REDIRECTED CELLS WITH MHC CHIMERIC RECEPTORS AND METHODS OF USE IN IMMUNOTHERAPY

Abstract

Chimeric receptors featuring major histocompatibility molecules grafted onto T cell receptor molecules and surrogate co-receptors featuring cell surface receptor ligands fused with signaling molecule domains. The chimeric receptors can be used to redirect cells, altering their specificity. T cells expressing chimeric receptors may bind to ICRs of target T cells for which their chimeric receptors are specific. Surrogate co-receptors may be used to help enhance TCR-CD3 signaling as part of this modular receptor system. The chimeric receptors and surrogate coreceptors may be used to help eliminate autoreactive T cells or program T cells to desired effector functions.

Claims

1. An engineered cell co-expressing on its surface: a. a chimeric receptor (MHCR) comprising a major histocompatibility complex (MHC) portion derived from a MHC protein directly or indirectly fused to a T cell receptor (TCR) portion derived from a TCR protein; and b. a surrogate coreceptor (SCR) comprising a cell surface receptor ligand portion directly or indirectly fused to a signaling molecule portion; wherein the MHCR is adapted to bind to a TCR of a target cell and the SCR is adapted to bind to a cell surface receptor of the target cell.

2-21. (canceled)

22. A chimeric receptor (MHCR) comprising a major histocompatibility complex (MHC) portion derived from a MHC protein directly or indirectly fused to a T cell receptor (TCR) portion derived from a TCR protein, wherein the MHCR is adapted to bind to a TCR of a target cell.

23. The MHCR of claim 22, wherein binding of the MHCR to the TCR of the target cell (i) initiates a signaling cascade effective for eliminating the target cell or (ii) instructs the target cell to differentiate to a specific effector function.

24. (canceled)

25. The MHCR of claim 22, wherein the MHC portion of the MHCR is N-terminal to the TCR portion of the MHCR.

26. The MHCR of claim 22, wherein the MHC portion is indirectly fused to the TCR portion via a linker.

27. The MHCR of claim 26, wherein the linker comprises a glycine-rich peptide.

28. The MHCR of claim 22, wherein the MHC protein, the TCR protein, or both the MHC protein and the TCR protein are mammalian proteins.

29. (canceled)

30. The MHCR of claim 22, wherein the TCR portion comprises at least a portion of a transmembrane domain and at least a portion of a cytoplasmic domain of a TCR protein, and the MHC portion comprises at least a portion of an extracellular domain of the MHC protein.

31. The MHCR of claim 22, wherein the MHCR further comprises a peptide antigen integrated into the MHC portion, or directly or indirectly fused to the MHC portion.

32. The MHCR of claim 22, wherein the MHC protein comprises HLA-A, HLA-B, HLA-C, Beta2-microglobulin, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB, H2-Aa, H2-B1, H2-K1, H2-EB beta, H2-EK alpha, H2-EK beta, a fragment thereof, or a combination thereof.

33. The MHCR of claim 22, wherein the MHC molecule comprises HLA-A, HLA-B, HLA-C, Beta2-microglobulin, HLA-DPA, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB, H2-Aa, H2-B1, H2-K1, H2-EB beta, H2-EK alpha, H2-EK beta, a peptide that is at least 90% identical to HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB, H2-Aa, H2-B1, H2-K1, H2-EB beta, H2-EK alpha, or H2-EK beta, a fragment thereof, or a combination thereof.

34. The MHCR of claim 22, wherein the TCR molecule comprises TRAC, TRBC1, TRBC2, TRDC, TRGC1, TRGC2, TCRA, TCB1, TCB2, TCC1, TCC2, TCC3, TCC4, a fragment thereof, or a combination thereof.

35. The MHCR of claim 22, wherein the TCR molecule comprises TRAC, TRBC1, TRBC2, TRDC, TRGC1, TRGC2, TCRA, TCB1, TCB2, TCC1, TCC2, TCC3, TCC4, a peptide that is at least 90% identical to TRAC, TRBC1, TRBC2, TRDC, TRGC1, TRGC2, TCRA, TCB1, TCB2, TCC1, TCC2, TCC3, or TCC4, a fragment thereof, or a combination thereof.

36. The MHCR of claim 22, wherein the MHCR is adapted to complex with a CD3 subunit.

37-49. (canceled)

50. A surrogate coreceptor (SCR) comprising a cell surface receptor ligand portion directly or indirectly fused to a signaling molecule portion via a transmembrane domain, wherein the SCR is adapted to bind to a cell surface receptor of a target cell.

51. The SCR of claim 50, wherein the cell surface receptor ligand portion is indirectly fused to the signaling molecule portion via a linker.

52. The SCR of claim 50, wherein the signaling molecule portion has kinase or phosphatase activity.

53. The SCR of claim 50, wherein the signaling molecule portion comprises a Src kinase.

54. The SCR of claim 50, wherein the cell surface receptor ligand portion of the SCR comprises a CD28 ligand, a CTLA-4 ligand, an ICOS ligand, an OX4O ligand, a PD-1 ligand, or a CD2 ligand.

55. The SCR of claim 54, wherein the CD28 ligand comprises CD80, CD86, or both CD80 and CD86.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The features and advantages of the present invention will become apparent from a consideration of the following detailed description presented in connection with the accompanying drawings in which:

[0027] FIG. 1A shows molecules involved in T cell activation. Engagement of the TCR with pMHC (MHC with a peptide antigen) initiates T cell activation.

[0028] FIG. 1B shows the molecular components of the ?lpha-beta-TCR-CD3 complex. The TCR transfers pMHC-specific information to the CD3 subunits and inside the T cell. Triangles represent the inner and outer leafs of the cell membrane. Red and blue dots and ovals represent the transmembrane charge interactions that drive subunit assembly of the complexes (from Kuhns et al., 2006, Immunity 24:133-139).

[0029] FIG. 2A shows a redirected T cell expressing a MHCR (pMHCR with peptide antigen) of the present invention. The MHCR in complex with CD3 subunits is bound to a target T cell's TCR.

[0030] FIG. 2B shows non-limiting examples of MHCR configurations (and the schematics are not limiting with respect to N-terminal and C-terminal orientation). TCR refers to the T cell receptor portion; MHC refers to the major histocompatibility portion, antigen refers to the antigen portion, and L refers to a linker. The present invention is not limited to these configurations. For example, in some embodiments the antigen portion is integrated into the MHC portion. In some embodiments, the MHC portion is N-terminal to the TCR portion (see orientation of sequences below).

[0031] FIG. 3A is a schematic view of a chimeric surrogate coreceptor (SCR), e.g., one comprising CD80/CD86-Lck.

[0032] FIG. 3B shows a redirected T cell expressing a MHCR (pMHCR) and two surrogate coreceptors (SCRs). The MHCR, bound to a target T cell's TCR, is complexed with CD3. The SCRs are bound to the target T cell's coreceptors (CD28, CTLA-4). Binding of the SCRs to coreceptors on the target T cell may help initiate CD3 signaling similar to that seen in normal T cell activation.

[0033] FIG. 4 shows expression of pMHCR-CD3 complexes on T cell hybridomas. 58?.sup.??.sup.? cells that lack endogenous TCRs were transduced with a pMHCR composed of MCC:I-E.sup.k. The proportional expression (diagonal) of I-E.sup.k and CD3 subunits suggests surface co-dependent expression of the epitopes.

[0034] FIG. 5 shows TCR-specific IL-2 production by pMHCR-CD3 expressing T cell hybridomas. 58?.sup.??.sup.? cells that lack endogenous TCRs were transduced with a pMHCR composed of MCC:I-E.sup.k as well as a CD80-Lck surrogate coreceptor (SCR). The cells were co-cultured with parental M12 B cells, or M12 cells stably transduced to express the MCC:I-E.sup.k-specific 284 TCR alone or with CD28. The increased IL-2 expression in the presence of CD28 indicates that the surrogate coreceptor (SCR) enhances pMHCR-CD3 signaling.

[0035] FIG. 6 shows TCR-specific killing of CD4 T cells by redirected CTLs. Purified CD8 T cells from B10.A mice were activated in vitro and transduced with a MCC:I-E.sup.k pMHCR (agonist) or an HB:I-E.sup.k pMHCR (null) as well as a CD80-Lck surrogate coreceptor. The redirected CTLs were then co-cultured at the indicated ratios with na?ve ex vivo 5c.c7 TCR transgenic CD4 T cells overnight. Killing was evaluated by flow cytometry using count beads relative to the 0:1 samples.

DETAILED DESCRIPTION OF THE INVENTION

Chimeric MHC Receptors (MHCRs)

[0036] The present invention features chimeric receptors (e.g., MHCRs) comprising at least a MHC portion (e.g., class I, class II, non-classical, a combination thereof, etc.) and a TCR portion (e.g., ??, ?? TCR, etc.) (see FIG. 2B(i)). For example, the MHCR may comprise a MHC portion and a TCR portion, a MHC and a TCR portion optionally separated by a linker (see FIG. 2B (iii) and (iv)). A linker may be any appropriate linker such as but not limited to a peptide linker. In some embodiments, the MHCR further comprises a peptide antigen (see FIG. 2B (ii)); a MHCR comprising a peptide antigen may herein be referred to as a pMHCR. Note that MHC portions and/or TCR portions may be from any appropriate species including but not limited to human, monkey, mouse, rat, rabbit, or the like, e.g., any other appropriate mammalian species. The components and configurations of the MHRCs of the present invention are not limited to those shown in FIG. 2B. For example, the MHCR may comprise a TCR portion and a MHC portion; a TCR portion and a MHC portion separated by a linker; a TCR portion and a MHC portion and an antigen portion; a TCR portion and a MHC portion and an antigen portion, wherein the TCR portion and MHC portion are separated by a linker; a TCR portion and a MHC portion and an antigen portion, wherein the MHC portion and antigen portion are separated by a linker; a TCR portion and a MHC portion and an antigen portion, wherein the TCR and MHC portion are separated by a linker and the MHC portion and the antigen portion are separate by a linker; etc.

