is fused to the N terminus a sequence corresponding to a TCR chain constant domain extracellular sequence, the first and second polypeptides being linked by a disulfide bond between cysteine residues substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 or the non-human equivalent thereof. (TRAC etc. nomenclature herein as per T cell receptor Factsbook, (2001) LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8)
The dTCR or scTCR form of the TCRs of the invention may have amino acid sequences corresponding to human TCR extracellular constant and variable region sequences, and a disulfide bond may link amino acid residues of the said constant domain sequences, which disulfide bond has no equivalent in native TCRs. The disulfide bond is between cysteine residues corresponding to amino acid residues whose carbon atoms are less than 0.6 nm apart in native TCRs, for example between cysteine residues substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 or the non-human equivalent thereof. Other sites where cysteines can be introduced to form the disulfide bond are the following residues in exon 1 of TRAC*01 for the TCR chain and TRBC1*01 or TRBC2*01 for the TCR chain:
In addition to the non-native disulfide bond referred to above, the dTCR or scTCR form of the TCRs of the invention may include a disulfide bond between residues corresponding to those linked by a disulfide bond in native TCRs.
The TCRs of the invention preferably do not contain a sequence corresponding to transmembrane sequence.
The TCRs of the invention preferably do not contain a sequence corresponding to cytoplasmic sequences of native TCRs.
The dTCR or scTCR form of the TCRs of the invention may comprise a first polypeptide wherein a sequence corresponding to a TCR chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR chain constant domain extracellular sequence, and
a second polypeptide wherein a sequence corresponding to a TCR chain variable region sequence fused to the N terminus a sequence corresponding to a TCR chain constant domain extracellular sequence,
the first and second polypeptides being linked by a disulfide between cysteine residues substituted for Thr 48 of exon 1 of TRAC*01 and Ser 57 of exon 1 of TRBC1*01 or TRBC2*01 or the non-human equivalent thereof.
One currently preferred embodiment of the invention provides a TCR comprising the alpha chain amino acid sequence of SEQ ID NO: 15 and beta chain amino acid sequence SEQ ID NO: 16. Another currently preferred embodiment of the invention provides a TCR comprising the alpha chain amino acid sequence of SEQ ID NO: 20 and beta chain amino acid sequence SEQ ID NO: 16
Also provided is a nucleic acid or nucleic acids encoding TCRs of the invention. Such a nucleic acid or nucleic acids 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 (GeneOptimizer). WO 2004/059556, owned by GeneArt, provides further details of the optimisation process.
Other currently preferred embodiments of the invention are provided by nucleic acids consisting of a full-length TCR chain DNA sequence and a full-length TCR chain DNA sequence. A nucleic acid complementary to any of the foregoing, or a corresponding RNA sequence 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:
In one particular embodiment a TCR of the invention is associated with at least one polyalkylene glycol chain. This association may be caused in a number of ways known to those skilled in the art. In a preferred embodiment the polyalkylene chain(s) is/are covalently linked to the TCR. In a further embodiment the polyethylene glycol chains of the present aspect of the invention comprise at least two polyethylene repeating units.
One aspect of the invention provides a multivalent TCR complex comprising at least two TCRs of the invention. In one embodiment of this aspect, at least two TCR molecules are linked via linker moieties to form multivalent complexes. Preferably the complexes are water soluble, so the linker moiety should be selected accordingly. Furthermore, it is preferable that the linker moiety should be capable of attachment to defined positions on the TCR molecules, so that the structural diversity of the complexes formed is minimised. One embodiment of the present aspect is provided by a TCR complex of the invention wherein the polymer chain or peptidic linker sequence extends between amino acid residues of each TCR which are not located in a variable region sequence of the TCR.
Since the complexes of the invention may be for use in medicine, the linker moieties should be chosen with due regard to their pharmaceutical suitability, for example their immunogenicity.
Examples of linker moieties which fulfil the above desirable criteria are known in the art, for example the art of linking antibody fragments.
There are two classes of linker that are preferred for use in the production of multivalent TCR molecules of the present invention. These are non-peptidic polymer chains or peptidic linker sequences. A TCR complex of the invention in which the TCRs are linked by a polyalkylene glycol chain provides one embodiment of the present aspect.
