WO2013057586A1

WO2013057586A1 - Compositions and methods for producing soluble t - cell receptors

Info

Publication number: WO2013057586A1
Application number: PCT/IB2012/002628
Authority: WO
Inventors: Even WALSENG; Sebastien Walchli; Lars-egil FALLANG; Johanna Olweus
Original assignee: Oslo Universitetssykehus Hf
Priority date: 2011-10-19
Filing date: 2012-10-19
Publication date: 2013-04-25

Abstract

The present invention relates to T-cell receptors. In particular, in some embodiments, the present invention provides a novel constructs and methods to produce a soluble form of the T-cell receptor.

Description

COMPOSITIONS AND METHODS FOR PRODUCING SOLUBLE T-CELL

RECEPTORS

FIELD OF THE INVENTION

The present invention relates to T-cell receptors. In particular, in some embodiments, the present invention provides a novel method to produce a soluble form of the T-cell receptor.

BACKGROUND OF THE INVENTION

T cells recognize peptide-loaded MHC molecules via their T-cell Receptor (TcR) which, upon binding to target, triggers dramatic cellular changes. TcRs are heterodimeric transmembrane proteins belonging to the immunoglobulin (Ig) super family consisting of two polypeptides; αβ-chains (conventional) or γδ (gamma delta) [1-3]. Each chain consists of a variable (V) and a constant (C) domain followed by a transmembrane domain and a short cytoplasmic tail. C and V domains have Ig-like structure and participate in the dimerization of the TcR. The cysteine bridge connecting the αβ -chains is located in the short stretch between the C domain and the transmembrane domain (Fig. 2A) and stabilizes the dimer.

TcRs and antibodies are both antigen-specific molecules. While TcRs are

transmembrane proteins and do not exist in soluble form, antibodies can be both secreted and membrane bound. Their antigen specificity depends on the complementary determining regions (CDRs), which are found on the V-domain and are three for the TcR. Only the CDR3 undergoes somatic recombination and is therefore believed to be the main contributor for antigen specificity. The CDR1 and 2 vary between V chains, but do not undergo somatic mutations and have been shown to contribute to the binding of Major Histocompatibility (MHC) molecule. Indeed, antibodies recognize both secreted or cell surface molecules, whereas TcRs are restricted to recognition of peptides generated from all degraded proteins but presented in the context of MHC molecules. Finally, most of the TcRs depend on co- receptor to properly MHC-peptide (pMHC) complexes, namely CD4 or CD8, but some are independent, it has thus been speculated that this independency to co-receptor was correlated to a higher affinity of the TcR itself. Thus TcRs have the important advantage of being able to detect any protein found in the cell regardless of its cellular localization. This independence to protein localization has prompted researchers to develop TcR-based tools to recognize pMHC. Soon after the characterization of TcR structure [4], attempts to create soluble TcRs (sTcRs) were undertaken [5]. sTcRs were mainly produced for crystallography as reviewed in [6, 7], but were also used to test binding kinetics [8] and for substrate identification [9]. These results were shown to be helpful in the design of full length TcR for cell therapy [10, 11]. The production of sTcR to be directly used as therapeutic molecules has been proven difficult [12- 15] though modified types of sTcRs, such as single-chain TcRs have been used [7, 14, 16-19].

Currently the main source of sTcR production is bacteria, where the two TcR chains are expressed separately, isolated in inclusion bodies and refolded together [8]. Although the production has proven efficient for crystal structure determination [20], the success rate for a correct refolding was estimated as low as one third [21]. The main problem residing in the instability of the dimer, as the membrane proximal cysteines forming the interchain disulfide bridge were excluded. Thus the correct dimer formation of bacterially produced TcR mainly relies on the stability of the Vab- and Cab- domains affinity.

Another approach was to produce a soluble single chain molecule such as the STAR™ technology, which is an hybrid soluble TcR-Ig molecules connected with a flexible linker [16]. Functional sTcRs have been produced this way, though the design of the product is an issue as it requires empirical testing and screening to reach a full functional and specific single chain TcR.

T cells can efficiently recognize and kill cells that are virally infected, thereby curing e.g. influenza. Thus, T cells can recognize intracellular proteins, vastly increasing the number of potential therapeutic targets relative to antibodies. However, the insolubility of T-cell receptors limits their use as therapeutic agents. Methods and compositions for producing soluble T-cell receptors are needed.

SUMMARY OF THE INVENTION

For example, embodiments of the present invention provide a soluble T-cell receptor or a nucleic acid encoding a soluble T-cell receptor, comprising: a T-cell receptor, wherein alpha and beta chains of the T-cell receptor lack transmembrane and cytosolic domains, and wherein the alpha and beta chains are linked with a ribosomal skipping sequence (e.g., 2A or an IRES). In some embodiments, one or more of said alpha or beta chains are linking to a protein selected from, for example, a tag (e.g., a detection or purification tag such as, for example, His-tag, FLAG tag, BirA, GFP, mCherry and the like), a toxin (e.g., diphtheria toxin or auristatin E) or a radioactive substance (e.g. an alpha- or beta-emitter) or an antibody fragment (e.g., directed at CD3 or CD28).

Further embodiments provide for the use of the soluble T-cell receptor in diagnostic, research and therapeutic applications (e.g., research, screening, diagnosis and treatment of cancer, autoimmune disease or viral infection).

In some embodiments, the present invention provides a nucleic acid construct encoding a soluble T-Cell receptor comprising a soluble T-cell receptor a-chain nucleic acid sequence and a soluble T-cell receptor β-chain nucleic acid sequence operably linked by a ribosome skipping sequence sequence, the T-cell receptor a-chain and β-chain each lacking T- cell receptor transmembrane and cytosolic domains. In some embodiments, the ribosome skipping sequence is selected from the group consisting of a 2A peptide nucleic acid sequence or an IRES sequence. In some embodiments, the 2A peptide nucleic acid sequence is a Picomavirus 2A peptide nucleic acid sequence. In some embodiments, the 2A peptide nucleic acid sequence comprises a consensus sequence encoding a polypeptide corresponding to SEQ ID NO: 69.

In some embodiments, the soluble T-cell receptor a-chain nucleic acid sequence and the soluble T-cell receptor β-chain nucleic acid sequence each comprise a T-cell receptor variable domain. In some embodiments, the soluble T-cell receptor a-chain nucleic acid sequence and the soluble T-cell receptor β-chain nucleic acid sequence each comprise at least a portion of the T-cell receptor a-chain nucleic acid sequence and a T-cell receptor β-chain nucleic acid sequence constant domains, and wherein the at least a portion of the constant domains do not contain an amino acid substitution. In some embodiments, the soluble T-cell receptor a-chain nucleic acid sequence and the soluble T-cell receptor β-chain nucleic acid sequence each comprise the entire T-cell receptor a-chain nucleic acid sequence and a T-cell receptor β-chain nucleic acid sequence constant and variable domains. In some embodiments, the soluble T-cell receptor a-chain nucleic acid sequence and the soluble T-cell receptor β- chain nucleic acid sequence each comprise a dimerization domain. In some embodiments, the dimerization domain is a leucine zipper domain operably linked to the c-terminal end of each of the soluble T-cell receptor a-chain nucleic acid sequence and the soluble T-cell receptor β- chain nucleic acid sequence. In some embodiments, the sTcR construct is encoded by a sequence that is at least 70%, 80%, 90%, 95%, 99% or 100% identical to SEQ ID NO:73 or 75. In some embodiments, one or both of the soluble T-cell receptor a-chain nucleic acid sequence and the soluble T-cell receptor β-chain nucleic acid sequence are operably linked to a nucleic acid sequence encoding an accessory polypeptide. In some embodiments, the accessory polypeptide is selected from the group consisting of an enzyme, an antimicrobial polypeptide, a cytokine and a fluorescent polypeptide. In some embodiments, the soluble T- cell receptor encoded by the construct is internalizable by cells displaying a ligand bound by the soluble T-cell receptor.

In some embodiments, the present invention provides a nucleic acid vector comprising the construct as described above. In some embodiments, the vector is selected from the group consisting of a plasmid vector and a viral vector.

In some embodiments, the present invention provides a host cell comprising a nucleic acid vector as described above. In some embodiments, the host cell is a mammalian host cell.

In some embodiments, the present invention provides methods for producing a soluble T-cell receptor comprising: introducing a nucleic acid construct according to any of Claims 1 to 11 or a vector according to any of Claims 12 to 13 into a host cell under conditions such that the soluble T-cell receptor a-chain and the soluble T-cell receptor β-chain are expressed; culturing the host cell to provide a host cell culture; and purifying the soluble T-cell receptor from the host cell culture.

In some embodiments, the present invention provides a soluble T-cell receptor produced by the methods described above, wherein the soluble T-cell receptor comprises the T-cell receptor a-chain and the T-cell receptor β-chain linked by a disulfide bond. In some embodiments, the disulfide bond is a native disulfide bond.