[0037] The MHC portion may comprise one or more MHC proteins (e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1), one or more fragments thereof, or combinations thereof. For reference, non-limiting MHC sequences (human, mouse) are listed below in Table 1.1 and Table 1.2. Note that MHC genes are highly polymorphic, and thus the present invention is not limited to the sequences in Table 1.1 And Table 1.2. The present invention includes MHC polymorphisms and any other appropriate variant of MHC proteins.

TABLE-US-00001 TABLE1.1 ExamplesofHumanMHCProteinSequences SEQ IDNO. Description AminoAcidSequence 1 UniprotP01891 MAVMAPRTLVLLLSGALALTQTWAGSHSMR HLA-Agene YFYTSVSRPGRGEPRFIAVGYVDDTQFVRF (MHCI) DSDAASQRMEPRAPWIEQEGPEYWDRNTRN VKAQSQTDRVDLGTLRGYYNQSEAGSHTIQ MMYGCDVGSDGRFLRGYRQDAYDGKDYIAL KEDLRSWTAADMAAQTTKHKWEAAHVAEQW RAYLEGTCVEWLRRYLENGKETLQRTDAPK THMTHHAVSDHEATLRCWALSFYPAEITLT WQRDGEDQTQDTELVETRPAGDGTFQKWVA VVVPSGQEQRYTCHVQHEGLPKPLTLRWEP SSQPTIPIVGIIAGLVLFGAVITGAVVAAV MWRRKSSDRKGGSYSQAASSDSAQGSDVSL TACKV 2 UniprotP13464 MRVTAPRTVLLLLWGAVALTETWAGSHSMR HLA-Bgene YFYTAMSRPGRGEPRFIAVGYVDDTQFVRF (MHCI) DSDAASPRTEPRAPWIEQEGPEYWDRNTQI FKTNTQTYRENLRIALRYYNQSEAGSHTWQ TMYGCDVGPDGRLLRGHNQYAYDGKDYIAL NEDLSSWTAADTAAQITQRKWEAAREAEQL RAYLEGLCVEWLRRHLENGKETLQRADPPK THVTHHPVSDHEATLRCWALGFYPAEITLT WQRDGEDQTQDTELVETRPAGDRTFQKWAA VVVPSGEEQRYTCHVQHEGLPKPLTLRWEP SSQSTIPIVGIVAGLAVLAVVVIGAVVATV MCRRKSSGGKGGSYSQAASSDSAQGSDVSL TA 3 UniprotQ29963 MRVMAPRTLILLLSGALALTETWACSHSMR HLA-Cgene YFDTAVSRPGRGEPRFISVGYVDDTQFVRF (MHCI) DSDAASPRGEPRAPWVEQEGPEYWDRETQK YKRQAQADRVNLRKLRGYYNQSEDGSHTLQ WMYGCDLGPDGRLLRGYDQSAYDGKDYIAL NEDLRSWTAADTAAQITQRKWEAAREAEQW RAYLEGTCVEWLRRYLENGKETLQRAEHPK THVTHHPVSDHEATLRCWALGFYPAEITLT WQRDGEDQTQDTELVETRPAGDGTFQKWAA VVVPSGEEQRYTCHVQHEGLPEPLTLRWEP SSQPTIPIVGIVAGLAVLAVLAVLGAVMAV VMCRRKSSGGKGGSCSQAASSNSAQGSDES LIACKA 4 UniprotP20036 MRPEDRMFHIRAVILRALSLAFLLSLRGAG HLADPA1 AIKADHVSTYAAFVQTHRPTGEFMFEFDED (MHCII) EMFYVDLDKKETVWHLEEFGQAFSFEAQGG LANIAILNNNLNTLIQRSNHTQATNDPPEV TVFPKEPVELGQPNTLICHIDKFFPPVLNV TWLCNGELVTEGVAESLFLPRTDYSFHKFH YLTFVPSAEDFYDCRVEHWGLDQPLLKHWE AQEPIQMPETTETVLCALGLVLGLVGIIVG TVLIIKSLRSGHDPRAQGTL 5 UniprotP04440 MMVLQVSAAPRTVALTALLMVLLTSVVQGR HLADPB1 ATPENYLFQGRQECYAFNGTQRFLERYIYN (MHCII) REEFARFDSDVGEFRAVTELGRPAAEYWNS QKDILEEKRAVPDRMCRHNYELGGPMTLQR RVQPRVNVSPSKKGPLQHHNLLVCHVTDFY PGSIQVRWFLNGQEETAGVVSTNLIRNGDW TFQILVMLEMTPQQGDVYTCQVEHTSLDSP VTVEWKAQSDSARSKTLTGAGGFVLGLIIC GVGIFMHRRSKKVQRGSA 6 UniprotP01909 MILNKALMLGALALTTVMSPCGGEDIVADH HLADQA1 VASYGVNLYQSYGPSGQYTHEFDGDEQFYV (MHCII) DLGRKETVWCLPVLRQFRFDPQFALTNIAV LKHNLNSLIKRSNSTAATNEVPEVTVFSKS PVTLGQPNILICLVDNIFPPVVNITWLSNG HSVTEGVSETSFLSKSDHSFFKISYLTLLP SAEESYDCKVEHWGLDKPLLKHWEPEIPAP MSELTETVVCALGLSVGLVGIVVGTVFIIR GLRSVGASRHQGPL 7 UniprotP01920 MSWKKALRIPGGLRAATVTLMLAMLSTPVA HLADQB1 EGRDSPEDFVYQFKAMCYFTNGTERVRYVT (MHCII) RYIYNREEYARFDSDVEVYRAVTPLGPPDA EYWNSQKEVLERTRAELDTVCRHNYQLELR TTLQRRVEPTVTISPSRTEALNHHNLLVCS VTDFYPAQIKVRWFRNDQEETTGVVSTPLI RNGDWTFQILVMLEMTPQHGDVYTCHVEHP SLQNPITVEWRAQSESAQSKMLSGIGGFVL GLIFLGLGLIIHHRSQKGLLH 8 UniprotP01903 MAISGVPVLGFFIIAVLMSAQESWAIKEEH HLADRAgene VIIQAEFYLNPDQSGEFMFDFDGDEIFHVD (MHCII) MAKKETVWRLEEFGRFASFEAQGALANIAV DKANLEIMTKRSNYTPITNVPPEVTVLTNS PVELREPNVLICFIDKFTPPVVNVTWLRNG KPVTTGVSETVFLPREDHLFRKFHYLPFLP STEDVYDCRVEHWGLDEPLLKHWEFDAPSP LPETTENVVCALGLTVGLVGIIIGTIFIIK GVRKSNAAERRGPL 9 UniprotQ30167 MVCLRLPGGSCMAVLTVTLMVLSSPLALAG HLADRB1gene DTRPRFLEEVKFECHFFNGTERVRLLERRV (MHCII) HNQEEYARYDSDVGEYRAVTELGRPDAEYW NSQKDLLERRRAAVDTYCRHNYGVGESFTV QRRVQPKVIVYPSKTQPLQHHNLLVCSVNG FYPGSIEVRWFRNGQEEKTGVVSTGLIQNG DWTFQTLVMLETVPQSGEVYTCQVEHPSVM SPLTVEWRARSESAQSKMLSGVGGFVLGLL FLGAGLFIYFRNQKGHSGLPFTGFLS