The first are hydrophilic polymers such as polyalkylene glycols. The most commonly used of this class are based on polyethylene glycol or PEG, the structure of which is shown below.
Wherein n is greater than two. However, others are based on other suitable, optionally substituted, polyalkylene glycols include polypropylene glycol, and copolymers of ethylene glycol and propylene glycol.
Such polymers may be used to treat or conjugate therapeutic agents, particularly polypeptide or protein therapeutics, to achieve beneficial changes to the PK profile of the therapeutic, for example reduced renal clearance, improved plasma half-life, reduced immunogenicity, and improved solubility. Such improvements in the PK profile of the PEG-therapeutic conjugate are believe to result from the PEG molecule or molecules forming a shell around the therapeutic which sterically hinders the reaction with the immune system and reduces proteolytic degradation. (Casey et al, (2000) Tumor Targetting 4: 235-244) The size of the hydrophilic polymer used my in particular be selected on the basis of the intended therapeutic use of the TCR complex. Thus for example, where the product is intended to leave the circulation and penetrate tissue, for example for use in the treatment of a tumour, it may be advantageous to use low molecular weight polymers in the order of 5 KDa. There are numerous review papers and books that detail the use of PEG and similar molecules in pharmaceutical formulations. For example, see Harris (1992) Polyethylene Glycol ChemistryBiotechnical and Biomedical Applications, Plenum, New York, N.Y. or Harris Zalipsky (1997) Chemistry and Biological Applications of Polyethylene Glycol ACS Books, Washington, D.C.
The polymer used can have a linear or branched conformation. Branched PEG molecules, or derivatives thereof, can be induced by the addition of branching moieties including glycerol and glycerol oligomers, pentaerythritol, sorbitol and lysine.
Usually, the polymer will have a chemically reactive group or groups in its structure, for example at one or both termini, and/or on branches from the backbone, to enable the polymer to link to target sites in the TCR. This chemically reactive group or groups may be attached directly to the hydrophilic polymer, or there may be a spacer group/moiety between the hydrophilic polymer and the reactive chemistry as shown below:
The spacer used in the formation of constructs of the type outlined above may be any organic moiety that is a non-reactive, chemically stable, chain, Such spacers include, by are not limited to the following:
(CH2)n wherein n=2 to 5
A TCR complex of the invention in which a divalent alkylene spacer radical is located between the polyalkylene glycol chain and its point of attachment to a TCR of the complex provides a further embodiment of the present aspect.
A TCR complex of the invention in which the polyalkylene glycol chain comprises at least two polyethylene glycol repeating units provides a further embodiment of the present aspect.
There are a number of commercial suppliers of hydrophilic polymers linked, directly or via a spacer, to reactive chemistries that may be of use in the present invention. These suppliers include Nektar Therapeutics (CA, USA), NOF Corporation (Japan), Sunbio (South Korea) and Enzon Pharmaceuticals (NJ, USA).
Commercially available hydrophilic polymers linked, directly or via a spacer, to reactive chemistries that may be of use in the present invention include, but are not limited to, the following:
A wide variety of coupling chemistries can be used to couple polymer molecules to protein and peptide therapeutics. The choice of the most appropriate coupling chemistry is largely dependant on the desired coupling site. For example, the following coupling chemistries have been used attached to one or more of the termini of PEG molecules (Source: Nektar Molecular Engineering Catalogue 2003):
As stated above non-PEG based polymers also provide suitable linkers for multimerising the TCRs of the present invention. For example, moieties containing maleimide termini linked by aliphatic chains such as BMH and BMOE (Pierce, products Nos. 22330 and 22323) can be used.
Peptidic linkers are the other class of TCR linkers. These linkers are comprised of chains of amino acids, and function to produce simple linkers or multimerisation domains onto which TCR molecules can be attached. The biotin/streptavidin system has previously been used to produce TCR tetramers (see WO/99/60119) for in-vitro binding studies. However, strepavidin is a microbially-derived polypeptide and as such not ideally suited to use in a therapeutic.
A TCR complex of the invention in which the TCRs are linked by a peptidic linker derived from a human multimerisation domain provides a further embodiment of the present aspect.