In some embodiments, the present invention provides a soluble T-cell receptor molecule comprising a soluble T-cell receptor a-chain polypeptide sequence and a soluble T- cell receptor β-chain polypeptide sequence linked by a disulfide bond, one of the soluble T- cell receptor a-chain polypeptide sequence and the soluble T-cell receptor β-chain polypeptide sequence comprising a processed C-terminal 2A peptide sequence. In some embodiments, the 2A peptide nucleic acid sequence is a Picornavirus 2A peptide nucleic acid sequence. In some embodiments, the 2A peptide nucleic acid sequence comprises a consensus sequence encoding a polypeptide corresponding to SEQ ID NO: 69. In some embodiments, the soluble T-cell receptor a-chain polypeptide sequence and the soluble T-cell receptor β-chain polypeptide sequence each comprise a T-cell receptor variable domain. In some

embodiments, the soluble T-cell receptor a-chain polypeptide sequence and the soluble T-cell receptor β-chain polypeptide sequence each comprise at least a portion of the T-cell receptor a-chain nucleic acid sequence and a T-cell receptor β-chain nucleic acid sequence constant domains, and wherein at least a portion of the constant domains do not contain an amino acid substitution. In some embodiments, the soluble T-cell receptor a-chain sequence and the soluble T-cell receptor β-chain sequence each comprise the entire T-cell receptor a-chain sequence and a T-cell receptor β-chain sequence constant and variable domains. In some embodiments, the T-cell receptor a-chain polypeptide sequence and the T-cell receptor β- chain polypeptide sequence each comprise a dimerization domain. In some embodiments, the dimerization domain is a leucine zipper domain operably linked to the c-terminal end of each of the T-cell receptor a-chain polypeptide sequence and the T-cell receptor β-chain polypeptide sequence. In some embodiments, the disulfide bond is a native disulfide bond. In some embodiments, the sTcR is encoded by a sequence that is at least 70%, 80%, 90%, 95%, 99% or 100% identical to SEQ ID NO:73 or 75 prior to processing into the heterodimeric protein.

In some embodiments, one or both of the T-cell receptor a-chain polypeptide sequence and the T-cell receptor β-chain polypeptide sequence are operably linked to an accessory polypeptide. In some embodiments, the accessory polypeptide is selected from the group consisting of an enzyme, an antimicrobial polypeptide, a cytokine and a fluorescent polypeptide. In some embodiments, wherein the disulfide bond is a native disulfide bond. In some embodiments, the soluble T-cell receptor is operably linked to an accessory molecule. In some embodiments, the accessory molecule is detectable label. In some embodiments, the accessory molecule is therapeutic molecule. In some embodiments, the soluble T-cell receptor is intemalizable by cells displaying a ligand bound by the soluble T-cell receptor. In some embodiments, wherein the soluble T-cell receptor binds to an intracellular ligand in the context of MHC molecules.

In some embodiments, the present invention provides a multimeric T cell receptor complex comprising a plurality of soluble T-cell receptors as described above. In some embodiments, the complex is multivalent. In some embodiments, the complexes comprise two or three or four or more soluble T-cell receptors associated with one another.

In some embodiments, the soluble T-cell receptor or multimeric complex described above is conjugated to or associated with an accessory molecule or conjugated to, associated with, or loaded into a particle, e.g., a nanoparticle or microparticle. In some embodiments, the present invention provides a pharmaceutical formulation comprising a soluble T-cell receptor or multimeric complex or conjugate thereof as described above in association with a pharmaceutically acceptable carrier.

In some embodiments, the present invention provide for the use of a soluble T-cell receptor or multimeric complex or conjugate thereof as described above for a diagnostic application or therapeutic administration to a subject.

In some embodiments, the present invention provides for the use of a soluble T-cell receptor or multimeric complex or conjugate thereof as described above in the treatment of cancer, an autoimmune disease or a viral infection.

In some embodiments, the present invention provides methods comprising

administering a therapeutically effective amount of a soluble T-cell receptor or multimeric complex or conjugate thereof as described above to a subject.

In some embodiments, the present invention provides methods for detecting an analyte of interest comprising contacting a test material with a soluble T-cell receptor or multimeric complex or conjugate thereof as described above and detecting the presence of the analyte via binding of the soluble T-cell receptor or multimeric complex to the analyte of interest.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

Fig. 1. Schematic illustration of the various structures of soluble T cell receptor generated.

Fig. 2. Schematic illustration of biotinylated and ligand-coupled tetrameric sTcR. Fig. 3A, B, C, D, E, F. Figure 3A is a schematic of a full length TcR (top) and a truncated sTcR of the present invention (bottom). Figure 3B is a schematic of an exemplary nucleic acid construct of the present invention encoding a TcR alpha and beta chains separated by a 2A peptide sequence. The alpha chain includes a FLAG sequence and the beta chain includes a fluorescent protein in combination with either a BirA sequence of HisTag sequence. Figure 3C is a graph depicting the results of a flow cytometry experiment. Figure 3D is a graph depicting the results of a flow cytometry experiment. Figure 3E is an image of the results of Western blot analysis of the sTcR in reducing and non-reducing conditions. lOul of supernatant was boiled and reduced (Red) or not (Non). The membrane was subsequently blotted using an anti-Flag ab (left) or anti-His ab (right). A band around 30kD was observed in reducing conditions whereas in addition to this band another higher molecular weight band (approx 60 kD) was seen in the non-reducing conditions. Figure 3F provides graphs depicting the results of a flow cytometry experiment.

Figure 4A, B, C, and D. Figure 4A is a graph of the the IC₅₀ of DMF5 SA-PE sTcR binding to its target. Figure 4B is a graph demonstrating that a saturating amount of sTcR (100 ng/mL) to show that SA-PE sTcR could detect 1 μΜ of MARTI peptide loaded on T2 cells. Figure 4C provides the results of a FACS analysis on peptide-loaded T2 cells with either the sTcR-cherry-beads or SA-PE Cherry-sTcR, a predicted increase in sensitivity was observed with the beads. Figure 4D shows staining of melanoma cancer cell lines with sTcR conjugated to SA-alexa-647, demonstrating the ability of the sTcR to recognize HLA-A2 positve melanoma cells.Figure 5 A, B and C. Figure 5A provides images of staining of HeLa cells with supernatant of producing cells containing sTcR (TcRa untagged and TcRb-His, Fig. 3B). The soluble heterodimer MARTI sTcR could easily be detected at the surface of the cells, but also in complex with SCT in intracellular vesicles. Figure 5B provides images of the HeLa cells stained with supernantants containing the mCherry-sTcR for 30 minutes. Figure 5C is an image demonstrating endicytosis of beads conjugated with sTcR by the target cells expressing SCT-Martl but not by the cells expressing SCT with an irrelevant peptide (not shown).

Figure 6 A, B, C, D, E, F. Figure 6 A is a graph showing ³H-thymidine incorporation in MART1-SCT expressing cells incubated for 3 days with TcR conjugated with SA-Saporin at a final concentration of 20 nM. Figure 6B is a graph demonstrating that 0.1 nM (50 ng/mL) of the SA-Saporin conjugate was sufficient to kill half of the cells. Figure 6C is a graph showing ³H-thymidine incorporation in the HLA-A2 positive melanoma cell line, Malme, following exposure to the same Saporin conjugated TcRs. Figure 6D is a graph showing ³H- thymidine incorporation in Hek cells (HLA-A2 positive and MARTI negative) incubated with MARTI sTcR conjugated with SA-saporin and Hek cells pulsed with with 10 μΜ MARTI . Figure 6E is a graph depicting binding of A94 sTcR its target in a CD8 co-receptor- independent manner. Figure 6F is a graph demonstrating that the A94 sTcR selectively kills EBV cells only if they are isolated from a HLA-A2 positive donor.

Figure 7 provides four graphs that provide flow cytometry data from a comparison of sTcRs with and without leucine zipper domains. The DMF5 and s591 (CD20p specific) TCRs were genetically modified by adding a complimentary Leucine zipper dimerization motif (Moll et al, 2001), linked by a 15aa GS sequence, to the C-terminal end of each of the chains. The modified and wt TCRs were then tandemly produced, and the supernatants were compared in terms of the ability to stain cells expressing either Ml-SCT (red line) or CD20p- SCT (blue line). The staining was done using anti-FLAG antibody, and Donkey-Anti Mouse PE secondary antibody, and the samples were analyzed by flow cytometry.

Figure 8 provides the amino acid and nucleic acid sequences (SEQ ID NO:73 and 74) for soluble DMF5-FLAG-BirA.

Figure 9 provides the amino acid and nucleic acid sequences (SEQ ID NO:75 and 76) for soluble A94-FLAG-BirA.

DEFINITIONS

As used herein, the term "T-cell receptor" (abbr. "TcR") refers to membrane-bound heterodimeric proteins that comprise a T-cell receptor a-chain and T-cell receptor β-chain, each chain comprising a variable region and a constant region, transmembrane domain, and cytosolic domain. The V and C regions are generally homologous to immunoglobulin V and C regions and comprise three complementarity-determing regions (CDRs). Both TcR chains are anchored in the plasma membrane of cell presenting the TcR.

As used herein, the term "soluble T-cell receptor" (abbr. "sTcR") refers to

heterodimeric truncated variants of native TcRs which comprise extracellular portions of the TcR a-chain and β-chain linked by a disulfide bond, but lack the transmembrane and cytosolic domains of the native protein. The terms "soluble T-cell receptor a-chain sequence and soluble T-cell receptor β-chain sequence" refer to TcR a-chain and β-chain sequences that lack the transmembrane and cytosolic domains. The sequence (amino acid or nucleic) of the sTcR a-chain and β-chains may be identical to the corresponding sequences in a native TcR or may comprise variant sTcR a-chain and β-chain sequences as compared to the corresponding native TcR sequences. The term "soluble T-cell receptor" as used herein encompasses sTcRs with variant or non-variant sTcR a-chain and β-chain sequences. The variations may be in the variable or constant regions of the sTcR a-chain and β-chain sequences and can include, but are not limited to, amino acid deletion, insertion, substitution mutations as well as changes to the nucleic acid sequence which do not alter the amino acid sequence.