TABLE-US-00002 TABLE1.2 ExamplesofMouseMHCProteinSequences SEQ IDNO. Description AminoAcidSequence 10 UniprotQ9TQ72 RSRALILGVLALTTMLSLCGGEDYIEADHV MHCIIantigenIE AFYGISVYQSPGDIGQYTFEFDGDELFYVD alpha(H2-Aa) LDKKETVWMLPEFGQLTSFDPQGGLQEIAT GKYNLEILIKDSNFTPAANEAPQATVFPKS PVLLGQPNTLICFVDNIFPPVINITWLRNS KSVTDGVYETSFLVNRDHSFHKLSYLTFIP SDDDIYDCKVEHWGLEEPVLKHWEPEIPAP MSELTETVICALGLSVGLVGIVVGTIFIIQ GLRSGGTSRH 11 UniprotO19440 MAQRTLFLLLAAALTMIETRAGPHSMRYFE MHCIantigen TAVFRPGLGEPRFISVGYVDNTQFVSFDSD (H2-B1) AENPRSEPRAPWMEQEGPEYWERETQIAKD NEQSFGWSLRNLIHYYNQSKGGFHTFQRLS GCDMGLDGRLLRGYLQFAYDGRDYITLNED LKTWMAADLVALITRRKWEQAGAAELYKFY LEGECVEWLRRYLELGNETLLRTDPPKAHV THHPRPAGDVTLRCWALGFYPADITLTWQL NGEELTQDMELVETRPAGDGTFQKWAAVVV PLGKEQNYTCHVYHEGLPEPLTLRWEPPPS TGSNMVNIAVLVVLGAVIIIEAMVAFVLKS SRKIAILPGPAGTKGSSAS 12 UniprotQ31191 MAPCTLLLLLAAALAPTQTRAARAAARGPV MHCIH2-Kgene RRSGSHRAPPPGPHSLSDADNPRFEPRAPW (Haplotyped) MEQEGPEYWEEQTQRAKSDEQWFRVSLRTA (H2-K1) QRYYNQSKGGSHTFQRMFGCDVGSDWRLLR GYQQFAYDGRDYIALNEDLKTWTAADTAAL ITRRKWEQAGDAEYYRAYLEGECVEWLRRY LELGNETLLRTDSPKAHVTYHPRSQVDVTL RCWALGFYPADITLTWQLNGEDLTQDMELV ETRPAGDGTFQKWAAVVVPLGKEQNYTCHV HHKGLPEPLTLRWKLPPPTVSNTVIIAVLV VLGAAIVTGAVVAFVMKMRRNTGGKGVNYA LAPGSQTSDLSLPDGKVMVH 13 UniprotP04230 MVWLPRVPCVAAVILLLTVLSPPMALVRDS H2ClassII RPWFLEYCKSECHFYNGTQRVRLLERYFYN histocompatibility LEENLRFDSDVGEFHAVTELGRPDAENWNS antigenE-Bbeta QPEFLEQKRAEVDTVCRHNYEISDKFLVRR chain RVEPTVTVYPTKTQPLEHHNLLVCSVSDFY PGNIEVRWFRNGKEEKTGIVSTGLVRNGDW TFQTLVMLETVPQSGEVYTCQVEHPSLTDP VTVEWKAQSTSAQNKMLSGVGGFVLGLLFL GAGLFIYFRNQKGQSGLQPTGLLS 14 UniprotP04224 MATIGALVLRFFFIAVLMSSQKSWAIKEEH MHCIIE-Kalpha TIIQAEFYLLPDKRGEFMFDFDGDEIFHVD chain(underlined IEKSETIWRLEEFAKFASFEAQGALANIAV portionis DKANLDVMKERSNNTPDANVAPEVTVLSRS portionusedin PVNLGEPNILICFIDKFSPPVVNVTWLRNG SEQIDNO:30) RPVTEGVSETVFLPRDDHLFRKFHYLTFLP STDDFYDCEVDHWGLEEPLRKHWEFEEKTL LPETKENVVCALGLFVGLVGIVVGIILIMK GIKKRNVVERRQGAL 15 GenBankID: MWLPRVPCVAAVILLLTVLSPPVALVRDSRPW M36939.1 FLEYCKSECHFYNGTQRVRLLVRYFYNLEENL MHCIIE-Kbeta RFDSDVGEFRAVTELGRPDAENWNSQPEFLEQ chain(underlined KRAEVDTVCRHNYEIFDNFLVPRRVEPTVTVY portionis PTKTQPLEHHNLLVCSVSDFYPGNIEVRWFRN usedinSEQIDNO: GKEEKTGIVSTGLVRNGDWTFQTLVMLETVPQ 31,32) SGEVYTCQVEHPSLTDPVTVEWKAQSTSAQNK MLSGVGGFVLGLLFLGAGLFIYFRNQKGQSGL QPTGLLS

[0038] Referring to Table 1.1, the HLA-A (MHC I) sequence (SEQ ID NO: 1) includes the signal peptide (amino acids 1-24); amino acids 25-308 are believed to make up the extracellular region, amino acids 309-332 are believed to make up the transmembrane region, and amino acids 333-365 are believed to make up the cytoplasmic region. The HLA-B (MHC I) sequence (SEQ ID NO: 2) includes the signal peptide (amino acids 1-24); amino acids 25-308 are believed to make up the extracellular region, amino acids 309-332 are believed to make up the transmembrane region, and amino acids 333-362 are believed to make up the cytoplasmic region. The HLA-C (MHC I) sequence (SEQ ID NO: 3) includes the signal peptide (amino acids 1-24); amino acids 25-308 are believed to make up the extracellular region, amino acids 309-333 are believed to make up the transmembrane region, and amino acids 334-366 are believed to make up the cytoplasmic region. The HLA DPA1 (MHC II) sequence (SEQ ID NO: 4) includes the signal peptide (amino acids 1-28); amino acids 29-222 are believed to make up the extracellular region, amino acids 223-245 are believed to make up the transmembrane region, and amino acids 246-260 are believed to make up the cytoplasmic region. The HLA DPB1 (MHC II) sequence (SEQ ID NO: 5) includes the signal peptide (amino acids 1-29); amino acids 30-225 are believed to make up the extracellular region, amino acids 226-246 are believed to make up the transmembrane region, and amino acids 247-258 are believed to make up the cytoplasmic region. The HLA DQA1 (MHC II) sequence (SEQ ID NO: 6) includes the signal peptide (amino acids 1-23); amino acids 24-216 are believed to make up the extracellular region, amino acids 217-239 are believed to make up the transmembrane region, and amino acids 240-254 are believed to make up the cytoplasmic region. The HLA DQB1 (MHC II) sequence (SEQ ID NO: 7) includes the signal peptide (amino acids 1-32); amino acids 33-230 are believed to make up the extracellular region, amino acids 231-251 are believed to make up the transmembrane region, and amino acids 252-261 are believed to make up the cytoplasmic region. The HLA DRA (MHC II) sequence (SEQ ID NO: 8) includes the signal peptide (amino acids 1-25); amino acids 26-216 are believed to make up the extracellular region, amino acids 217-239 are believed to make up the transmembrane region, and amino acids 240-254 are believed to make up the cytoplasmic region. The HLA DRB1 (MHC II) sequence (SEQ ID NO: 9) includes the signal peptide (amino acids 1-29); amino acids 30-227 are believed to make up the extracellular region, amino acids 228-250 are believed to make up the transmembrane region, and amino acids 251-266 are believed to make up the cytoplasmic region. The MHC E-K alpha chain (SEQ ID NO: 14) includes the signal peptide (aa 1-25), the extracellular domain (aa 26-216), the transmembrane domain (aa 217-24), and a cytoplasmic portion (aa 243-255).

[0039] As previously discussed, the MHCR of the present invention comprises at least a MHC portion and a TCR portion. In some embodiments, a MHC portion comprises one or more MHC proteins (e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, MHC E-K alpha, MHC E-K beta, etc.), fragments thereof, or combinations thereof. For example, in some embodiments, the MHC portion comprises a fragment of any of SEQ ID NO: 1-15.

[0040] In some embodiments, the MHC portion comprises a peptide that is at least 80% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 85% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 90% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 95% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 99% identical to a MHC protein or a fragment thereof.

[0041] In some embodiments, a fragment of a MHC protein is from 10 to 25 aa in length. In some embodiments, a fragment of a MHC protein is from 10 to 50 aa in length. In some embodiments, a fragment of a MHC protein is from 10 to 100 aa in length. In some embodiments, a fragment of a MHC protein is from 10 to 150 aa in length. In some embodiments, a fragment of a MHC protein is from 10 to 200 as in length. In some embodiments, a fragment of a MHC protein is from 10 to 250 as in length. In some embodiments, a fragment of a MHC protein is from 10 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 10 to 350 as in length. In some embodiments, a fragment of a MHC protein is from 25 to 50 as in length. In some embodiments, a fragment of a MHC protein is from 25 to 100 aa in length. In some embodiments, a fragment of a MHC protein is from 25 to 150 as in length. In some embodiments, a fragment of a MHC protein is from 25 to 200 as in length. In some embodiments, a fragment of a MHC protein is from 25 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 25 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 25 to 350 as in length. In some embodiments, a fragment of a MHC protein is from 50 to 100 as in length. In some embodiments, a fragment of a MHC protein is from 50 to 150 as in length. In some embodiments, a fragment of a MHC protein is from 50 to 200 aa in length. In some embodiments, a fragment of a MHC protein is from 50 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 50 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 50 to 350 aa in length. In some embodiments, a fragment of a MHC protein is from 100 to 150 as in length. In some embodiments, a fragment of a MHC protein is from 100 to 200 as in length. In some embodiments, a fragment of a MHC protein is from 100 to 250 as in length. In some embodiments, a fragment of a MHC protein is from 100 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 100 to 350 as in length. In some embodiments, a fragment of a MHC protein is from 150 to 200 as in length. In some embodiments, a fragment of a MHC protein is from 150 to 250 as in length. In some embodiments, a fragment of a MHC protein is from 150 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 150 to 350 as in length. In some embodiments, a fragment of a MHC protein is from 200 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 200 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 200 to 350 as in length. In some embodiments, a fragment of a MHC protein is from 250 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 250 to 350 as in length. In some embodiments, a fragment of a MHC protein is more than 350 as in length.

[0042] A TCR portion may comprise one or more TCR proteins (e.g., TCRA, TCRB), one or more fragments thereof, or combinations thereof. For reference, non-limiting TCR sequences (human and mouse) are listed below in Table 2.1 and Table 2.2. The present invention is not limited to the TCR sequences in Table 2.1 and Table 2.2.