There are a number of human proteins that contain a multimerisation domain that could be used in the production of multivalent TCR complexes. For example the tetramerisation domain of p53 which has been utilised to produce tetramers of scFv antibody fragments which exhibited increased serum persistence and significantly reduced off-rate compared to the monomeric scFv fragment. (Willuda et al. (2001) J. Biol. Chem. 276 (17): 14385-14392) Haemoglobin also has a tetramerisation domain that could potentially be used for this kind of application.
A multivalent TCR complex of the invention comprising at least two TCRs provides a final embodiment of this aspect, wherein at least one of said TCRs is associated with a therapeutic agent.
In one aspect a TCR (or multivalent complex thereof) of the present invention may alternatively or additionally comprise a reactive cysteine at the C-terminal or N-terminal of the alpha or beta chains thereof.
In one aspect the TCR of the invention may be associated with a therapeutic agent or detectable moiety. For example, said therapeutic agent or detectable moiety may be covalently linked to the TCR.
In one embodiment of the invention said therapeutic agent or detectable moiety is covalently linked to the C-terminus of one or both TCR chains.
In one aspect the scTCR or one or both of the dTCR chains of TCRs of the present invention may be labelled with an detectable moiety, for example a label that is suitable for diagnostic purposes. Such labelled TCRs are useful in a method for detecting a VYGFVRACL-HLA-A*0201 complex which method comprises contacting the TCR ligand with a TCR (or a multimeric high avidity TCR complex) which is specific for the TCR ligand; and detecting binding to the TCR ligand. In tetrameric TCR complexes formed for example, using biotinylated heterodimers, fluorescent streptavidin can be used to provide a detectable label. Such a fluorescently-labelled TCR tetramer is suitable for use in FACS analysis, for example to detect antigen presenting cells carrying the VYGFVRACL-HLA-A*0201 complex for which these TCRs are specific.
Another manner in which the soluble TCRs of the present invention may be detected is by the use of T cell specific antibodies (e.g. anti-CD3 antibodies), in particular monoclonal antibodies. There are many commercially available anti-T cell antibodies, such as F1 and F1, which recognise the constant domains of TCR and chains, respectively.
In a further aspect a TCR (or multivalent complex thereof) of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent which may be, for example, a cytotoxic moiety for use in cell killing, or an immune effector molecule such as an interleukin or a cytokine. A multivalent TCR complex of the invention may have enhanced binding capability for a TCR ligand compared to a non-multimeric wild-type or T cell receptor heterodimer of the invention. Thus, the multivalent TCR complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent TCR complexes having such uses. These TCRs or multivalent TCR complexes may therefore be provided in a pharmaceutically acceptable formulation for use in vivo.
The invention also provides a method for delivering a therapeutic agent to a target cell, which method comprises contacting potential target cells with a TCR or multivalent TCR complex in accordance with the invention under conditions to allow attachment of the TCR or multivalent TCR complex to the target cell, said TCR or multivalent TCR complex being specific for the VYGFVRACL-HLA-A24 complex and having the therapeutic agent associated therewith.
In particular, the soluble TCR or multivalent TCR complex of the present invention can be used to deliver therapeutic agents to the location of cells presenting a particular antigen. This would be useful in many situations and, in particular, against tumours. A therapeutic agent could be delivered such that it would exercise its effect locally but not only on the cell it binds to. Thus, one particular strategy envisages anti-tumour molecules linked to TCRs or multivalent TCR complexes according to the invention specific for tumour antigens.
Many therapeutic agents could be employed for this use, for instance radioactive compounds, enzymes (perforin for example) or chemotherapeutic agents (cis-platin for example). To ensure that toxic effects are exercised in the desired location the toxin could be inside a liposome linked to streptavidin so that the compound is released slowly. This will prevent damaging effects during the transport in the body and ensure that the toxin has maximum effect after binding of the TCR to the relevant antigen presenting cells.
Other suitable therapeutic agents include:
Antibody fragments and variants/analogues which are suitable for use in the compositions and methods described herein include, but are not limited to, the following.
As is known to those skilled in the art, it is possible to produce fragments of a given antibody which retain substantially the same binding characteristics as those of the parent antibody. The following provides details of such fragments:
MinibodiesThese constructs consist of antibodies with a truncated Fc portion. As such they retain the complete binding domains of the antibody from which are derived.