As used herein, the term "ribosome skipping sequence" refers to any sequence that can be introduced between two or more gene sequences under the control of the same promotor so that the gene sequences are translated as separate polypeptides (i.e., translated as biscistronic or multicistronic sequences). Examples of suitable ribosome skipping sequences include both 2 A peptide sequences and IRES sequences. 2 A peptide sequences are relatively short peptides (of the order of 20 amino acids long, depending on the virus of origin) containing the consensus motif Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro (SEQ ID NO:69). Examples of suitable 2A peptide sequences, include, but are not limited to, the following sequences:

Picornaviruses;

EMC-B -119aa- GIFNAHYAGYFADLLIHDIETNPGP (SEQ ID NO: 1)

EMC-D GIFNAHYAGYFADLLIHDIETNPGP (SEQ ID NO: 2)

EMC-PV21 RIFNAHYAGYFADLLIHDIETNPGP (SEQ ID NO: 3)

MENGO HVFETHYAGYFSDLLIHDVETNPGP (SEQ ID NO: 4)

TME-GD7 -109aa- KAVRGYHADYYKQRLIHDVEMNPGP (SEQ ID NO: 5)

TME-DA RAVRAYHADYYKQRLIHDVEMNPGP (SEQ ID NO: 6)

TME-BEAN KAVRGYHADYYRQRLIHDVETNPGP (SEQ ID NO: 7)

Theiler's-Like Virus KHVREYHAAYYKQRLMHDVETNPGP (SEQ ID NO: 8)

Ljungan virus (174F) -MHSDEMDFAGGKFLNQCGDVETNPGP (SEQ ID NO: 9)

Ljungan virus (145SL) -MHNDEMDYSGGKFLNQCGDVESNPGP (SEQ ID NO: 10)

Ljungan virus (87-012) -MHSDEMDFAGGKFLNQCGDVETNPGP(SEQ ID NO: 11)

Ljungan virus (Ml 146) -YHDKDMDYAGGKFLNQCGDVETNPGP (SEQ ID NO: 12)

FMD-A10 LLNFDLLKLAGDVESNPGP(SEQ ID NO: 13)

FMD-A12 LLNFDLLKLAGDVESNPGP(SEQ ID NO: 14)

FMD-C1 LLNFDLLKLAGDVESNPGP (SEQ ID NO: 15)

FMD-OIG LLNFDLLKLAGDMESNPGP(SEQ ID NO: 16)

FMD-OIK LTNFDLLKLAGDVESNPGP(SEQ ID NO: 17)

FMD-0 (Taiwan) LLNFDLLKLAGDVESNPGP (SEQ ID NO: 18)

FMD-O/SK LLSFDLLKLAGDVESNPGP(SEQ ID NO: 19)

FMD-SAT3 MCNFDLLKLAGDVESNPGP(SEQ ID NO: 21)

FMD-SAT2 LLNFDLLKLAGDVESNPGP(SEQ ID NO: 21)

ERAV CTNYSLLKLAGDVESNPGP(SEQ ID NO: 22)

ERBV GATNFSLLKLAGDVELNPGP(SEQ ID NO: 23)

ERV-3 GATNFDLLKLAGDVESNPGP(SEQ ID NO: 24)

PTV-1 GPGATNFSLLKQAGDVEENPGP(SEQ ID NO: 25)

PTV-2 GPGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26)

PTV-3 GPGASSFSLLKQAGDVEENPGP(SEQ ID NO: 27)

PTV-4 GPGASNFSLLKQAGDVEENPGP (SEQ ID NO: 28)

PTV-5 GPGAANFSLLRQAGDVEENPGP(SEQ ID NO: 29)

PTV-6 GPGATNFSLLKQAGDVEENPGP(SEQ ID NO: 30)

PTV-7 GPGATNFSLLKQAGDVEENPGP(SEQ ID NO: 31)

PTV-8 GPGATNFSLLKQAGDIEENPGP(SEQ ID NO: 32) PTV-9 GPGATNFSLLKQAGDVEENPGP(SEQ ID NO: 33)

PTV-10 GPGATNFSLLKQAGDVEENPGP(SEQ ID NO: 34)

PTV-11 GPGATNFSLLKRAGDVEENPGP (SEQ ID NO: 35)

Insect Viruses;

CrPV -FLRKRTQLLMSGDVESNPGP(SEQ ID NO: 36)

DCV -EAARQMLLLLSGDVETNPGP(SEQ ID NO: 37)

ABPV -GSWTDILLLLSGDVETNPGP(SEQ ID NO: 38)

ABPV isolate Poland 1 -GSWTDILLLLSGDVETNPGP(SEQ ID NO: 39)

ABPV isolate Hungary 1 -GSWTDILLLWSGDVETNPGP(SEQ ID NO: 40)

IFV -TRAEIEDELIRAGIESNPGP(SEQ ID NO: 41)

TaV -RAEGRGSLLTCGDVEENPGP (SEQ ID NO: 42)

EEV -QGAGRGSLVTCGDVEENPGP (SEQ ID NO: 43)

APV -NYPMPEALQKIIDLESNPPP (SEQ ID NO: 44)

KBV -GTWESVLNLLAGDIELNPGP (SEQ ID NO: 45)

PnPV (a) -AQGWVPDLTVDGDVESNPGP(SEQ ID NO: 46)

PnPV (b) -IGGGQKDLTQDGDIESNPGP(SEQ ID NO: 47)

Ectropis obliqua picorna-like virus

(a) -AQGWAPDLTQDGDVESNPGP(SEQ ID NO: 48)

(b) -IGGGQRDLTQDGDIESNPGP(SEQ ID NO: 49)

Providence virus

(a) -VGDRGSLLTCGDVESNPGP (SEQ ID NO: 50)

(b) -SGGRGSLLTAGDVEKNPGP(SEQ ID NO: 51)

(c) -GDPIEDLTDDGDIEKNPGP (SEQ ID NO: 52)

Type C Rotaviruses;

Bovine Rotavirus -SKFQIDRILISGDIELNPGP (SEQ ID NO: 53)

Porcine Rotavirus -AKFQIDKILISGDVELNPGP(SEQ ID NO: 54) Human Rotavirus -SKFQIDKILISGDIELNPGP(SEQ ID NO: 55)

Reovirus ( cypovirus 1 );

Bombyx mori -FRSNYDLLKLCGDIESNPGP(SEQ ID NO: 56)

Lymantria dispar -FRSNYDLLKLCGDVESNPGP (SEQ ID NO: 57) Dendrolimus punctatus -FRSNYDLLKLCGDVESNPGP (SEQ ID NO: 58)

Tr pansoma spp. Repeated Sequences;

T. brucei TSR1 -SSIIRTKMLVSGDVEENPGP(SEQ ID NO: 59)

(CAB95325.1) -SSIIRTKMLLSGDVEENPGP(SEQ ID NO: 60)

(CAB95342.1) -SSIIRTKMLLSGDVEENPGP(SEQ ID NO: 61)

(CAB95559.1) -SSIIRTKILLSGDVEENPGP(SEQ ID NO: 62) T. cruzi AP Endonuclease -CDAQRQKLLLSGDIEQNPGP(SEQ ID NO: 63) Prokaryotic Sequences;

T. maritima aguA -YIPDFGGFLVKADSEFNPGPX (SEQ ID NO: 64)

B. bronchiseptica -VHCAGRGGPVRLLDKEGNPGP (SEQ ID NO: 65)

Eukaryotic ( cellular) Sequences;

Mouse mor-lF -DLELETVGSHQADAETNPGPX (SEQ ID NO: 66)

D. melanogaster mod(mdg4) -TAADKIQGSWKMDTEGNPGPX (SEQ ID NO: 67)

A. nidulans Ca channel MIDI -PITNRPRNSGLIDTEINPGP(SEQ ID NO: 68) Suitable IRES sequences are known in the art and include, but are not limited to, IRES sequences from the following viruses: Poliovirus, Rhinovirus, Encephalomyocarditis virus, Foot-and-mouth disease virus, Hepatitis A virus, Hepatitis C virus, Classical swine fever virus, Bovine viral diarrhea virus and the like.

As used herein, the terms "protein," "polypeptide," and "peptide" refer to a molecule comprising amino acids joined via peptide bonds. In general "peptide" is used to refer to a sequence of 20 or less amino acids and "polypeptide" is used to refer to a sequence of greater than 20 amino acids.

As used herein, the term "wild-type" (or "native") when used in reference to a protein refers to proteins encoded by the genome of a cell, tissue, or organism, other than one manipulated to produce synthetic proteins.

As used herein, the term "vector," when used in relation to recombinant DNA technology, refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, retrovirus, virion, etc. , which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

As used herein, the term "host cell" refers to any eukaryotic cell {e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, insect cells, yeast cells), and bacteria cells, and the like, whether located in vitro or in vivo {e.g., in a transgenic organism).

As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines {e.g., with an immortal phenotype), primary cell cultures, finite cell lines {e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.

The term "isolated" when used in relation to a nucleic acid, as in "an isolated oligonucleotide" refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acids are nucleic acids present in a form or setting that is different from that in which they are found in nature. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA that are found in the state in which they exist in nature.

The terms "in operable combination," "in operable order," and "operably linked" as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.

A "subject" is an animal such as vertebrate, preferably a mammal such as a human, a bird, or a fish. Mammals are understood to include, but are not limited to, murines, simians, humans, bovines, cervids, equines, porcines, canines, felines etc.).

An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations.