TABLE-US-00003 TABLE2.1 ExamplesofHumanTCRProteinSequences SEQ IDNO. Description AminoAcidSequence 16 UniprotP01848 PNIQNPDPAVYQLRDSKSSDKSVCLFTDFD Tcellreceptor SQTNVSQSKDSDVYITDKTVLDMRSMDFKS alphachain NSAVAWSNKSDFACANAFNNSIIPEDTFFP constantregion SPESSCDVKLVEKSFETDTNLNFQNLSVIG (TRAC,TCRA) FRILLLKVAGFNLLMTLRLWSS 17 UniprotP01850 EDLNKVFPPEVAVFEPSEAEISHTQKATLV Tcellreceptor CLATGFFPDHVELSWWVNGKEVHSGVSTDP beta-1chain QPLKEQPALNDSRYCLSSRLRVSATFWQNP constantregion RNHFRCQVQFYGLSENDEWTQDRAKPVTQI (TRBC1) VSAEAWGRADCGFTSVSYQQGVLSATILYE ILLGKATLYAVLVSALVLMAMVKRKDF 18 UniprotA0A5B9 DLKNVFPPEVAVFEPSEAEISHTQKATLVC Tcellreceptor LATGFYPDHVELSWWVNGKEVHSGVSTDPQ beta-2chain PLKEQPALNDSRYCLSSRLRVSATFWQNPR constantregion NHFRCQVQFYGLSENDEWTQDRAKPVTQIV (TRBC2,TCRBC2) SAEAWGRADCGFTSESYQQGVLSATILYEI LLGKATLYAVLVSALVLMAMVKRKDSRG 19 UniprotB7Z8K6 SQPHTKPSVFVMKNGTNVACLVKEFYPKDI Tcellreceptor RINLVSSKKITEFDPAIVISPSGKYNAVKL deltachain GKYEDSNSVTCSVQHDNKTVHSTDFEVKTD constantregion STDHVKPKETENTKQPSKSCHKPKAIVHTE (TRDC) KVNMMSLTVLGLRMLFAKTVAVNFLLTAKL FFL 20 UniprotP0CF51 DKQLDADVSPKPTIFLPSIAETKLQKAGTY Tcellreceptor LCLLEKFFPDVIKIHWQEKKSNTILGSQEG gamma-1chain NTMKTNDTYMKFSWLTVPEKSLDKEHRCIV constantregion RHENNKNGVDQEIIFPPIKTDVITMDPKDN (TRGC1) CSKDANDTLLLQLTNTSAYYMYLLLLLKSV VYFAIITCCLLRRTAFCCNGEKS 21 UniprotP03986 DKQLDADVSPKPTIFLPSIAETKLQKAGTY Tcellreceptor LCLLEKFFPDIIKIHWQEKKSNTILGSQEG gamma-2chain NTMKTNDTYMKFSWLTVPEESLDKEHRCIV constantregion RHENNKNGIDQEIIFPPIKTDVTTVDPKDS (TRGC2,TCRGC2) YSKDANDVITMDPKDNWSKDANDTLLLQLT NTSAYYMYLLLLLKSVVYFAIITCCLLGRT AFCCNGEKS

TABLE-US-00004 TABLE2.2 ExamplesofMouseTCRProteinSequences SEQ IDNO. Description AminoAcidSequence 22 UniprotP01849 PYIQNPEPAVYQLKDPRSQDSTLCLFTDFD Tcellreceptoralpha SQINVPKTMESGTFITDKTVLDMKAMDSKS chainconstantregion NGAIAWSNQTSFTCQDIFKETNATYPSSDV (TCRA-mouse) PCDATLTEKSFETDMNLNFQNLSVMGLRIL (underlinedportion LLKVAGFNLLMTLRLWSS referstosequencealso usedinSEQIDNO:30) 23 UniprotP01852 EDLRNVTPPKVSLFEPSKAEIANKQKATLV Tcellreceptorbeta-1 CLARGFFPDHVELSWWVNGKEVHSGVSTDP chainconstantregion QAYKESNYSYCLSSRLRVSATFWHNPRNHF (TCB1-mouse) RCQVQFHGLSEEDKWPEGSPKPVTQNISAE AWGRADCGITSASYQQGVLSATILYEILLG KATLYAVLVSTLVVMAMVKRKNS 24 UniprotP01851 EDLRNVTPPKVSLFEPSKAEIANKQKATLV Tcellreceptorbeta-2 CLARGFFPDHVELSWWVNGKEVHSGVSTDP chainconstantregion QAYKESNYSYCLSSRLRVSATFWHNPRNHF (TCB2-mouse) RCQVQFHGLSEEDKWPEGSPKPVTQNISAE (underlinedportion AWGRADCGITSASYHQGVLSATILYEILLG referstosequenceused KATLYAVLVSGLVLMAMVKKKNS inSEQIDNO:31,32) 25 UniprotP01853 DKRLDADISPKPTIFLPSVAETNLHKTGTY Tcellreceptorgamma LCLLEKFFPDVIRVYWKEKNGNTILDSQEG chainconstantregion DTLKTKGTYMKFSWLTVPERAMGKEHSCIV C10.5(TCC1-mouse) KHENNKGGADQEIFFPSIKKVATTCWQDKN DVLQFQFTSTSAYYTYLLLLLKSVIYLAII SFSLLRRTSVCGNEKKS 26 UniprotP03985 DKKLDADISPKPTIFLPSVAETNLHKTGTY Tcellreceptorgamma LCVLEKFFPDVIRVYWKEKKGNTILDSQEG chainconstantregion DMLKTNDTYMKFSWLTVPERSMGKEHRCIV C7.5(TCC2-mouse) KHENNKGGADQEIFFPTIKKVAVSTKPTTC WQDKNDVLQLQFTITSAYYTYLLLLLKSVI YLAIISFSLLRRTSVCCNEKKS 27 UniprotP06334 PSDKRLDADISPKPTIFLPSVAETNLHKTG Tcellreceptorgamma TYLCILEKFFPDVIRVYWKDKNGNTILDSQ chainconstantregion EGDTLKTKGTYMKFSWLTVPERSMGKEHRC DFL12(TCC3-mouse) IVKHENNKGGADQEIFFPSIKKVATTCWQD KNDVLQLQFMSTSAYYTYLLLLLKSVIYLA IISFSLLRRTSVCCNEKRS 28 UniprotP06335 DKRTDSDFSPKPTIFLPSAAETNLHKAGTY Tcellreceptorgamma LCLLEKFFPKVIRVYWKEKDGEKILESQEG chainconstantregion NTIKTNDRYMKFSWLTVTEDSMAKEHSCIV 5/10-13(TCC4-mouse) KHENNKRGVDQEILFPPIGKAFTTINVNPR DSVLRHENVNNATDLEDCMKGRKDMLQLQV TTTYAFYTYLILFFKSMVHLAFVVFCLFRR AAMSCDDQRS

[0043] Referring to the TRAC protein (SEQ ID NO: 16) in Table 2, amino acids 118-137 are believed to make up the transmembrane domain, and amino acids 138-142 are believed to make up the cytoplasmic domain. Referring to the TRBC1 protein (SEQ ID NO: 17) in Table 2, amino acids 151-171 are believed to make up the transmembrane domain. Referring to the TRBC2 protein (SEQ ID NO: 18) in Table 2, amino acids 145-167 are believed to make up the transmembrane domain. Referring to the TRDC protein (SEQ ID NO: 19) in Table 2, amino acids 130-152 are believed to make up the transmembrane domain. Referring to the TRGC1 protein (SEQ ID NO: 20) in Table 2, amino acids 139-161 are believed to make up the transmembrane domain. Referring to the TRGC2 protein (SEQ ID NO: 21) in Table 2, amino acids 157-177 are believed to make up the transmembrane domain, and amino acids 178-189 are believed to make up the cytoplasmic domain.

[0044] As previously discussed, the MHCR of the present invention comprises at least a MHC portion and a TCR portion. In some embodiments, a TCR portion comprises one or more TCR proteins (e.g., TRAC, TRBC1, TRBC2, TRDC, TRCG1, TRCG2, TCRA-mouse, TCB1-mouse, TCB2-mouse, TCC1-mouse, TCC2-mouse, TCC3 mouse, TCC4 mouse, etc.), fragments thereof, or combinations thereof. For example, in some embodiments, the TCR portion comprises a fragment of any of SEQ ID NO: 16-28. (In some embodiments, the fragment is from 5 to 10 as in length. In some embodiments, the fragment is from 10 to 20 aa in length, in some embodiments, the fragment is from 10 to 30 as in length. IN some embodiments, the fragment is from 10 to 40 aa in length. In some embodiments, the fragment is from 10 to 50 as in length, etc.

[0045] In some embodiments, the TCR portion comprises a peptide that is at least 80% identical to a TCR protein (e.g., any of SEQ ID NO: 16-28), or a fragment thereof. In some embodiments, the TCR portion comprises a peptide that is at least 85% identical to a TCR protein (e.g., any of SEQ ID NO: 16-28), or a fragment thereof. In some embodiments, the TCR portion comprises a peptide that is at least 90% identical to a TCR protein (e.g., any of SEQ ID NO: 16-28), or a fragment thereof. In some embodiments, the TCR portion comprises a peptide that is at least 95% identical to a TCR protein (e.g., any of SEQ ID NO: 16-28), or a fragment thereof. In some embodiments, the TCR portion comprises a peptide that is at least 99% identical to a TCR protein (e.g., any of SEQ ID NO: 16-28), or a fragment thereof.