Fab fragmentsThese comprise a single immunoglobulin light chain covalently-linked to part of an immunoglobulin heavy chain. As such, Fab fragments comprise a single antigen combining site. Fab fragments are defined by the portion of an IgG that can be liberated by treatment with papain. Such fragments are commonly produced via recombinant DNA techniques. (Reeves et al., (2000) Lecture Notes on Immunology (4th Edition) Published by Blackwell Science)
F(ab)2 fragmentsThese comprise both antigen combining sites and the hinge region from a single antibody. F(ab)2 fragments are defined by the portion of an IgG that can be liberated by treatment with pepsin. Such fragments are commonly produced via recombinant DNA techniques. (Reeves et al., (2000) Lecture Notes on Immunology (4th Edition) Published by Blackwell Science)
Fv fragmentsThese comprise an immunoglobulin variable heavy domain linked to an immunoglobulin variable light domain. A number of Fv designs have been produced. These include dsFvs, in which the association between the two domains is enhanced by an introduced disulfide bond. Alternatively, scFvs can be formed using a peptide linker to bind the two domains together as a single polypeptide. Fvs constructs containing a variable region of a heavy or light immunoglobulin chain associated to the variable and constant domain of the corresponding immunoglobulin heavy or light chain have also been produced. FV have also been multimerised to form diabodies and triabodies (Maynard et al., (2000) Annu Rev Biomed Eng 2 339-376)
NanobodiesThese constructs, marketed by Ablynx (Belgium), comprise synthetic single immunoglobulin variable heavy domain derived from a camelid (e.g. camel or llama) antibody.
Domain AntibodiesThese constructs, marketed by Domantis (Belgium), comprise an affinity matured single immunoglobulin variable heavy domain or immunoglobulin variable light domain.
The defining functional characteristic of antibodies in the context of the present invention is their ability to bind specifically to a target ligand. As is known to those skilled in the art it is possible to engineer such binding characteristics into a range of other proteins. Examples of antibody variants and analogues suitable for use in the compositions and methods of the present invention include, but are not limited to, the following.
Protein scaffold-based binding polypeptidesThis family of binding constructs comprise mutated analogues of proteins which contain native binding loops. Examples include Affibodies, marketed by Affibody (Sweden), which are based on a three-helix motif derived from one of the IgG binding domains of Staphylococcus aureus Protein A. Another example is provided by Evibodies, marketed by EvoGenix (Australia) which are based on the extracellular domains of CTLA-4 into which domains similar to antibody binding loops are grafted. A final example, Cytokine Traps marketed by Regeneron Pharmaceuticals (US), graft cytokine receptor domains into antibody scaffolds. (Nygren et al., (2000) Current Opinion in Structural biology 7:463-469) provides a review of the uses of scaffolds for engineering novel binding sites in proteins. This review mentions the following proteins as sources of scaffolds: CP1 zinc finger, Tendamistat, Z domain (a protein A analogue), PST1, Coiled coils, LACI-D1 and cytochrome b562. Other protein scaffold studies have reported the use of Fibronectin, Green fluorescent protein (GFP) and ankyrin repeats.
As is known to those skilled in the art antibodies or fragments, variants or analogues thereof can be produced which bind to various parts of a given protein ligand. For example, anti-CD3 antibodies can be raised to any of the polypeptide chains from which this complex is formed (i.e. , , , , and CD3 chains) Antibodies which bind to the CD3 chain are the preferred anti-CD3 antibodies for use in the compositions and methods of the present invention.
Soluble TCRs or multivalent TCR complexes of the invention may be linked to an enzyme capable of converting a prodrug to a drug. This allows the prodrug to be converted to the drug only at the site where it is required (i.e. targeted by the sTCR).
It is expected that the VYGFVRACL (SEQ ID NO: 1)-HLA-A24 specific TCRs disclosed herein may be used in methods for the diagnosis and treatment of cancer.
For cancer treatment, the localisation in the vicinity of tumours or metastasis would enhance the effect of toxins or immunostimulants. For vaccine delivery, the vaccine antigen could be localised in the vicinity of antigen presenting cells, thus enhancing the efficacy of the antigen. The method can also be applied for imaging purposes.