As used herein, the term "purified" or "to purify" refers to the removal of undesired components from a sample. As used herein, the term "substantially purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An "isolated polynucleotide" is therefore a substantially purified polynucleotide.

As used herein, the terms "nanoparticle" and "nanoscale particles" are used interchangeably and refer to a nanoscale particle with a size that is measured in nanometers, for example, a nanoscopic particle that has at least one dimension of less than about 1000 nm. Examples of nanoparticles include nanobeads, nanofibers, nanohorns, nano-onions, nanorods, and nanoropes.

As used herein, the term "microparticle" and "microscale particles" are used interchangeably and refers to a microscale particle with a size that is measured in

micrometers, for example, a microscopic particle that has at least one dimension of less than about 10 micrometers, 5 micrometers, or 2 micrometers. Examples of microparticles include microbeads, microfibers, microhorns, micro-onions, microrods, and microropes.

DESCRIPTION OF THE INVENTION

Although antibodies are extremely powerful molecules to detect foreign antigens or transformed cells, they can only see secreted or extracellular proteins, which restrict their field of action. T-cell receptors (TcRs) are another type of antigen detecting proteins, which are heterodimeric transmembrane proteins that recognize peptides presented in the context of an MHC molecule. Unlike antibodies, TcRs are always cell associated with an extracellular portion recognizing the antigen complex and an intracellular part involved in the signaling that will finally trigger the activation of the T cell. TcRs have the advantage of recognizing all cellular proteins, including the intracellular ones. Artificial constructs that encode for soluble TcRs containing the antigen recognition part were designed and shown to keep their specificity. The present invention encompasses a new method of cloning and expressing sTcRs. In some embodiments, a construct is provided where the ribosome skipping sequence 2A is introduced between the TcR-a and-β chains lacking the transmembrane and intracellular domains. This construct is expressable in human cells and the supernatant of these cells can directly be used to identify ligands displayed on target cells. In further embodiments, the sTcR may be conjugated to accessory molecules such as fluorochromes, toxins and nanoparticles, for example, through use of an intermediary hapten such as biotin. The sTcRs are useful both for diagnostic and therapeutic purposes. For example, a sTcR against the tumor associated antigen MARTI has been demonstrated to stain and kill target cells in a MHC- antigen dependent manner. The methods described herein can readily be utilized to make additional sTcRs targeting a wide range of cancer specific targets potentially undetectable by conventional antibodies.

Embodiments of the present invention utilize the unsurpassed specificity of the immune system to create novel molecules for therapeutic and diagnostic applications. The immune receptors of T cells (T-cell receptors - TcRs) comprise a library of specificities that in principle can recognize any protein fragment with a high degree of selectivity. Soluble TcRs can be used therapeutically as "generic" drugs in infectious disease and cancer when conjugated to toxic agents. For example, in some embodiments, soluble TcRs specific for a particular antigen are administered to all individuals with the same MHC antigen (tissue type antigen) and cancer type.

A T-cell Receptor (TcR) is a heterodimeric membrane protein that recognizes a peptide in the context of an MHC molecule. Embodiments of the present invention describe how to produce a soluble version of the T-cell receptor. Soluble TcRs (sTcR), unlike antibodies, are artificial constructs. They are able to recognize the same peptide-MHC complex as the original TcR in the T cell that they were cloned from. Soluble T-cell Receptors and Expression Systems

The present invention provides soluble T-cell receptors. The soluble T-cell receptors preferably comprise TcRa and β chains from a T-cell receptor molecule, preferably at least the variable domains of the TcRa and β chains, more preferably at the variable domains and a portion of the constant domains of the TcRa and β chains, and most preferably the full variable and constant domains of the TcRa and β chains. The present invention is not limited to any particular TcRa and β chains. A wide variety of TcRa and β chain sequences from known TcRs are known in the art and readily accessible to the skilled artisan for use in the present invention. Moreover, additional TcR sequences can be readily identified and cloned for use in the present invention by methods known in the art. In some preferred embodiments, the TcRa and β chains are truncated so that they lack the transmembrane domain and cytosolic domain of wild-type TcRa and β chain sequences. In some preferred embodiments, the truncated TcRa and β chain sequences do not contain any additional mutations such as amino acid substitutions. In some embodiments, the TcRa and β chain sequences comprise the wild-type amino acid residues responsible for disulfide bond formation in the extracellular portion of the assembled TcR molecule. In some embodiments, the TcRa and β chain sequences are operably linked to one or more accessory polypeptides. Examples of such sTcRs are schematically depicted in Figure 1. Examples of nucleic acid sequences of sTcR constructs (and the corresponding amino acid sequences) of the present invention are provided in Figures 8 and 9.

In some embodiments, the present invention provides a nucleic acid construct for expression of a sTcR. In some preferred embodiments, the construct comprises a

picornovirus 2A sequence positioned between the TcRa and β chain sequences. Suitable 2A sequences are obtainable from Picornaviridae. The 2A sequences are relatively short peptides (of the order of 20 amino acids long, depending on the virus of origin) containing the consensus motif Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro (SEQ ID NO:69). The 2A sequences act co-translationally, by preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping to the next codon, and the nascent peptide cleaving between the Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the 'upstream' protein, while the proline is added to the N-terminus of the 'downstream' protein. Due to its mode of action, the 2A peptide is described as a 'cis-acting hydrolase element' (CHYSEL). In preferred embodiments, use of this construct allows efficient, equimolar expression of a secreted TcR (sTcR) in one and the same mammalian host cell (e.g., a human cell line). Prior to the present invention, this was not done for a sTcR and is a very effective method producing high yields of stable protein, as it bypasses the problems associated with non-mammalian sTcR production systems, e.g. poor solubility and stability and inefficient proper folding of the sTcR.

In some embodiments, the nucleic acid constructs further comprise a dimerization domain. In some embodiments, the dimerization is a leucine zipper. Suitable leucine zipper motifs are described, for example in Moll JR, Ruvinov SB, Pastan I, Vinson C. Designed heterodimerizing leucine zippers with a ranger of pis and stabilities up to 10(-15) M. Protein Sci. 2001 Mar;10(3):649-55). In some preferred embodiments, the leucine zipper motifs are linked to the C-terminal end of the TcRa and β chain sequences.

While use of substantially unmodified TcRa and β chain sequences is preferred, the

TcRa and β chain sequences may be modified as is known in the art. Examples of modification of TcRa and β chain sequences which are within the scope of the present invention include mutation of the variable and/or constant regions of the TcRa and β chain sequences. In some embodiments, the modifications serve to stabilize the sTcR and can include codon optimization of the constant regions as well as addition of cysteine residues to the constant regions. In other embodiments, residues which are normally glycosylated may be modified to alter glycosylation of the sTcR as desired. In still further embodiments, the residues responsible for disulfide bond formation in the wild-type TcRa and β chain sequences are substituted and additional cysteine residues are introduced at desired positions. In still further embodiments, the variable region of the TcRa and β chain sequences may be modified to alter affinity, e.g., to increase or decrease affinity as desired. In some

embodiments, one or more of the CDRs of the variable region is modified or replaced to provide a desired binding affinity. In some embodiments, the variable regions of the TcRa and β chain sequences are the result of an in vitro affinity maturation process. In general, random mutations inside the CDRs are introduced using radiation, chemical mutagens or error-prone PCR. In addition, the genetic diversity can be increased by chain shuffling.

Iterative rounds of mutation and selection using display methods like phage display and panning can be used to produce affinities in the low nanomolar range. In some embodiments, the nucleic acid constructs are provided in a suitable vector. Suitable vectors include, but are not limited to, vectors useful for mammalian cell expression such as plasmid vectors, retroviral vectors, adenoviral vectors, transposon vectors and the like. In some embodiments of the present invention, the constructs comprise a vector, such as a plasmid or viral vector, into which the construct of the invention has been inserted, in a forward or reverse orientation. In still other embodiments, the construct is assembled in appropriate phase with translation initiation and termination sequences. In preferred embodiments of the present invention, the appropriate DNA sequence is inserted into the vector using any of a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuc lease site(s) by procedures known in the art.

Large numbers of suitable vectors are known to those of skill in the art, and are commercially available. Such vectors include, but are not limited to, pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). Any other plasmid or vector may be used as long as they are replicable and viable in the host. In some preferred embodiments of the present invention, mammalian expression vectors comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences. In other embodiments, DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Promoters useful in the present invention include, but are not limited to, the LTR or SV40 promoter, the E. coli lac or tip, the phage lambda P_L and P_R, T3 and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, and mouse metallothionein-I promoters and other promoters known to control expression of gene in prokaryotic or eukaryotic cells or their viruses. In other embodiments of the present invention, recombinant expression vectors include origins of replication and selectable markers permitting transformation of the host cell (e.g.,

dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli).

In some embodiments of the present invention, transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. In still other embodiments of the present invention, the vector may also include appropriate sequences for amplifying expression.

In further embodiments, the present invention provides host cells containing the above-described constructs. In some embodiments of the present invention, the host cell is a higher eukaryotic cell (e.g., a mammalian or insect cell). In other embodiments of the present invention, the host cell is a lower eukaryotic cell (e.g., a yeast cell). Specific examples of host cells include, but are not limited to, HEK 293 (Human Embryonic Kidney) cells, Chinese hamster ovary (CHO) cells, COS-7 lines of monkey kidney fibroblasts [23], , C127, 3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. In some embodiments, introduction of the construct into the host cell can be accomplished by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (See e.g., Davis et al. (1986) Basic Methods in Molecular Biology).