[0046] In some embodiments, a fragment of a TCR protein is from 10 to 25 as in length. In some embodiments, a fragment of a TCR protein is from 10 to 50 aa in length. In some embodiments, a fragment of a TCR protein is from 10 to 100 aa in length. In some embodiments, a fragment of a TCR protein is from 10 to 150 aa in length. In some embodiments, a fragment of a TCR protein is from 25 to 50 as in length. In some embodiments, a fragment of a TCR protein is from 25 to 100 as in length. In some embodiments, a fragment of a TCR protein is from 25 to 150 as in length. In some embodiments, a fragment of a TCR protein is from 50 to 100 as in length. In some embodiments, a fragment of a TCR protein is from 50 to 150 as in length. In some embodiments, a fragment of a TCR protein is from 100 to 150 aa in length. In some embodiments, a fragment of a TCR protein is more than 150 aa in length.

[0047] In some embodiments, the MHCR comprises a peptide antigen. Any appropriate peptide antigen may be used. The peptide antigen in the pMHCR complex directs the specificity of the pMHCR molecule, therefore the pMHCR molecule will be specific for T cells with TCRs that are specific for that peptide antigen/pMHCR. A non-limiting example of a peptide antigen that may be used with the MHCR is moth cytochrome c peptide (as 88-103, ANERADLIAYLKQATK (SEQ ID NO: 29)). The peptide antigens used in the Examples (see below) are peptides commonly used as model antigens in mouse models. Any appropriate peptide antigen may be used, and the present invention is not limited to the peptide antigens disclosed herein. For example, in some embodiments, the peptide antigen comprises any immunodominant peptide antigen identified to bind a class I or class II MHC. In some embodiments, the peptide antigen comprises any immunodominant peptide antigen identified to bind a class I or class II MHC and elicit a response. A response may include but is not limited to an autoimmune response, an allergic response, an asthma response, or an inappropriate Treg response. The peptide antigen may be any appropriate length.

[0048] In some embodiments, the MHCR comprises at least a portion of a MHC molecule that allows for binding to an appropriate TCR. In some embodiments, the MHCR comprises at least a portion of a MHC molecule that allows for binding to an appropriate TCR and at least a portion of a TCR molecule (e.g., a portion of a TCR molecule that allows for appropriate signaling and/or complexing subunits such as CD3 subunits). In some embodiments, the MHCR comprises a transmembrane domain that is at least partially derived from (i) a MHC molecule, (ii) a TCR molecule, or (iii) both the MHC molecule and TCR molecule. In some embodiments, the MHCR comprises a transmembrane domain, wherein a portion (or all) of the transmembrane domain is not derived from a MHC molecule or a TCR molecule. In some embodiments, the MHCR comprises an extracellular domain that is at least partially derived from (i) a MHC molecule, (ii) a TCR molecule, or (iii) both the MHC molecule and TCR molecule. In some embodiments, the MHCR comprises an extracellular domain, wherein a portion of the extracellular domain is not derived from a MHC molecule or a TCR molecule.

[0049] As an example, in some embodiments, the MHCR comprises at least a portion of the extracellular domain of a MHC molecule (e.g., the extracellular domain of HLA-DRA) and at least a portion of the transmembrane domain of a TCR molecule and at least a portion of the cytoplasmic domain of a TCR molecule. As another example, in some embodiments, the MHCR comprises at least a portion of the extracellular domain of a TCR molecule.

[0050] The present invention also features redirected cells, such as redirected T cells, expressing MHCRs of the present invention, e.g., as described above. Without wishing to limit the present invention to any theory or mechanism, the MHCRs are generally adapted to recognize and bind to appropriate (specific) TCRs. In some embodiments, the MHCR is expressed in a CD8+ T cell (e.g., a cytotoxic T cell, T.sub.C cells, CTLs). In some embodiments, the MHCR is expressed in a CD4+ T cell (e.g., a T helper cell, T.sub.H cell or a regulatory T cell (Treg cell)). The present invention is not limited to the expression of MHCRs in T cells, nor is the present invention limited to expression of MHCRs in CD8+ or CD4+ T cells, e.g., the MHCRs may be expressed in CD8+/CD4+ thymocytes, ??T cells, NK cells, NK T cells, etc. In some embodiments, the MHCR of the redirected T cell complexes or is adapted to complex with CD3 subunits (e.g., forming a MHCR-CD3 complex).

[0051] In some embodiments, the MHCR comprises a MHC portion derived from an extracellular portion of a MHC protein and a TCR portion derived from a transmembrane domain of a TCR protein. In some embodiments, the MHC portion and TCR portion are directly linked. In some embodiments, the MHC portion and TCR portion are separated by a linker. In some embodiments, the linker comprises a glycine-rich linker.

[0052] The present invention is not limited to the MHC portions and TCR portions described herein. For example, the MHC portion may comprise any MHC peptide, e.g., an extracellular domain (or a portion thereof) of any MHC peptide. The TCR portion may comprise any TCR peptide, e.g., a transmembrane domain (or portion thereof) of any TCR peptide. Further, the present invention is not limited to antigens, signaling molecules, and cell surface receptor ligands described herein, e.g., the present invention may be applicable to a wide range of MHC molecules, TCR molecules, antigens, signaling molecules cell surface receptor ligands, etc.

Surrogate Coreceptors (SCRs)

[0053] The present invention also features chimeric surrogate coreceptors (SCR). e.g., receptors that recruit signaling molecules (e.g., kinases such as but not limited to Src kinases (e.g., Lck), phosphatases, etc.). In some embodiments, the SRCs recruit signaling molecules (e.g., kinases) to the MHCR and/or CD3 subunits. The present invention also features cells expressing a SCR. In some embodiments, redirected cells, e.g., redirected T cells, express both a MHCR and a SCR. In some embodiments, cells express more than one type of SCR. Without wishing to limit the present invention to any theory or mechanism, it is believed that certain SCRs may enhance signaling through the pMHCR-CD3 complex.

[0054] In some embodiments, the SCR comprises a cell surface receptor ligand (e.g., T cell surface receptor ligand) fused to a signaling molecule (e.g., kinase (e.g., Lck or other appropriate kinase), phosphatase, etc.). In some embodiments, the cell surface receptor ligand and the kinase are separated by a linker, e.g., a peptide linker or any other appropriate linker. The signaling molecule is not limited to a kinase or a phosphatase.

[0055] In some embodiments, the cell surface receptor ligand (e.g., T cell surface receptor ligand) comprises CD80, CD86, fragments thereof, or combinations thereof. The present invention is not limited to CD80 and CD86; any other appropriate cell surface receptor ligand (or a fragment thereof) may be used. For example, in some embodiments, the cell surface receptor ligand comprises a CD28 ligand, a CTLA-4 ligand, an ICOS ligand, an OX4O ligand, a PD-1 ligand (e.g., PD-1L), a CD2 ligand, etc.

[0056] As an example, in some embodiments, when a T cell is expressing a pMHCR (a MHCR with a peptide antigen), the pMHCR may complex with CD3 subunits, forming a pMHCR-CD3 complex. If the cell is also expressing a CD80-Lck SCR, then when the pMHCR binds a TCR on a target T cell, the CD80-Lck may also bind to CD28 on the same target T cell. Without wishing to limit the present invention to any theory or mechanism, it is believed that then the CD80-Lck SCR should recruit Lck to the pMHCR-CD3 complex to phosphorylate the pMHCR-CD3 ITAMs for robust signaling.

[0057] In some embodiments, the SCR is engineered (e.g., a particular cell surface receptor ligand of the SCR is selected) to target a specific set of target cells. For example, T follicular helper cells express a molecule called PD-1 and these cells provide help to B cells to make autoantibodies in autoimmune diseases such as Lupus. The ligand for PD-1 is PD-1L. so a SCR comprising PD-1L and Lck may be co-expressed with a pMHCR recognized by the TCR of the T follicular helper cell. This may allow for targeting of this specific T follicular helper cell population.

[0058] The present invention also features methods of use of said MHCRs, SCRs, and/or said redirected cells, for example for immunotherapy. In some embodiments, the redirected cells may eliminate autoreactive T cells, regulatory T cells (Tregs) that protect tumor cells by suppressing anti-tumor T cell responses, or any other appropriate T cell. For example, in some embodiments, the MHCR is an auto-antigen MHCR, and the MHCR's target is an autoreactive T cell.

EXAMPLES

Example 1: Redirected T Cells Targeting CD4 T Helper Cells

[0059] Example 1 describes a non-limiting experimental approach to target CD4 T cells. A prototype pMHCR was engineered with a peptide antigen: the moth cytochrome c peptide (SEQ ID NO: 29) was fused to the mouse class II MHC I-E.sub.k (MCC:I-E.sup.k; e.g., see SEQ ID NO: 31). This pMHCR was expressed (e.g., retrovirally expressed) in T cell hybridomas. It was determined that this pMHCR (e.g., pMHCR-CD3 complex) was expressed on the surface of T cell hybridomas (see FIG. 4). IL-2 production was induced after interactions with cognate TCRs (e.g., 5c.c7, 284), yet an irrelevant peptide (control peptide antigen) in the pMHCR-CD3 complex rendered it non-stimulatory (data not shown).