One embodiment is provided by a cell presenting a TCR of the invention. Another related embodiment is provided by a cell transfected to present a TCR of the invention. The cell presenting, or transfected to present, the TCRs of the invention may preferable be a human T cell or a human haematopoietic stem cell. Cells presenting the TCRs of the invention are useful in treatment of cancer by adoptive therapy methods. These methods provide a means of directing cells, such as T cells, to a population of target cells in a patient, said method comprising administering to a cancer patient a plurality of cells presenting TCRs of the invention that are specific for the VYGFVRACL-HLA-A24 ligand on the population of target cells.
Further embodiments of the invention are provided by a pharmaceutical composition comprising:
a TCR or a multivalent TCR complex of the invention (optionally associated with a therapeutic agent), or a plurality of cells presenting at least one TCR of the invention, or a nucleic acid or nucleic acids encoding a TCR 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 TCR or a multivalent TCR complex of the invention (optionally associated with a therapeutic agent), or a plurality of cells presenting at least one TCR of the invention, or a nucleic acid or nucleic acids encoding a TCR of the invention. In a related embodiment the invention provides for the use of a TCR or a multivalent TCR complex of the invention (optionally associated with a therapeutic agent), or a plurality of cells presenting at least one TCR of the invention, or a nucleic acid or nucleic acids encoding a TCR of the invention in the preparation of a composition for the treatment of cancer.
Therapeutic or imaging TCRs 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 substances 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.
A scTCR or dTCR (which preferably is constituted by constant and variable sequences corresponding to human sequences) of the present invention may be provided in substantially pure form, or as a purified or isolated preparation. For example, it may be provided in a form which is substantially free of other proteins.
High affinity TCRs having the property of specifically binding to VYGFVRACL-HLA-A*2402, and (i) comprising at least one TCR chain variable region and/or at least one TCR chain variable region and (ii) having a KD for the said VYGFVRACL-HLA-A*2402 complex of less than or equal to 1 M, may be identified by a method comprising:
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 invention is further described in the following examples, which do not limit the scope of the invention in any way.
FIGS. 4a and 4b provide the DNA sequences alpha and beta chains of a soluble disulfide-linked form of the parental VYG-A24 TCR which is specific for the VYGFVRACL-HLA-A*2402 complex. The beta chain sequence was identified from a phage library by the method referred to in Example 6 below. The alpha chain is that of a known TCR specific for the ILAKFLHWL-HLA-A*0201 complex. The DNA and amino acid sequence of this alpha chain alpha was first published in WO 2005/116075. These DNA sequences can be synthesised de-novo by a number of contract research companies, for example GeneArt (Regensburg, Germany). Restriction enzyme recognition sites are also added to these DNA sequences in order to facilitate ligation of these DNA sequences into pGMT7-based expression plasmids, which contain the T7 promoter for high level expression in E. coli strain BL21-DE3(pLysS) (Pan et al., Biotechniques (2000) 29 (6): 1234-8)
The DNA sequences encoding each TCR chain cut with NdeI and HindIII are ligated into separate pGMT7 vectors, which are also cut with NdeI and HindIII. (See FIG. 7a for the plasmid map of pGMT7, and FIG. 7b for the DNA sequence of this vector (SEQ ID NO: 17)
Restriction Enzyme Recognition Sites as Introduced into DNA Encoding the Soluble Parental VYG-A24 TCR Chains:
Ligated plasmids are transformed into competent E. coli strain XL1-blue cells and plated out on LB/agar plates containing 100 mg/ml ampicillin. Following incubation overnight at 37 C., single colonies are picked and grown in 10 ml LB containing 100 mg/ml ampicillin overnight at 37 C. with shaking. Cloned plasmids are purified using a Miniprep kit (Qiagen) and the insert is sequenced using an automated DNA sequencer (Lark Technologies).
FIGS. 5a and 5b show respectively the soluble disulfide-linked parental VYG-A24 TCR and chain extracellular amino acid sequences produced from the DNA sequences of FIGS. 4a and 4b.
The soluble disulfide-linked parental VYG-A24 TCR produced as described in Example 1 can be used a template from which to produce the TCRs of the invention which have an increased affinity for the VYGFVRACL (SEQ ID NO: 43)-HLA-A*0201 complex.