In some embodiments of the present invention, following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. In other embodiments of the present invention, cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. In still other embodiments of the present invention, microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

The present invention also provides methods for recovering and purifying sTcRs from recombinant cell cultures including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. In other embodiments of the present invention, protein-refolding steps can be used as necessary, in completing

configuration of the mature protein. In still other embodiments of the present invention, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The present invention further provides nucleic acid constructs encoding sTcRs which are fused or operably linked to one or more accessory polypeptides and the sTcRs encoded thereby. Suitable accessory polypeptides include, but are not limited to purification tags, cellular toxins, enzymes, fluorescent polypeptides and the like. Suitable purification tags include, but are not limited to the hexahistidine tag, HA (influenza hemagglutinin protein) tag, FLAG tag and BirA tag (for subsequent biotinylation). Suitable enzymatic accessory polypeptides include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase and β-lactamase.

Suitable cellular toxins include, but are not limited to, antimicrobial polypeptides, ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNAase and RNAase. Suitable flourescent polypeptides include, but are not limited to, green floursescent protein (GFP) and GFP-like proteins, red fluorescent protein (RFP), mCherry, and the like. In some

emboidments, the accessory protein may be a cytokine or antibody fragment, for example, an antibody fragment specific for a cell surface receptor such as CD3 or CD28. Figure 1 provides schematic depictions of sTcRs with a His-tag, FLAG-tag and His-tag, GFP fusion protein and a His-tag, and FLAG-tag and BirA site for subsequent biotinylation.

In some embodiments, the present invention provides a multimer of two or three or four or more sTcR molecules associated (e.g. covalently or otherwise linked) with one another, preferably via a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin and extravidin, each of which has four binding sites for biotin. Thus, biotinylated sTcR molecules can be formed into multimers of T cell receptors having a plurality of sTcR binding sites. The number of sTcR molecules in the multimer will depend upon the quantity ofsTcR in relation to the quantity of linker molecule used to make the multimers, and also on the presence or absence of any other biotinylated molecules. Preferred multimers are dimeric, trimeric or tetrameric sTcRcomplexes.

Soluble single-chain TcRs have previously been produced in mammalian cells by others, containing a flexible linker between the variable a-domain and the variable β domain of the TcR connected to either an Ig domain or a constant domain of the TcR itself. One of the obstacles with this type of protein resides in the design: it is not trivial to produce such a molecule and preserve the specificity of the original TcR.

Heterodimeric complete TcRs, on the other hand, have only been produced in non- mammalian systems. Here, the low production efficiency is the issue. It has been reported that as little as 30% of the TcR produced could indeed be purified and refolded [23]. In addition, the system does not permit the glycosylation of the protein that occurs in human cells.

It is contemplated that the nucleic acid constructs and production methods of the present invention have several advantages for producing sTcRs. Utilization of the 2A picornavirus sequence between the two chains of the TcR allows production of an unmodified complete TcR containing all the domains except for the transmembrane- and cytosolic domains. By avoiding any modifications to the TcR, such as adding linkers between the variable regions and additional cysteine bridges to name a couple, the risk of rendering the TcR not functional is avoided. The data presented in the following Examples demonstrates that the use of the 2A sequence promotes correct folding and highly efficient production of sTcRs. Furthermore, mammalian production of proteins ensures a correct folding of the proteins, whereas bacterial or insect cell chaperones might indeed not be efficient/specialized enough to fold the sTcR correctly. Furthermore, human cell lines, such as Hek (and unlike the commonly used Hamster cells) will glycosylate the sTcR correctly. It is now well accepted that glycosylation is important for the stability as well as the specificity of TcR. It is expected that the same requirements are valid for sTcR. Finally, in some embodiments, the sTcR is minimally modified. This will reduce the chance of triggering unwanted side effects such as immune response to sTcR, which could well occur in products coming from bacteria or highly modified at the protein sequence level with e.g. murine sequences. Soluble T-cell Receptor Conju2ates

In some embodiments, the sTcRs are conjugated to an accessory molecule following expression and purification. Suitable accessory molecules that may be conjugated to the sTcRs of the present invention include, but are not limited to, accessory polypeptides as described above, as well as haptens, detectable labels, and therapeutic agents. The sTcRs may likelwise be conjugated to or loaded into a variety or nano- and microparticles.

Suitable haptens include, but are not limited to, pyrazoles, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas, particularly phenyl ureas, and even more particularly phenyl thioureas; rotenone and rotenone derivatives, also referred to herein as rotenoids; oxazole and thiazoles, particularly oxazole and thiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans, exemplified by Podophyllotoxin and Podophyllotoxin derivatives; and combinations thereof. Specific examples of haptens include, but are not limited to, 2,4- Dintropheyl (DNP), Biotin, Fluorescein deratives (FITC, TAMRA, Texas Red, etc.), Digoxygenin (DIG), 5-Nitro-3-pyrozolecarbamide (nitropyrazole, NP), 4,5,-Dimethoxy-2-nitrocinnamide

(nitrocinnamide, NCA), 2-(3,4-Dimethoxyphenyl)-quinoline-4-carbamide (phenylquinolone, DPQ), 2, l,3-Benzoxadiazole-5-carbamide (benzofurazan, BF), 3-Hydroxy-2-quinoxalinecarbamide

(hydroxyquinoxaline, HQ), 4-(Dimethylamino)azobenzene-4' -sulfonamide (DABSYL), Rotenone isoxazoline (Rot), (E)-2-(2-(2-oxo-2,3-dihydro-lH-benzo[b] [l,4]diazepin-4-yl)phenozy)acetamide (benzodiazepine, BD), 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid (coumarin 343, CDO), 2-Acetamido-4-methyl-5-thiazolesulfonamide (thiazolesulfonamide, TS), and p- Mehtoxypheny lpyrazopodophy llamide (Podo) .

Suitable detectable labels include, but are not limited to, but are not limited to, 4-acetamido-4'- isothiocyanatostilbene-2,2'disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS), 4-amino-N-[3- vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, anthranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7- amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5"-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;

diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)aminofluorescein (DTAF), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC); 2',7'-difluorofluorescein (OREGON

GREEN™); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); Ν,Ν,Ν',Ν'- tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives.

Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 nm [24, 25], as well as GFP, Lissamine, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Invitrogen Detection Technologies, Molecular Probes (Eugene, Oreg.) and including the ALEXA FLUOR™ series of dyes (for example, as described in U.S. Pat. Nos. 5,696, 157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes

(dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339,

5, 187,288, 5,248,782, 5,274, 113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOT™ (obtained, for example, from QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.; see also, U.S. Pat. Nos. 6,815,064, 6,682,596 and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the bandgap of the semiconductor material used in the semiconductor nanocrystal. This emission can be detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can be coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, [26], [27], and U.S. Pat. No. 6,274,323.

Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S.

Pat. Nos. 6,927,069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225, 198; 6,207,392; 6, 114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can be produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can be produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 nm, 655 nm, 705 nm, or 800 nm emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Invitrogen. Additional detectable labels include, for example, radioisotopes (such as ³H, ³⁵ S and ³²P), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd³⁺, and liposomes.

Suitable therapeutic agents include, but are not limited to, 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 sTcR to the relevant antigen presenting cells.

Other suitable therapeutic agents include: small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted under physiological conditions to release cytotoxic agents. Examples of such agents include cis-platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolmide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e. proteins or fragments thereof with the ability to kill mammalian cells. Examples include ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNAase and RNAase; radio- nuclides, i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of alpha or beta particles, or gamma rays. Examples include iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody directed enzyme pro-drugs; immuno-stimulants, i.e. moieties which stimulate immune response. Examples include cytokines such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc, antibodies or fragments thereof such as anti-CD3 antibodies or fragments thereof, complement activators, xenogeneic protein domains, allogeneic protein domains, viral/bacterial protein domains and viral/bacterial peptides.

The sTcRs of the present invention may further be conjugated or loaded into a nano- or microscale particle. Suitable nano- and microscale particles include, but are not limited to,inorganic particles (e.g., ceramic nanoparticles, gold nanoparticles, iron oxide nanoparticles, silica nanoparticles, etc.), polymeric particles (e.g., gelatin, chitosan, poly(lactic-co-glycolic acid) copolymer, polylactic acid, polyglycolic acid, poly(alkylcyanoacrylate), poly(methylmethacrylate) and

poly(butyl)cyanoacrylate), solid lipid particles, liposomes, nanocrystals, nanotubes and dendrimers. In some embodiments, the particles are coated with a suitable polymer such as poly(ethylene glycol) either before or after to conjugation or loading with the sTcR. Uses of Soluble T-cell Receptors and Conju2ates

The sTcR and conjugates of the present invention can bind to any cell containing the relevant peptide-MHC complex for which the sTcR is specific (i.e., a target cell). Such target cells include for instance cancer cells or infected cells (e.g., infected by a virus). Thus, the sTcR and conjugates of the present invention can be utilized in a variety of applications.

This binding capability of the sTcR can be utilized, for example, in diagnostic applications, therapeutic applications, and for the purification and/or isolation of target cells. In some embodiments, the variable domain of the sTCR confers the specificity of the molecule, while the toxicity is mediated via agents fused or covalently attached to the sTcR or sTcR conjugate. Such agents can for instance be directly fused to the TcR (fusion protein), biochemically coupled (streptavidin), or attached via particles encapsulating the toxic agents (nanoparticles).

In some embodiments, the sTcR can be administered either as monomers or multimers of different avidities (tetramers, pentamers or attached to particles of different sizes). The conjugation to form higher complexes can be achieved in several ways such as streptavidin- biotin, via TCR-specific antibodies, or tags attached to the TcR.