[0060] Lck fusions were generated with known ligands for T cell surface receptors. For example, all T cells express CD28. Lck fusions with CD28 ligands (e.g., CD80, CD86) were engineered to generate surrogate coreceptors (SCRs), e.g., CD80-Lck (see SEQ ID NO: 33, SEQ ID NO: 38), e.g., CD86-Lck (see SEQ ID NO: 34, SEQ ID NO: 39). When the pMHCR-CD3 complex was co-expressed with SCR CD80-Lck in hybridomas, these cells produced significantly more IL-2 in response to cells expressing the 2B4 TCR ligand+CD28 than they did in response to cells expressing only the 284 TCR ligand (see FIG. 5). This suggested that signaling through the pMHCR-CD3 complex could be augmented through the use of a SCR.

[0061] MCC:IE.sup.k pMHCR-CD3 and the SCR CD80-Lck or HB:IE.sup.k pMHCR-CD3 (e.g., see SEQ ID NO: 32) and the SCR CD80-Lck were expressed in in vitro differentiated CD8 cytotoxic T cells (CTLs) and their ability to kill 5c.c7 TCR transgenic CD4 T cells expressing the TCR specific for the MCC:IE.sup.k pMHCR was evaluated. Surface expression of the pMHCRs on the redirected CTLs was observed, suggesting that these chimeric receptor modules compete with the endogenous TCR for assembly with the endogenous CD3 subunits (data not shown). CTLs expressing the MCC:IE.sup.k pMHCR robustly killed the target CD4 T cells while those expressing the null HB:IE.sup.k pMHCR did not (see FIG. 6). This suggests that CD8 T cells can be redirected to target and eliminate antigen-specific CD4 T cells.

Example 2: Redirected T Cells Targeting CD4 T Helper Cells in Allergic Asthma

[0062] Example 2 describes a non-limiting experimental approach to target CD4 T helper cells involved in allergic asthma, e.g., to help eliminate na?ve Der p 1-specific CD4 T cells from the repertoire prior to House Dust Mite (HDM) sensitization. Without wishing to limit the present invention to any theory or mechanism, it is believed that eliminating allergen-specific CD4 T cells from the repertoire may help prevent the onset of T.sub.H2 immunity upon HDM sensitization.

[0063] A pMHCR (pMHCR-CD3 complex) will be retrovirally expressed in in vitro activated CTLs. The pMHCR will bear a pMHCR comprising either the immunodominant HDM-derived Der p 1 epitope (aa 17-127) in the context of I-A.sup.b (Derp1:IA.sup.b) or the immunodominant West Nile Virus peptide from the envelope protein (aa641-655) in the context of I-A.sup.b (E641:IA.sup.b). The E641:IA.sup.b pMHCR cells will serve as a non-specific control population.

[0064] The in vitro activated CTLs will also be transduced with a CD80-Lck SCR to enhance signaling. These redirected CTLs will then be transferred intravenously into C57Bl/6 mice to target and eliminate Derp1:IA.sup.b- or E641:IA.sup.b-specific na?ve CD4 T cells from the endogenous repertoire. After a certain length of time, e.g., 1 week, the elimination of antigen-specific CD4 T cells will be evaluated. This will be performed via tetramer enrichment experiments using a Derp1:IA.sup.b tetramer and a E641:IA.sup.b tetramer. The presence of the redirected CD8 T cells will also be assessed by flow cytometry by gating on CD3.sup.+CD8.sup.+IA.sup.b+ T cells since mouse T cells do not express class II MHC.

[0065] After determining if the redirected CTLs eliminate the target population, mice that received redirected CTLs one-week prior will be sensitized with HDM (e.g., intranasally, e.g., with HDM extracts). This will be done even if endogenous CD4 T cells specific for Derp1:IA.sup.b are detected, but only if redirected T cells are still present in the mice. This may help to determine if activation of the CD4 T cells made them more susceptible to targeting by the redirected CTLs.

Example 3: Redirected T Cells Targeting CD4 T Helper Cells in Lungs after Sensitization

[0066] Example 3 describes a non-limiting experimental approach to target CD4 T helper cells in lungs of HDM-sensitized mice. Without wishing to limit the present invention to any theory or mechanism, it is believed that eliminating allergen-specific CD4 T cells from the lungs of HDM-sensitized mice may help attenuate T.sub.H2 immunity.

[0067] Der p 1-specific CD4 T cells will be targeted similarly to Example 2, but only after HDM sensitization. In brief, mice will be sensitized with HDM according to the protocol described above. They will then receive redirected Derp1:IA.sup.b or E641:IA.sup.b pMHCR-CD3 CTLs on day 14. Various surrogate co-receptors will be employed to explore the efficacy of the technology and approach. For example, the CD80-Lck fusion SCR will be used, as well as others, e.g., a TIM-4-Lck SCR (since the TIM-1 expressed on CD4 T cells is genetically linked with asthma and this combination for targeting might enhance effectiveness). One week after transfer of redirected CTLs, cytokine and cellular analysis will be performed as described above in Example 2 so as to assess the impact of these cells on the lung cytokine milieu and cellularity. The status of the redirected CTLs will also be evaluated.

Example 4: Attenuation of Der p 1-Specific CD4 T Cell Function In Situ

[0068] Example 4 describes a non-limiting experimental approach to redirect Tregs against Der p 1-specific CD4 T cells. Without wishing to limit the present invention to any theory or mechanism, it is believed that this may help attenuate function of said CD4 T cells and help diminish T.sub.H2 immunity.

[0069] In vitro generated induced Tregs (iTregs) expressing a MHCR will be tested for efficacy in reducing HDM-induced airway hypersensitivity. Induced Tregs (iTregs) will be generated in vitro and transduced with pMHCR and SCRs as described in Examples 2 and 3 above. These cells will then either be transferred prior to HDM sensitization as in Example 2 or after sensitization as in Example 3. Evaluation of the lung cytokine milieu and cellularity will then be performed as described above.

[0070] Table 3 shows examples of protein sequences for reagents the above examples. Table 4 shows the nucleotide sequences for the proteins in Table 3. Note that in SEQ ID NO: 30, a portion is derived from SEQ ID NO: 14 and a portion is derived from SEQ ID NO: 22. In SEQ ID NO: 31, a portion is derived from SEQ ID NO: 15, a portion is derived from SEQ ID NO: 23, and a portion is derived from SEQ ID NO: 29 (and other residues may correspond to a glycine-rich linking region). In SEQ ID NO: 32, a portion is derived from SEQ ID NO: 15 and a portion is derived from SEQ ID NO: 23 (and other residues may correspond to a glycine-rich linking region).