As is known to those skilled in the art the necessary codon changes required to produce these mutated chains can be introduced into the DNA encoding these chains by site-directed mutagenesis. (QuickChange Site-Directed Mutagenesis Kit from Stratagene)
Briefly, this can be achieved by using primers that incorporate the desired codon change(s) and the pGMT7 plasmids containing the relevant parental VYG-A24 TCR chain DNA as a template for the mutagenesis:
Mutagenesis can be carried out using the following conditions: 50 ng plasmid template, 1 l of 10 mM dNTP, 5 l of 10Pfu DNA polymerase buffer as supplied by the manufacturer, 25 pmol of fwd primer, 25 pmol of rev primer, 1 l pfu DNA polymerase in total volume 50 l. After an initial denaturation step of 2 mins at 95 C, the reaction can be subjected to 25 cycles of denaturation (95 C, 10 secs), annealing (55 C 10 secs), and elongation (72 C, 8 mins). The resulting product can be digested with DpnI restriction enzyme to remove the template plasmid and transformed into E. coli strain XL1-blue. Mutagenesis can be verified by sequencing.
The pGMT7 expression plasmids containing the parental VYG-A24 TCR -chains and parental VYG-A24 TCR -chains as prepared in Example 1 were transformed separately into E. coli strain BL21pLysS, and single ampicillin-resistant colonies were grown at 37 C. in TYP (ampicillin 100 g/ml) medium to OD600 of 0.4 before inducing protein expression with 0.5 mM IPTG. Cells were harvested three hours post-induction by centrifugation for 30 minutes at 400 rpm in a Beckman J-6B. Cell pellets were re-suspended in a buffer containing 50 mM Tris-HCl, 25% (w/v) sucrose, 1 mM NaEDTA, 0.1% (w/v) NaAzide, 10 mM DTT, pH 8.0. After an overnight freeze-thaw step, re-suspended cells were sonicated in 1 minute bursts for a total of around 10 minutes in a Milsonix XL2020 sonicator using a standard 12 mm diameter probe. Inclusion body pellets wee recovered by centrifugation for 30 minutes at 13000 rpm in a Beckman J2-21 centrifuge. Three detergent washes wee then carried out to remove cell debris and membrane components. Each time the inclusion body pellet was homogenised in a Triton buffer (50 mM Tris-HCl, 0.5% Triton-X100, 200 mM NaCl, 10 mM NaEDTA, 0.1% (w/v) NaAzide, 2 mM DTT, pH 8.0) before being pelleted by centrifugation for 15 minutes at 13000 rpm in a Beckman J2-21. Detergent and salt was then removed by a similar wash in the following buffer: 50 mM Tris-HCl, 1 mM NaEDTA, 0.1% (w/v) NaAzide, 2 mM DTT, pH 8.0. Finally, the inclusion bodies are divided into 30 mg aliquots and frozen at 70 C. Inclusion body protein yield was quantitated by solubilising with 6M guanidine-HCl and measurement with a Bradford dye-binding assay (PerBio).
Approximately 30 mg of TCR chain and 60 mg of TCR chain solubilised inclusion bodies were thawed from frozen stocks, samples were then mixed and the mixture diluted into 15 ml of a guanidine solution (6 M Guanidine-hydrochloride, 10 mM Sodium Acetate, 10 mM EDTA), to ensure complete chain de-naturation. The guanidine solution containing fully reduced and denatured TCR chains was then injected into 1 litre of the following refolding buffer: 100 mM Tris pH 8.5, 400 mM L-Arginine, 2 mM EDTA, 5 mM reduced Glutathione, 0.5 mM oxidised Glutathione, 5M urea, 0.2 mM PMSF. The redox couple (2-mercaptoethylamine and cystamine (to final concentrations of 6.6 mM and 3.7 mM, respectively) were added approximately 5 minutes before addition of the denatured TCR chains. The solution was left for 5 hrs 15 minutes. The refolded TCR was dialysed in Spectrapor 1 membrane (Spectrum; Product No. 132670) against 10 L 10 mM Tris pH 8.1 at 5 C.3 C. for 18-20 hours. After this time, the dialysis buffer was changed to fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5 C.3 C. for another 20-22 hours.
sTCR was separated from degradation products and impurities by loading the dialysed refold onto a POROS 50HQ anion exchange column and eluting bound protein with a gradient of 0-500 mM NaCl over 50 column volumes using an Akta purifier (Pharmacia). Peak fractions were stored at 4 C. and analysed by Coomassie-stained SDS-PAGE before being pooled and concentrated. Finally, the sTCR was purified and characterised using a Superdex 200HR gel filtration column pre-equilibrated in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40). The peak eluting at a relative molecular weight of approximately 50 kDa was pooled and concentrated prior to characterisation by BIAcore surface plasmon resonance analysis.