An advantage of sTcR is that they can recognize intracellular targets (in the context of MHC molecules) whereas conventional antibodies can see targets expressed at the cell surface only.

In some embodiments, sTcR targeted against MHC associated with a tumor related peptide is used to detect target cells difficult to distinguish morphologically or by

conventional methods. This can be performed in tissue sections (biopsies) or in single cell suspension (peripheral blood cells, bone marrow). Finally sTcR can also be used to detect micrometastasis in body fluids (Cerebrospinal fluid, ascites, urine etc).

In some embodiments, the sTcRs may be used in the treatment of cancer in a subject.

Patient T cells genetically modified to express cancer specific TcRs have been successfully used to treat malignant melanoma in patients [28]. This strategy involves, however, a labor intensive procedure that has to be performed for each patient. In contrast, a soluble TcR is generic for all patients expressing the same MHC-peptide complex, suitable for intravenous delivery. Indeed, the use of the sTcR can be compared with that of therapeutic monoclonal antibodies. Therapeutic monoclonal antibodies represent one of the fastest growing classes of drugs today. Two widely used and very successful examples of therapeutic monoclonal antibodies are directed towards the surface proteins CD20 (leukemia lymphoma - Rituximab) and Her-2-Neu (breast cancer - Herceptin), respectively. However, an advantage of sTcR is that they can also recognize intracellular targets, whereas conventional antibodies can see targets expressed at the cell surface only. This opens new possibilities as the majority of the cellular proteins are intracellular, indicating potential future use of a great number of non explored targets. Thus, an intracellular protein that is mutated or overexpressed in a given type of cancer becomes a sTcR target. Suitable target cancers include hematological cancers and solid tumors.

In further embodiments, sTcRs are targeted against autoreactive cells of the immune system in order to block their activity.

In some embodiments, the sTcRs are used to treat subjects with infections, preferably viral infections. Viral infections cannot be cured by the use of antibiotics. A number of viral infections not cured by the immune system become chronic and may cause severe disease.

Examples include, for example, infections caused by hepatitis B and C, Herpes virus, HIV and papilloma virus. Since the virus is intracellular, soluble TcRs (designed as described above) can specifically recognize and bind to virally infected cells and eliminate these as opposed to monoclonal antibodies. Thus, sTcR specific for a virus are used to aid the immune system in detecting and destroying infected cells.

In some embodiments, soluble TcR are used to purify and isolate target cells for further diagnostic or research purposes. For instance, in some embodiments, cells are isolated form bone marrow, peripheral blood or body fluids. In some embodiments, isolation is performed by coupling soluble TcR to fluorescent molecules, either directly in a monomelic form, or in a multimerized form (e.g., Streptavidin-fluorochrome) for separation by FACS

(flow cytometric sorting) or by magnetic beads. EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

The following examples describe a novel method of producing sTcRs. The examples demonstrate that an alternative to avoid default in chain pairing can be accomplished by expressing sTcRs in a mammalian system without modifications of the V- and C-chains other than removing the transmembrane and the intracellular domains. It is contemplated that such a sTcR is correctly folded and paired as the production system (mammalian cells) is the same as where TcRs are naturally produced. A TcR lacking the transmembrane and the cytosolic domains was designed. In order to simplify the production method that was previously performed using a two plasmid system, the two TcR coding sequences were places on one plasmid. The ribosomal skipping sequence 2A found in the picornavirus was utilized to separate the TcR ensuring equimolar amount production of the alpha and the beta chains.

The high affinity TcR DMF5 was selected as a model in this study. This TcR is specific for HLA-A2 in complex with MART-1 peptide26-35 (MARTlp, ELAGIGILTV (SEQ ID NO:70)), it is CD8 co-receptor independent and it has previously been shown to be producible as a sTcR from bacteria [29]. A functional DMF5 sTcR was produced in a mammalian system that could simply be harvested in the supernatant of the producing cells. The results further demonstrate that the sTcR was a functional heterodimer that could specifically bind target cells. C-terminal tagging of the TcR-a and/or -β chains were tested to add extra features in order to improve binding capacities or detection of the sTcR.

Surprisingly, when tagged with a fluorescent protein, the sTcR could not only reveal target cells but was also internalized. Whether this internalization could be further exploited was tested by adding the Saporin toxic subunit to the sTcR. This was sufficient to specifically kill MARTlp/HLA-A2 positive cells. Finally, an additional example of successfully produced sTcRs specific for another relevant target, namely, CD20, is provided, demonstrating that this simple, yet very potent method can generate antigen specific molecules able to identify cells and to deliver cargo capable of killing cells.

Materials and Methods

Cloning

The sTcR constructs were cloned as follows using a 2-steps procedure similar to the one described previously [30].

HLA-A2 -peptide SCT constructs were based on the murine MHC equivalent as explained in REF [31]. Briefly, the synthesized gene (MWG) consisted of the antigenic peptide, β2ιη and heavy chain of HLA-A2 linked by means of 15 and 20aa G4S linkers, respectively. In addition, cysteines were introduced in position 84 of the heavy chain and in the peptide- 2m linker to both ensure the opening of the peptide binding groove, and to create a disulfide trap for increased binding of the peptide in the groove. To simplify the peptide exchange, Xhol and Afel restriction enzyme sites flanked the peptide sequence. The gene was cloned into the Gateway compatible vector pENTR-D (Invitrogen) for easy cloning into the Gateway compatible pMP71 retroviral expression vector (Walchli et al. 2011). Retrovirus was produced as previously explained [30], and HLA-A2 negative Sup-Tl cells were transduced and subsequently FACS sorted based on HLA-A2 expression. HLA-A2 construct was previously described [32] and was used as a template for site direct mutagenesis to create a DK mutant using the following primers:

Soluble protein expression, purification and Western blot

For production of the sTcR, we transfected Hek-P using XtremeGene 9 as

recommended by the manufacturer, and collected the supernatants 72 hours post-transfection.

For FLAG purification of the sTCR, we immunoprecipitated the supernatant by passing it over an M2-sepharose column (Sigma), and eluted with FLAG peptide as recommended by the manufacturer (Sigma). The eluate was concentrated by means of a Vivaspin ultrafiltration spin column with 30.000MWCO (Sartorius Stedim Biotech).

Biotinylation and multimerization

Biotinylation of the BirA sequence containing sTcR proteins were done as

recommended by the manufacturer (Avidity). Multimerization was completed by incubating

235nM sTcR with 45nM of SA-PE (Invitrogen) for 40min/RT.

Results

Generation of a 2 A peptide linked soluble TcR.

The possibility of producing a sTcR by removing the transmembrane and cytosolic parts of the two chains (Fig. 3A) and linking them together by a picorna virus 2A sequence (Fig. 3B) was tested. As an initial test, the MART-1 ₍₂₇_35) (ELAGIGILTV (SEQ ID NO:70)) specific TcR DMF5 was selected because it has a high affinity for its pMHC target and shows a partial CD8-independency [33]. To simplify detection and purification, the first DMF5- sTcR variant was His tagged on the proximal end of its TcRb chain while keeping the alpha chain unmodified (Fig. 3B). It was produced in Hek293 cells by harvesting the supernatant 72 hours post-transfection.

The sTcR was first tested using HLA-A2 positive T2 cells loaded with MARTI peptide or an irrelevant peptide. Loaded cells were incubated with the sTcR containing supernatants and binding of sTcR was detected by using an anti-His antibody and analysis by Flow cytometry. As expected, only the cells loaded with MARTI peptide were detected, suggesting that the specificity of the original full length DMF5 TcR was maintained (Fig. 3C). Cells constitutively expressing a MHC-I single-chain trimer, SCT [34] encoding the MARTI peptide or an irrelevant peptide as a negative control were stained. Here again, the MARTI peptide was specifically detected by DMF5 sTcR as revealed by anti-His staining (Fig. 3D). Taking together, these data indicated that the sTcR was produced and released into the medium as a functional molecule.

In order to verify that the produced molecule indeed was a heterodimer, a double tagged construct of the DMF5 sTcR was designed using a FLAG tag on the C-terminus of the TcRa and keeping the His tag on the TcRb (Fig. 3B). This construct was expressed and supernatant was separated on a SDS-PAGE in either reducing or non reducing conditions. Proteins were detected by Western blot using either an anti-His antibody to detect the b-chain (Fig. 3E, left) or an anti-FLAG antibody to detect the a-chain (Fig. 3E, right). As shown, for both antibodies one band migrated around 30 kD in reducing conditions (expected size of a TcR chain) whereas an additional band of double molecular weight was detected in non- reducing conditions, suggesting that the sTcR was mainly expressed as a heterodimeric molecule. Furthermore this dimer was reduction sensitive, thus probably linked by disulfide bond. Since both antibodies gave a similar signal, the higher band was probably a heterodimer. Furthermore, this sTcR could be specifically detected on cells using anti-FLAG antibody and anti-His, supporting the presence of an active heterodimer (Fig. 3F). HPLC analysis finally confirmed the presence of a majority (i.e., >90 ) of sTcR in dimeric form on the supernatant. Taken together, these results confirm that a mammalian cell system can produce, without external modification or purification, a specific and fully functional dimeric sTcR transcribed from a single coding sequence.

Multimerization of the sTcR significantly increases the sensitivity.