TABLE-US-00005 TABLE3 PeptidesequencesforreagentsinExamples. SEQ IDNO. Description AminoAcidSequence 30 I-E.sup.k?-TCR? MATIGALLLRFFFIAVLMSSCIKSWAIKEEHTIIQ Note:underlined AEFYLLPDKRGEFMFDFDGDEIFHVDIEKSETIWR portionisfromSEQ LEEFAKFASFEAQGALANIAVDKANLDVMKERSNN IDNO:14(MHC TPDANVAPEVTVLSRSPVNLGEPNILICFIDKFSP portion),boldportion PVVNVTWFRNGRPVTEGVSETVFLPRDDHLFRKFH isfromSEQIDNO: YLTFLPSTDDFYDCEVDHWGLEEPLRKHWEFEEKT 22(TCRportion) LLPETKECDATLTEKSFETDMNLNFQNLSVMGLRI LLLKVAGFNLLMTLRLWSS 31 MCC:I-E.sup.k?-TCR? MVWLPRVPCVAAVILLLTVLSPPVALVRDSGSANE (note:italicportion RADLIAYLKQATKEFRSGGGGSLVPRGSGGGGSVD showspeptideantigen RPWFLEYCKSECHFYNGTORVRLLVRYFYNLEENL sequence,underlined RFDSDVGEFRAVTELGRPDAENWNSQPEFLEQKRA portionisfromSEQ EVDTVCRHNYEIFDNFLVPRRVEPTVTVYPTKTQP IDNO:15(MHC LEHHNLLVCSVSDFYPGNIEVRWFRNGKEEKTGIV portion),andbold STGLVRNGDWTFQTLVMLETVPQSGEVYTCQVEHP portionisfromSEQ SLTDPVTVEWKAQSTSAQNKCGITSASYHQGVLSA IDNO:24(TCRportion) TILYEILLGKATLYAVLVSGLVLMAMVKKKNSAAA 32 HB:I-E.sup.k?-TCR? MVWLPRVPCVAAVILLLTVLSPPVALVRDSGSGKK Note:italicportion VITAFNEGLKEFRSGGGGSLVPRGSGGGGSVDRPW showspeptideantigen FLEYCKSECHFYNGTQRVRLINRYFYNLEENLRFD sequence,underlined SDVGEFRAVTELGRPDAENWNSCPEFLEQKRAEVD portionisfromSEQID TVCRHNYEIFDNFLVPRRVEPTVTVYPTKTQPLEH NO:15(MHCportion), HNLLVCSVSDFYPGNIEVRWFRNGKEEKTGIVSTG andboldportionis LVRNGDWTFQTLVMLETVPQSGEVYTCQVEHPSLT fromSEQIDNO:24 DPVTVEWKAQSTSAQNKCGITSASYHQGVLSATIL (TCRportion) YEILLGKATLYAVLVSGLVLMAMVKKKNSAAA 33 CD80-Lck(mCD80- MACNCQLMQDTPLLKFPCPRLILLFVLLIRLSQVS mLckfusion) SDVDEQLSKSVKDKVLLPCRYNSPHEDESEDRIYW QKHDKVVLSVIAGKLKVWPEYKNRTLYDNTTYSLI ILGLVLSDRGTYSCVVQKKERGTYEVKHLALVKLS IKADFSTPNITESGNPSADTKRITCFASGGFPKPR FSWLENGRELPGINTTISQDPESELYTISSQLDFN TTRNHTIKCLIKYGDAHVSEDFTWEKPPEDPPDSK NTLVLFGAGFGAVITVVVIVVIIKCFCKHRSCFRR NEASRETNNSLTFGPEEALAEQTVFLTISHYPIVP LDSKISLPIRNGSEVRDPLVTYEGSLPPASPLQDN LVIALHSYEPSHDGDLGFEKGEQLRILEQSGEWWK AQSLTTGQEGFIPFNFVAKANSLEPEPWFFKNLSR KDAERQLLAPGNTHGSFLIRESESTAGSFSLSVRD FDQNQGEVVKHYKIRNLDNGGFYISPRITFPGLHD LVRHYTNASDGLCTKLSRPCQTQKPQKPWWEDEWE VPRETLKLVERLGAGQFGEVWMGYYNGHTKVAVKS LKQGSMSPDAFLAEANLMKQLQHPRLVRLYAVVTQ EPIYIITEYMENGSLVDFLKTPSGIKLNVNKLLDM AAQIAEGMAFIEEQNYIHRDLRAANILVSDTLSCK IADFGLARLIEDNEYTAREGAKFPIKWTAPEAINY GTFTIKSDVWSFGILLTEIVTHGRIPYPGMTNPEV IQNLERGYRMVRPDNCPEELYHLMMLCWKERPEDR PTFDYLRSVLDDFFTATEGQYQPQPGT 34 CD86-Lck(mCD86- MDPRCTMGLAILIFVTVLLISDAVSVETQAYFNGT mLckfusion) AYLPCPFTKAQNISLSELVVFWQDQQKLVLYEHYL GTEKLDSVNAKYLGRTSFDRNNWTLRLHNVQIKDM GSYDCFIQKKPPTGSIILQQTLTELSVIANFSEPE IKLAQNVTGNSGINLTCTSKQGHPKPKKMYFLITN STNEYGDNMQISQDNVTELFSISNSLSLSFPDGVW HMTVVCVLETESMKISSKPLNFTQEFPSPQTYWKE ITASVTVALLLVMLLIIVCHKKPNQPSRPSNTASK LERDSNADRETINLKELEPQIASAKPNAECTSHYP IVPLDSKISLPIRNGSEVRDPLVTYEGSLPPASPL QDNLVIALHSYEPSHDGDLGFEKGEQLRILEQSGE WWKAQSLTTGQEGFIPFNFVAKANSLEPEPWFFKN LSRKDAERQLLAPGNTHGSFLIRESESTAGSFSLS VRDFDQNQGEVVKHYKIRNLDNGGFYISPRITFPG LHDLVRHYTNASDGLCTKLSRPCQTQKPQKPWWED EWEVPRETLKLVERLGAGQFGEVWMGYYNGHTKVA VKSLKQGSMSPDAFLAEANLMKQLQHPRLVRLYAV VTQEPIYIITEYMENGSLVDFLKTPSGIKLNVNKL LDMAAQIAEGMAFIEEQNYIHRDLRAANILVSDTL SCKIADFGLARLIEDNEYTAREGAKFPIKWTAPEA INYGTFTIKSDVWSFGILLTEIVTHGRIPYPGMTN PEVIQNLERGYRMVRPDNCPEELYHLMMLCWKERP EDRPTFDYLRSVLDDFFTATEGQYQPQPGT