A surface plasmon resonance biosensor (Biacore 3000) was used to analyse the binding of parental VYG-A24 TCR to VYGFVRACL-HLA-A*2402. This was facilitated by producing single VYGFVRACL-HLA-A*2402 complexes (described below) which were immobilised to a streptavidin-coated binding surface in a semi-oriented fashion, allowing efficient testing of the binding of a soluble T-cell receptor to up to four different pMHC (immobilised on separate flow cells) simultaneously. Manual injection of HLA complex allows the precise level of immobilised class I molecules to be manipulated easily.
Biotinylated class I HLA-A*2402 molecules were refolded in vitro from bacterially-expressed inclusion bodies containing the constituent subunit proteins and synthetic peptide, followed by purification and in vitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15). HLA-A*2402-heavy chain was expressed with a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains of the protein in an appropriate construct. Inclusion body expression levels of 75 mg/litre bacterial culture were obtained. The MHC light-chain or 2-microglobulin was also expressed as inclusion bodies in E. coli from an appropriate construct, at a level of 500 mg/litre bacterial culture.
E. coli cells were lysed and inclusion bodies are purified to approximately 80% purity. Protein from inclusion bodies was denatured in 6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA, and was refolded at a concentration of 30 mg/litre heavy chain, 30 mg/litre 2 m into 0.4 M L-Arginine-HCl, 100 mM Tris pH 8.1, 3.7 mM cystamine, 6.6 mM -cysteamine, 4 mg/ml of the VYGFVRACL peptide required to be loaded by the HLA-A*0201 molecule, by addition of a single pulse of denatured protein into refold buffer at 5 C. Refolding was allowed to reach completion at 4 C. for at least 1 hour.
Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently. The protein solution was then filtered through a 1.5 m cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCl gradient. HLA-A*0201-peptide complex eluted at approximately 250 mM NaCl, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
Biotinylation tagged pMHC molecules were buffer exchanged into 10 mM Tris pH 8.1, 5 mM NaCl using a Pharmacia fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgCl2, and 5 g/ml BirA enzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15). The mixture was then allowed to incubate at room temperature overnight.
The biotinylated pHLA-A*2402 molecules were purified using gel filtration chromatography. A Pharmacia Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min. Biotinylated pHLA-A*0201 molecules eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated VYGFVRACL-HLA-A*2402 molecules were stored frozen at 20 C. Streptavidin was immobilised by standard amine coupling methods.
Such immobilised complexes are capable of binding both T-cell receptors and the coreceptor CD8, both of which may be injected in the soluble phase. Specific binding of TCR is obtained even at low concentrations (at least 40 g/ml), implying the TCR is relatively stable. The pMHC binding properties of sTCR are observed to be qualitatively and quantitatively similar if sTCR is used either in the soluble or immobilised phase. This is an important control for partial activity of soluble species and also suggests that biotinylated pMHC complexes are biologically as active as non-biotinylated complexes.
The interactions between the soluble parental VYG-A24 TCRs containing a novel inter-chain bond and its cognate pMHC or an irrelevant pMHC combination, the production of which is described above, were analysed on a Biacore 3000 surface plasmon resonance (SPR) biosensor. SPR measures changes in refractive index expressed in response units (RU) near a sensor surface within a small flow cell, a principle that can be used to detect receptor ligand interactions and to analyse their affinity and kinetic parameters. The probe flow cells were prepared by immobilising the individual HLA-peptide complexes in separate flow cells via binding between the biotin cross linked onto 2 m and streptavidin which have been chemically cross linked to the activated surface of the flow cells. The assay was then performed by passing sTCR over the surfaces of the different flow cells at a constant flow rate, measuring the SPR response in doing so.