The possibility of increasing the sensitivity of the staining by multimerizing the sTcR was tested next. To do this, a Bir-A sequence (GGLNDIFEAQKIEWH (SEQ ID NO:71)) for subsequent biotinylation was introduced at the end of the TcR β-chain and kept a FLAG-tag on the a-chain (Fig. 3B). This sTcR was purified by its FLAG tag, biotinylated, and finally tetramerized to streptavidin containing molecules or carrier. A tetramer composed of Phycoerythrin (PE) conjugated with streptavidin (SA, SA-PE sTcR) was produced first. To test the functionality of the SA-PE sTcR, FACS analysis was performed on both SCT expressing cells and peptide loaded T2 cells. Since SCT mimics a saturated condition where all the MHC molecules are loaded with the same peptide, we could determine that the IC₅₀ of DMF5 SA-PE sTcR binding to its target was around 10 ng/mL (Fig. 4A). Next, we used saturating amount of sTcR (100 ng/mL) to show that SA-PE sTcR could detect 1 μΜ of MARTI peptide loaded on T2 cells (Fig. 4B). The sensitivity of the detection was further increased when the biotinylated sTcR was coupled to SA conjugated MACS MicroBeads.

In order to be able to detect the beads without additional use of antibodies, a version of the sTcR was utilized that was cloned with a Cherry-tag at the C-terminal part of the TcR - chain downstream of the BirA-tag (Fig. 3B). Importantly, the addition of the fluorescent protein did not affect the production or the binding of the monomer (data not shown). When performing FACS analysis on peptide-loaded T2 cells with either the sTcR-cherry-beads or SA-PE Cherry-sTcR, a predicted increase in sensitivity was observed with the beads (Fig. 4C). Whereas the SA-PE Cherry gave a very weak signal at the second dilution (1 μΜ peptide) the beads showed strong signal even at 100 nM, but disappeared below this concentration. Finally, we explored the ability of the sTcR in complex SA-alexa-647 to stain melanoma cell lines. To do this we incubated the sTcR-SA-alexa-647 with HLA-A2 positive melanoma cell lines for 30 minutes at 37° and examined the result using FACS. As seen in figure 4D (top panel) the sTcR-SA-alexa-647 stain two of the cell lines very well, demonstrating that the sTcR couples to SA-alexa-647 indeed can recognize cancer cell lines without any addition of neither HLA-A2 or peptide. The bottom panel shows the expression of HLA-A2 in the same cells, showing that they all are HLA-A2 positive.

Taken together, these data show that mammalian cell produced sTcR can

accommodate a variety of modifications and can be used as a tool to stain MARTlp/HLA-A2 expressing cells.

Specific sTcR internalization

MHC peptide complex is constitutively internalized and recycled. Tests were preformed to determine of this mechanism could be utilized to transport sTcR inside the cell. A saturated system was first used where HLA-A2 negative cells were modified to overexpress SCT. As shown in Figure 5A, HeLa cells, which are HLA-A2 negative, were transfected to express a MARTI or an irrelevant peptide on SCT constructs fused to Cherry fluorescent protein (red). These cells were incubated with supernatant of producing cells containing sTcR (TcRa untagged and TcRb-His, Fig. 3B) and were further stained with anti-His antibodies (green). The soluble heterodimer MARTI sTcR could easily be detected at the surface of the cells, but also in complex with SCT in intracellular vesicles (Fig. 5A). Importantly, this internalization was peptide specific, suggesting that the binding of sTcR to its cognate MHC had adequate affinity to be transported into the cell. Next the extent to which the sTcR was capable of carrying cargo was examined. AMART1 sTcR construct was designed where mCherry was fused to the C-terminal end of the TcRb between the constant part and the His tag and the TcRa remained untagged

(mCherry-sTcR, Fig. 3B). SupTl cells, which are HLA-A2 negative, were transduced to constitutively express MARTI SCT (untagged) and sorted to obtain a pure population (HLA- A2 positive population, data not shown). These cells were incubated with supernantants containing the mCherry-sTcR for 30 minutes (Fig. 5B, red) at 37°C. To distinguish between the surface bound and the internalized sTcR, the cells were incubated on ice and stained with an anti-His antibody (Fig. 5B, white), thus the detection of His tag only occurred at the cell surface since no permeabilization step was performed. The cells were subsequently fixed and examined by confocal microscopy. The nucleus was visualized using DAPI-Hoechst (blue). As expected, a clear co-localization of the His-tag and the mCherry on the surface was detected (merge, pink), whereas punctuate intracellular structures were solely mCherry- positive, indicating that the mCherry-TcR had been internalized. A supernatant of mCherry- sTcR was prepared where the TcRa contained a FLAG tag and a BirA sequence and conjugated it to MACS MicroBeads. As seen in Fig. 5C, the beads conjugated with sTcR were clearly endocytosed by the target cells expressing SCT-Martl but not by the cells expressing SCT with an irrelevant peptide (not shown). Taken together these results show that not only can the sTcR be internalized by itself, but also in combination with a fused medium-sized protein or bound nano-sized bead. This demonstrates that this system can be efficiently exploited to transport molecules inside cells.

MARTI sTcR conjugated to toxins can specifically kill target cells.

Plant or bacterial toxins are efficient mammalian cells killers. If their active subunit can be specifically targeted to cancer cells, they can be converted into attractive cargos for therapeutic purposes. Saporin is an efficient plant toxin which, upon ER to cytosol translocation, inactivates ribosome molecules [35]. The biotinylated MARTI -sTcRwas conjugated to streptavidin conjugated-Saporin (SA-Saporin) active subunit. The SA-Saporin lacks a targeting domain and is thus unable to enter into mammalian cells by itself. Knowing that MARTl-sTcR was able to carry GFP into the cells, a test was conducted to dtermine if it would be able to transport Saporin too.

MART1-SCT expressing cells were used again as they are saturated with the target peptide. The killing of cells expressing either MARTI -SCT or an irrelevant peptide-SCT was analyzed as a control. The cells were incubated for 3 days with TcR conjugated with SA- Saporin at a final concentration of 20 nM and ³H-thymidine incorporation was determined (Fig. 6A). As shown, a specific killing of the Martl-SCT expressing cells was observed, whereas no effect was observed in the negative control. Kinetics were performed and demonstrated that 0.1 nM (50 ng/mL) were sufficient to kill half of the cells (Fig. 6B). From this data it was concluded that the toxin, when combined to a sTcR, was specifically carried into the cytosol of target cells expressing the correct combination of pMHC, while cells expressing a different pMHC complex remained unaffected (Fig. 6A).

The same assay was performed on an HLA-A2 positive melanoma cell line, Malme, using the same Saporin conjugated TcRs. A significant decrease was observed in ³H- thymidine incorporation (Fig. 4C). As expected, this effect was dramatically increased when cells were pulsed with MARTI peptide which thus almost saturated the membrane exposed HLA-A2. As a control, Hek cells (HLA-A2 positive and MARTI negative) were incubated with MARTI sTcR conjugated with SA-saporin. As expected, these cells were not affected (Fig. 4D). However, pulsing Hek cells with 10 μΜ MARTI peptide rendered them sensitive to the sTcR-toxin complex (Fig. 4D). These experiments demonstrate that the sTcR was able to specifically target HLA-A2 positive melanoma cell lines.

Other proteins can be targeted by Mammalian produced sTcR

The universality of the sTcR production method was tested by making another TcR soluble. A method to isolate alloreactive TcRs was previosly published andshows that these TcRs are specific for the pMHC complex they were raised against [32]. The present method was tested on one alloreactive TcR, A94, which is specific for HLA-A2/CD20p (peptide SLFLGILSV (SEQ ID NO:72)). This TcR was chosen because, it has shown a high affinity for its target (Walchli, Fallang et al., manuscript in preparation), and like DMF5 [33], A94 binds to its target in a CD8 co-receptor-independent manner (Fig. 6E). The soluble version of A94 TcR was produced with the TcRa FLAG tagged and the TcRb carrying a BirA sequence (Fig. 3B). Unlike DMF5 sTcR, A94 sTcR combined with PE-SA was not very efficient at staining HLA-A2/CD20p positive cells (data not shown). However, its ability to selectively kill EBV cells, which are CD20 protein positive was only observed if they had been isolated from a HLA-A2 positive donor (Fig. 6F). As expected SupTl expressing SCT-CD20p were also specifically killed by sTcR A94, but not when transfected with SCT-irrelevant peptide (data not shown). Taken together these data show that sTcR a94 had kept its original specificity and was able to selectively kill cells expressing the good HLA/peptide combination. Furthermore, the mammalian expression method presented herein was validated for a different target and thus seems universal.

Comparison ofwt and Leucine -Zipper modified soluble TCR

The DMF5 and s591 (CD20p specific) TCRs were genetically modified by adding a complimentary Leucine zipper dimerization motif [22]. Designed heterodimerizing leucine zippers with a ranger of pis and stabilities up to 10(-15) [22], linked by a 15aa GS sequence, to the C-terminal end of each of the chains. The modified and wt TCRs were then tandemly produced, and the supematants were compared in terms of the ability to stain cells expressing either Ml-SCT (red line) or CD20p-SCT (blue line). The staining was done using anti-FLAG antibody, and Donkey-Anti Mouse PE secondary antibody, and the samples were analyzed by flow cytometry.

As is apparent in Figure 7, the modification increases the staining of the target cells for both the DMF5 and the previously negative s591. This is likely due to the zipper's ability to force heterodimerization of the TCR chains, thereby increasing protein production and secretion into the supernatant. The increase in protein production has been verified by Western blotting (not shown). Therefore, this modification may be used to increase the success rate of solubilising different TCRs with varying interchain affinity, and also increase the overall yield of protein production.