TABLE-US-00006 TABLE4 ExamplesofDNAsequences forencodingtheproteinsinTable3. SEQ IDNO. Description GeneSequence 35 I-E.sup.k?-TCR? aataagcttctcgagcgccaccATGGCCACAATTGGAGCCCTGCTGTT fusion AAGATTTTTCTTCATTGCTGTTCTGATGAGCTCCCAGAAGTCATGGGC TATCAAAGAGGAACACACCATCATCCAGGCGGAGTTCTATCTTTTACC AGACAAACGTGGAGAGTTTATGTTTGACTTTGACGGCGATGAGATTTT CCATGTAGACATTGAAAAGTCAGAGACCATCTGGAGACTTGAAGAATT TGCAAAGTTTGCCAGCTTTGAGGCTCAGGGTGCACTGGCTAATATAGC TGTGGACAAAGCTAACCTGGATGTCATGAAAGAGCGTTCCAACAACAC TCCAGATGCCAACGTGGCCCCAGAGGTGACTGTACTCTCCAGAAGCCC TGTGAACCTGGGAGAGCCCAACATCCTCATCTGTTTCATTGACAAGTT CTCCCCTCCAGTGGTCAATGTCACCTGGTTCCGGAATGGACGGCCTGT CACCGAAGGCGTGTCAGAGACAGTGTTTCTCCCGAGGGACGATCACCT CTTCCGCAAATTCCACTATCTGACCTTCCTGCCCTCCACAGATGATTT CTATGACTGTGAGGTGGATCACTGGGGTTTGGAGGAGCCTCTGCGGAA GCACTGGGAGTTTGAAGAGAAAACCCTCCTCCCAGAAACTAAAGAGtg tgatgtcacgttgaccgagaaaaGCTTTGAAACAGATATgaacctaaa ctttcaaaacctgtcaGTTATGGGACTCCGAATCCtcctgctgaaagt agcgggatttaacCTGCTCATGACGCTgaggctgtggtccagttgagg atccgcta 36 MCC:I-E.sup.k?- aatCTCGAGCGCCACCATGGTGTGGCTCCCCAGAGTTCCCTGTGTGGC TCR? fusion AGCTGTGATCCTGTTGCTGACAGTGCTGAGCCCTCCAGTGGCTTTGGT CAGAGACTCCGGATCCGCCAACGAGAGGGCCGACCTGATCGCCTACCT GAAGCAGGCCACCAAGGAATTCAGATCCGGAGGCGGAGGCTCCCTGGT GCCTCGGGGCTCCGGAGGCGGAGGCTCCGTCGACAGACCATGGTTTTT GGAATACTGTAAATCTGAGTGTCATTTCTACAACGGGACGCAGCGCGT GCGGCTTCTGGTAAGATACTTCTACAACCTGGAGGAGAACCTGCGCTT CGACAGCGACGTGGGCGAGTTCCGCGCGGTGACCGAGCTGGGGCGGCC AGACGCCGAGAACTGGAACAGCCAGCCGGAGTTCCTGGAGCAAAAGCG GGCCGAGGTGGACACGGTGTGCAGACACAACTATGAGATCTTCGATAA CTTCCTTGTGCCGCGGAGAGTTGAGCCTACGGTGACTGTGTACCCCAC AAAGACGCAGCCCCTGGAACACCACAACCTCCTGGTCTGCTCTGTGAG TGACTTCTACCCTGGCAACATTGAAGTCAGATGGTTCCGGAATGGCAA GGAGGAGAAAACAGGAATTGTGTCCACGGGCCTGGTCCGAAATGGAGA CTGGACCTTCCAGACACTGGTGATGCTGGAGACGGTTCCTCAGAGTGG AGAGGTTTACACCTGCCAGGTGGAGCATCCCAGCCTGACCGACCCTGT CACGGTCGAGTGGAAAGCACAGTCCACATCTGCACAGAACAAGtgtgg aatcactagtgcatcctatcatcagggggttctgtctgcaaccatcct atgagatcctactggggaaggccaccctatatgctgtgctggtcagtg gcctagtgctgatgGCCATGGTCAAGAAAAAAAATTCCgcggccgcat gatgagatctgagctccatagaggcg 37 HB:I-E.sup.k?- aatCTCGAGCGCCACCATGGTGTGGCTCCCCAGAGTTCCCTGTGTGGC TCR? fusion AGCTGTGATCCTGTTGCTGACAGTGCTGAGCCCTCCAGTGGCTTTGGT CAGAGACTCCGGATCCGGCAAGAAGGTGATCACCGCCTTCAACGAGGG CCTGAAGGAATTCAGATCCGGAGGCGGAGGCTCCCTGGTGCCTCGGGG CTCCGGAGGCGGAGGCTCCGTCGACAGACCATGGTTTTTGGAATACTG TAAATCTGAGTGTCATTTCTACAACGGGACGCAGCGCGTGCGGCTTCT GGTAAGATACTTCTACAACCTGGAGGAGAACCTGCGCTTCGACAGCGA CGTGGGCGAGTTCCGCGCGGTGACCGAGCTGGGGCGGCCAGACGCCGA GAACTGGAACAGCCAGCCGGAGTTCCTGGAGCAAAAGCGGGCCGAGGT GGACACGGTGTGCAGACACAACTATGAGATCTTCGATAACTTCCTTGT GCCGCGGAGAGTTGAGCCTACGGTGACTGTGTACCCCACAAAGACGCA GCCCCTGGAACACCACAACCTCCTGGTCTGCTCTGTGAGTGACTTCTA CCCTGGCAACATTGAAGTCAGATGGTTCCGGAATGGCAAGGAGGAGAA AACAGGAATTGTGTCCACGGGCCTGGTCCGAAATGGAGACTGGACCTT CCAGACACTGGTGATGCTGGAGACGGTTCCTCAGAGTGGAGAGGTTTA CACCTGCCAGGTGGAGCATCCCAGCCTGACCGACCCTGTCACGGTCGA GTGGAAAGCACAGTCCACATCTGCACAGAACAAGtgtggaatcactag tgcatcctatcatcagggggttctgtctgcaaccatcctctatgagat cctatggggaaggccaccctatatgctgtgctggtcagtggcctagtg ctgatgGCCATGGTCAAGAAAAAAAATTCCgcggccgcatgatgagat ctgagctccatagaggcg 38 CD80-Lck acgtctagatacctcgaggccaccATGGCTTGCAATTGTCAGttgatg (mCD80- caggatacaccactcctcaagtttccatgtccaaggctcattcttctc mLckfusion) tttgtgctgctgattcgtctttcacaagtgtcttcagatgttgatgaa caactgtccaagtcagtgaaagataaggtattgctgccttgccgttac aactctcctcatgaagatgagtctgaagaccgaatctactggcaaaaa catgacaaagtggtgctgtctgtcattgatgggaaactaaaagtgtgg cccgagtataagaaccggactttatatgacaacactacctactctctt atcatcctgggcctggtcctttcagaccggggcacatacagctgtgtc gttcaaaagaaggaaagaggaacgtatgaagttaaaaacttggcttta gtaaagttgtccatcaaagctgacttctctacccccaacataactgag tctggaaacccatctgcagacactaaaaggattacctgctttgcttcc gggggtttcccaaagcctcgcttctcttggtggaaaatggaagagaat tacctggcatcaatacgacaatttcccaggatcctgaatctgaattgt acaccattagtagccaactagatttcaatacgactcgcaaccacacca ttaagtgtctcattaaatatggagatgctcacgtgtcagaggacttca cctgggaaaaacccccagaagaccctcctgatagcaagaacacacttg tgctctttggggcaggattcggcgcagtaataacagtcgtcgtcatcg ttgtcatcatcaaatgcttctgtaagcacagaagctgtttcagaagaa atgaggcaagcagagaaacaaacaacagccttaccttcgggcctgaag aagcattagctGAACAGACCGTCTTCCTTaccactagtCACTATCCCA TAGTCccactggacagcaagatctcgctgcccatccggaattggctct gaagtgcgggacccactggtcacctatgagggatactcccaccagcat ccccgctgcaagacaacctggttatcgccctgcacagttatgagccct cccatgatggagacttgggctttgagaagggtgaacagctccgaatcc tggagcagagcggtgagtggtggaaggctcagtccctgacgactggcc aagaaggattcattcccttcaacttcgtggcgaaagcaaacagcctgg agcctgaaccttggttcttcaagaatctgagccgtaaggacgccgagc ggcagcttttggcgcccgggaacacgcatggatccttcctgatccggg aaagcgaaagcactgcggggtccttttccctgtcggtcagagacttcg accagaaccagggagaagtggtgaaacattacaagatccgtaacctag acaacggtggcttctacatctcccctcgtatcacttttcccggattgc acgatctagtccgccattacaccaacgcctctgatgggctgtgcacaa agttgagccgtccttgccagacccagaagccccagaaaccatggtggg aggacgaatgggaagttcccagggaaacactgaagttggtggagcggc tgggagctggccagttcggggaagtgtggatggggtactacaacggac acacgaaggtggcggtgaagagtctgaaacaagggagcatgtcacccg acgccttcctggctgaggctaacctcatgaagcagctgcagcacccgc ggctagtccggctttatgcagtggtcacccaggaacccatctacatca tcacggaatacatggagaacgggagcctagtagattttctcaagactc cctcgggcatcaagagaatgtcaacaaacttttggacatggcagccca gattgcagagggcatggcgttcatcgaagaacagaattacatccatcg ggacctgcgcgccgccaacatcaggtgtctgacacgctgagagcaaga ttgcagactttggcctggcgcgcctcattgaggacaatgagtacaccc ccgggagggggccaaatttcccattaagtggacagcaccagaagccat taactatggaccttcaccatcaagtcagacgtgtggtccttcgggatc ttgcttacagagatcgtcacccacggtcgaatcccttacccaggaatg accaaccctgaagtcattcagaacctggagagaggctaccgcatggtg agacctgacaactgtccggaagagctgtaccacctcatgatgctgtgc tggaaggagcgcccagaggaccggcccacgtttgactaccttcggagt gttctggatgacttcttcacagccacagagggcCAGTACCAGCCCCAG CCTggtacctagtgagaattctacatg 39 CD86-Lck tactctagatacctcgaggccaccATGGACCCCAGATGCACCatgggc (mCD86- ttggcaatccttatctttgtgacagtcttgctgatctcagatgctgtt mLckfusion) tccgtggagacgcaagcttatttcaatgggactgcatatctgccgtgc ccatttacaaaggctcaaaacataagcctgagtgagctggtagtattt tggcaggaccagcaaaagttggttctgtacgagcactatttgggcaca gagaaacttgatagtgtgaatgccaagtacctgggccgcacgagcttt gacaggaacaactggactctacgacttcacaatgttcagatcaaggac atgggctcgtatgattgttttatacaaaaaaagccacccacaggatca attatcctccaacagacattaacagaactgtcagtgatcgccaacttc agtgaacctgaaataaaactggctcagaatgtaacaggaaattctggc ataaatttgacctgcacgtctaagcaaggtcacccgaaacctaagaag atgtattttctgataactaattcaactaatgagtatggtgataacatg cagatatcacaagataatgtcacagaactgttcagtatctccaacagc ctctctctttcattcccggatggtgtgtggcatatgaccgttgtgtgt gttctggaaacggagtcaatgaagatttcctccaaaccttctcaattt cactcaagagtttccatctcctcaaacgtattggaaggagattacagc ttcagttactgtggccctcctccttgtgatgctgctcatcattgtatg tcacaagaagccgaatcagcctagcaggcccagcaacacagcctctaa gttagagcgggatagtaacgctgacagagagactatcaacctgaagga acttgaaccccaaattgcttcagcaaaaccaaatgcagagtgtactag tCACTATCCCATAGTCccactggacagcaagatctcgctgcccatccg gaatggctctgaagtgcgggacccactggtcacctatgagggatctct cccaccagcatccccgctgcaagacaacctggttatcgccctgcacag ttatgagccctcccatgatggagacttgggctttgagaagggagaaca gctccgaatcctggagcagagcggtgagtggtggaaggctcagtccct gacgactggccaagaaggcttcattcccttcsacttcgtggcgaaagc aaacagcctggagcctgaaccttggttcttcaagaatctgagccgtaa ggacgccgagcggcagcttttggcgcccgggaacacgcatggatcctt cctgatccgggaaagcgaaagcactgcggggtccttttccctgtcggt cagagacttcgaccagaaccagggagaagtggtgaaacattacaagat ccgtaacctagacaacggtggcttctacatctcccctcgtatcacttt tcccggattgcacgatctagtccgccattacaccaacgcctctgatgg gctgtgcacaaagttgagccgtccttgccagacccagaagccccagaa accatggtgggaggacgaatgggsagttcccagggaaacactgaagtt ggtggagcggctgggagctggccagttcggggaagtgtggatggggta ctacaacggacacacgaaggtggcggtgaagagtctgaaacaagggag catgtcccccgacgccttcctggctgaggctaacctcatgaagcagct gcagcacccgcggctagtccggctttatgcagtgggtcacccaggaac ccatctacatcatcacggaatacatggagaacgggagcctagtagatt ttctcaagactccctcgggcatcaagttgaatgtcaacaaacttttgg acatggcagcccagattgcagagggcatggcgttcatcgaagaacaga attacatccatcgggacctgcgcgccgccaacatcctggtgtctgaca cgctgagctgcaagattgcagactttggcctggcgcgcctcattgagg acaatgagtacacggcccgggagggggccaaatttcccattaagtgga cagcaccagaagccattaactatgggaccttcaccatcaagtcagacg tgtggtccttcgggatcttgcttacagagatcgtcacccacggtcgaa tcccttacccaggaatgaccaaccctgaagtcattcagaacctggaga gaggctaccgcatggtgagacctgacaactgtccggaagagctgtacc acctcatgatgctgtgctggaaggagcgcccagaggaccggcccacgt ttgactaccttcggagtgttctggatgacttcttcacagccacagagg gcCAGTACCAGCCCCAGCCTggtacctagtgagaattctacatg

[0071] The disclosures of the following U.S. Patents are incorporated in their entirety by reference herein: U.S. Pat. Application No. 20140219975; U.S. Pat. No. 8,450,112; U.S. Pat. No. 7,741,465; U.S. Pat. No. 6,319,494; CA 2209300; CA 2104957; EP 0574512; U.S. Pat. No. 6,407,221; U.S. Pat. No. 6,268,411; U.S. Pat. Application No. 20040258697; EP 1292621; EP 2659893; WO 2011101681; WO 2005054292; EP 1379670; U.S. Pat. No. 6,056,952; U.S. Pat. No. 6,410,319; U.S. Pat. No. 8,524,234; U.S. Pat. No. 7,871,817.

[0072] As used herein, the term about refers to plus or minus 10% of the referenced number.

[0073] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[0074] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims. In some embodiments, the figures presented in this patent application are drawn to scale, including the angles, ratios of dimensions, etc. In some embodiments, the figures are representative only and the claims are not limited by the dimensions of the figures. In some embodiments, descriptions of the inventions described herein using the phrase comprising includes embodiments that could be described as consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase consisting of is met.