Serial dilutions of the parental VYG-A24 TCR were prepared and injected at constant flow rate of 5 l min-1 over two different flow cells; one coated with 1000 RU of specific VYGFVRACL-HLA-A*2402 complex, the second coated with 1000 RU of non-specific HLA-A24-peptide complex. Response was normalised for each concentration using the measurement from the control cell. Normalised data response was plotted versus concentration of TCR sample and fitted to a hyperbola in order to calculate the equilibrium binding constant, KD. (Price Dwek, Principles and Problems in Physical Chemistry for Biochemists (2nd Edition) 1979, Clarendon Press, Oxford).
For high affinity TCRs KD can be determined by experimentally measuring the dissociation rate constant, kd, and the association rate constant, ka. The equilibrium constant KD was calculated as kd/ka. TCR can be injected over two different cells one coated with 300 RU of specific HLA-A*2402-VYGFVRACL complex, the second coated with 300 RU of a non-specific peptide-HLA complex. Flow rate is set at 50 l/min. Typically 250 l of TCR at 3 M concentration are injected. Buffer is then flowed over until the response returns to baseline. Kinetic parameters are calculated using Biaevaluation software. The dissociation phase is also fitted to a single exponential decay equation enabling calculation of half-life.
The interaction between a soluble disulfide-linked parental VYG-A24 TCR (consisting of the soluble disulfide-linked and TCR chains detailed in SEQ ID Nos: 15 and 16 respectively) and the VYGFVRACL-HLA-A*2402 complex was analysed using the above methods and demonstrated a KD of 4 M. (See FIG. 9 for Biacore response curve)
The methods substantially as described in Examples 1 to 3 can be used to produce a soluble VYG-A24 TCR-WT human IL-2 fusion protein. Briefly, the DNA encoding the desired linker and WT human IL-2 are added into the 3 end of the DNA sequence of the soluble disulfide-linked VYG-A24 TCR beta chain. FIG. 8 provides the amino acid sequence of a fusion protein comprising a disulfide-linked parental VYG-A24 TCR beta chain fused to WT human IL-2 via linker sequence. (SEQ ID NO: 18) The linker and IL-2 portion of this fusion protein are indicated in italics and the introduced cysteine residue in the TCR beta is indicated by shading. The DNA encoding this construct can then be ligated into pGMT7. The soluble parental VYG-A24 TCR-IL-2 fusion protein can then be expressed by combining this beta chain fusion protein with the soluble disulfide-linked parental VYG-A24 TCR alpha chain detailed in FIG. 5a (SEQ ID NO: 15) using the methods substantially as described in Example 3.
A phage displayed TCR library was created using the procedures described in WO 2004/044004. Briefly, the library was based on a soluble disulfide-linked A6 TCR specific for HLA-A2-LLFGYPVYV. The diversity of the A6 TCR library was created by using mutagenic primers which introduced mutations in the CDR3 regions of the displayed A6 TCRs. In order to display the A6 TCR library phagemid vectors were constructed for expression of fusion proteins comprising the heterodimeric A6 TCR containing a non-native disulfide inter-chain bond with the gill phage coat protein. E. coli XL-1-Blue cells containing a phagemid encoding the soluble A6 TCR chain and an A6 TCR chain fused to the phage gIII protein were used to express the phage displayed TCRs. The presence of functional VYGFVRACL-HLA-A*2402 binding TCRs displayed on the phage particles were detected using a phage ELISA method, The DNA encoding the phage-displayed TCRs which were selected by the ELISA was then used to construct soluble disulfide-linked TCRs. The binding of these soluble TCRs to HLA-A24-VYGFVRACL was then assessed using the Biacore method of Example 4 herein. The soluble disulfide-linked parental VYG-A24 TCR was constructed using a TCR chain isolated from this library (SEQ ID NO; 16) associated with a soluble disulfide-linked analogue of a wild-type ILAKFLHWL-HLA-A*0201 binding TCR Chain (SEQ ID NO: 15). The TCR chain shown in FIG. 10b (SEQ ID NO: 20) was also isolated from this library, displayed as an TCR specific for the VYGFVRACL-HLA-A*2402 complex, in combination with the TCR chain shown in FIG. 5b (SEQ ID NO 16).