In summary, these examples describe use of an expression system to efficiently generate a functional sTcR dimer able to specifically target an antigen presented as a peptide in complex with HLA I. By adding various tags to the chains of the sTcR we have successfully purified, stained or combined it with the desired cargo. The sTcR is functional both as a monomer and as a multimer conjugated both to SA or even SA-containing nano- particles. As expected, multimerization yielded an obvious increase in the staining of cells compared to the monomer. Surprisingly, in addition to very efficient surface staining of cells, sTcRs were also endocytosed, and capable of carrying large cargo (such as the 28 kD mCherry) into target cells. By adding the active subunit of the toxin Saporin, we also demonstrated that the cargo was not only kept in endocytotic vesicles, but further translocated to the cytosol. This is important and opens a new avenue in the use of therapeutic sTcR as a specific targeting agent to detect altered or infected cells or to search and destroy selected antigen-expressing cells.

In contrast to antibodies which detect molecules present on the cell surface, TcRs are capable of detecting any protein in the context of MHC. This is a substantial advantage of TcRs over antibodies: any disease marker hidden within the cell can potentially be detected. Recent success stories have shown the power of therapeutic monoclonal antibodies such as Rituximab and Herceptin and the development of a sTcR as carrier agent will enlarge the spectrum of specific drug targeting. Furthermore, the ability of TcR to detect any protein can be exploited for diagnostics. Indeed our experiments show that endogenous pMHC complexes could be detected.

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All publications and patents mentioned in the above specification are herein incorporated by reference as if expressly set forth herein. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in relevant fields are intended to be within the scope of the following claims.

Claims

We claim: 1. A nucleic acid construct encoding a soluble T-Cell receptor comprising a soluble T-cell receptor a-chain nucleic acid sequence and a soluble T-cell receptor β-chain nucleic acid sequence operably linked by a ribosome skipping sequence sequence, said T-cell receptor a-chain and β-chain each lacking T-cell receptor transmembrane and cytosolic domains.

2. The nucleic acid construct of Claim 1 , wherein said ribosome skipping sequence is selected from the group consisting of a 2A peptide nucleic acid sequence or an IRES sequence.

3. The nucleic acid construct of Claim 1, wherein said soluble T-cell receptor a- chain nucleic acid sequence and said soluble T-cell receptor β-chain nucleic acid sequence each comprise a T-cell receptor variable domain.

4. The nucleic acid construct of Claim 1, wherein said soluble T-cell receptor a- chain nucleic acid sequence and said soluble T-cell receptor β-chain nucleic acid sequence each comprise at least a portion of the T-cell receptor a-chain nucleic acid sequence and a T- cell receptor β-chain nucleic acid sequence constant domains, and wherein said at least a portion of said constant domains do not contain an amino acid substitution.

5. The nucleic acid construct of Claim 3, wherein said soluble T-cell receptor a- chain nucleic acid sequence and said soluble T-cell receptor β-chain nucleic acid sequence each comprise the entire T-cell receptor a-chain nucleic acid sequence and a T-cell receptor β- chain nucleic acid sequence constant and variable domains.

6. The nucleic acid construct of any of Claims 1 to 5, wherein said soluble T-cell receptor a-chain nucleic acid sequence and said soluble T-cell receptor β-chain nucleic acid sequence each comprise a dimerization domain.

7. The nucleic acid construct of Claim 6, wherein said dimerization domain is a leucine zipper domain operably linked to the c-terminal end of each of said soluble T-cell receptor a-chain nucleic acid sequence and said soluble T-cell receptor β-chain nucleic acid sequence.

8. The nucleic acid construct of any of Claims 1 to 7, wherein one or both of said soluble T-cell receptor a-chain nucleic acid sequence and said soluble T-cell receptor β-chain nucleic acid sequence are operably linked to a nucleic acid sequence encoding an accessory polypeptide.

9. The nucleic acid construct of Claim 8, wherein said accessory polypeptide is selected from the group consisting of an enzyme, an antimicrobial polypeptide, a cytokine and a fluorescent polypeptide.

10. The nucleic acid construct of any of Claims 1 to 9, wherein said 2A peptide nucleic acid sequence comprises a consensus sequence corresponding to SEQ ID NO: 69.

11. The nucleic acid construct of any of Claims 1 to 10, wherein said soluble T-cell receptor encoded by said construct is internalizable by cells displaying a ligand bound by said soluble T-cell receptor.

12. A nucleic acid vector comprising the construct of any of Claims 1 to 11.

13. The nucleic acid vector of Claim 12, wherein said vector is selected from the group consisting of a plasmid vector and a viral vector.

14. A host cell comprising the nucleic acid vector of Claims 12 or 13.

15. The host cell of Claim 13, wherein said host cell is a mammalian host cell.

16. A method for producing a soluble T-cell receptor comprising: introducing a nucleic acid construct according to any of Claims 1 to 11 or a vector according to any of Claims 12 to 13 into a host cell under conditions such that said soluble T- cell receptor a-chain and said soluble T-cell receptor β-chain are expressed;

culturing said host cell to provide a host cell culture; and

purifying said soluble T-cell receptor from said host cell culture.

17. A soluble T-cell receptor produced by the method of Claim 15, wherein said soluble T-cell receptor comprises said T-cell receptor a-chain and said T-cell receptor β-chain linked by a disulfide bond.

18. The soluble T-cell receptor of Claim 17, disulfide bond is a native disulfide bond.

19. A soluble T-cell receptor molecule comprising a soluble T-cell receptor a- chain polypeptide sequence and a soluble T-cell receptor β-chain polypeptide sequence linked by a disulfide bond, one of said soluble T-cell receptor a-chain polypeptide sequence and said soluble T-cell receptor β-chain polypeptide sequence comprising a processed C-terminal 2A peptide sequence.

20. The soluble T-cell receptor of Claim 19, wherein said soluble T-cell receptor a- chain polypeptide sequence and said soluble T-cell receptor β-chain polypeptide sequence each comprise a T-cell receptor variable domain.

21. The soluble T-cell receptor of Claim 19, wherein said soluble T-cell receptor a- chain polypeptide sequence and said soluble T-cell receptor β-chain polypeptide sequence each comprise at least a portion of the T-cell receptor a-chain nucleic acid sequence and a T- cell receptor β-chain nucleic acid sequence constant domains, and wherein at least a portion of said constant domains do not contain an amino acid substitution.

22. The soluble T-cell receptor of Claim 21, wherein said soluble T-cell receptor a- chain sequence and said soluble T-cell receptor β-chain sequence each comprise the entire T- cell receptor a-chain sequence and a T-cell receptor β-chain sequence constant and variable domains.

23. The soluble T-cell receptor of Claims 19-22, wherein said T-cell receptor a- chain polypeptide sequence and said T-cell receptor β-chain polypeptide sequence each comprise a dimerization domain.

24. The nucleic acid construct of Claim 23, wherein said dimerization domain is a leucine zipper domain operably linked to the c-terminal end of each of said T-cell receptor a- chain polypeptide sequence and said T-cell receptor β-chain polypeptide sequence.

25. The soluble T-cell receptor of any of Claims 19 to 24, wherein one or both of said T-cell receptor a-chain polypeptide sequence and said T-cell receptor β-chain polypeptide sequence are operably linked to an accessory polypeptide.

26. The soluble T-cell receptor of Claim 25, wherein said accessory polypeptide is selected from the group consisting of an enzyme, an antimicrobial polypeptide, a cytokine and a fluorescent polypeptide.

27. The soluble T-cell receptor of any of Claims 19 to 26, wherein said disulfide bond is a native disulfide bond.

28. The soluble T-cell receptor of any of Claims 19 to 27, wherein said soluble T- cell receptor is operably linked to an accessory molecule.

29. The soluble T-cell receptor of Claim 28, wherein said accessory molecule is detectable label.

30. The soluble T-cell receptor of Claim 28, wherein said accessory molecule is therapeutic molecule.

31. The soluble T-cell receptor of any of Claims 17 to 30, wherein said soluble T- cell receptor is intemalizable by cells displaying a ligand bound by said soluble T-cell receptor.

32. The soluble T-cell receptor of any of Claims 17 to 31, wherein said soluble T- cell receptor binds to an intracellular ligand in the context of MHC molecules.

33. A multimeric T cell receptor complex comprising a plurality of soluble T-cell receptors as claimed in claim 17-32.

34. The multimeric T cell receptor complex of Claim 33, wherein said complex is multivalent.

35. The multimeric T cell receptor complex of Claim 33, comprising two or three or four or more soluble T-cell receptors associated with one another.

36. A soluble T-cell receptor or multimeric complex of any of Claims 17 to 35 conjugated to an accessory molecule or particle.

37. A pharmaceutical formulation comprising a soluble T-cell receptor or multimeric complex of any of Claims 17 to 36 in association with a pharmaceutically acceptable carrier.

38. Use of a soluble T-cell receptor or multimeric complex of any of Claims 17 to 37 for a diagnostic application or therapeutic administration to a subject.

39. Use of the soluble T-cell receptor or multimeric complex of any one of claims 17 to 37 in the treatment of cancer, an autoimmune disease or a viral infection.

40. A method comprising administering a therapeutically effective amount of a soluble T-cell receptor or multimeric complex of any of Claims 17 to 37 to a patient.

41. A method for detecting an analyte of interest comprising contacting a test material with a soluble T-cell receptor or multimeric complex of any of Claims 17 to 36 and detecting the presence of said analyte via binding of said soluble T-cell receptor or multimeric complex to said analyte of interest.