METHODS OF USE IN IMMUNOTHERAPY

CROSS REFERENCE

ooeij This application claims priorit to U .S. Provisional Patent Application No, 62/186,865 fifed June 30, 2015, the specficatiori(s) of which is/are incorporated herein in their entirety by reference.

REFERENCE TO SEQUENCE LISTING

{0002] Appiicant asserts that the information recorded in the form of an Annex C/ST.25 text file submitted under Rule 13ter.1(a), entitled UNIA_15_0 _PCT_ST25.txt is identical to that forming part of the international application as filed. The content of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

|θβ@3{ The present invention reiaies to T cells and T cell receptors, more particularly to redirected T cells with engineered receptors, more particularly to redirected cells expressing a chimeric receptor comprising a major histocompatibility complex (MHC) molecule, including redirected cells further comprising a surrogate coreceptor, e.g., as components of a modular chimeric receptor system.

GOVERNMENT SUPPORT

fiMM>4] This invention was made with government support unde Grant Ho, R01 AI1G1Q53 awarded by iH. The government has certain rights in the invention.

BACKGROUND OF THE INVENTIO

|0M5| T cells normally recogniz and respond to peptide antigens embedded within major histocompatibility compiex molecules (p HCs) of antigen presenting celis (APCs) via their TCR-CD3 complex (see FIG. 1A). This eight-subunit TCR-CD3 complex is composed of the TCR, which is the receptor module that binds the pfV!HC, and the CD3ye, 003δε, and ΟΌ ζζ signaling modules that connec the TCR to the Intracellular signaling machinery (see FiG. 18). The intracellular domains of the CDS subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs) that are phosphorylated by the Src kinases, e.g., Lck, Fyn. CD3y, CD36, and CD3£ each contain one ITAfVI while CD3( contains three ITAMs for a total of ten in a single complex. The TCR-CD3 complex does not appear to have any intrinsic Src kinase activity. In fact, coreceptors (e.g., CD4, CD8) appear to sequester Lck away from the TC -CD3 complex until both a coreceptor and a TCR bind a pMHC, The Lck associated with the coreceptor is then brought into close proximity to the CDS ITAMs to phosphoryiate tyrosines within these motifs and initiate signaling.

|Θ0Ο6| Ectopic T ceil receptors (ICRs) have been introduced into T celis in an effort to reprogram or after T ceil specificity. However, in some cases, the introduction of ectopic ICRs has been found to lead to cross-pairing events with endogenous TCRs, resulting in novel TCRs with autoimmune specificities. This lead to the use of chimeric antigen receptors (CARs), which are typically designed with (a) an extracellular domain consisting of a single-chain variable fragment (scFv) of a monoclonal antibody directed against a target antigen; (b) a transmembrane domain that does not mediate interactions with other protein subunits; and (c) an intracellular domain consisting of the 003ζ intracellular signaling domain as well as signaling domains from a variety of other signaling molecules (e.g., CD28, CD27, ICG5, 4-1 BB, OX40). Without wishing to !imit the present invention to any theory or mechanism, it is believed that CARs do not sufficiently take advantage of the modularity of the existing signaling apparatus, which is optimized to direct T ceii activation and effector functions. CARs are likely to be delivering incomplete signals that could have unintended consequences or side effects.

|θθθ7) The present invention features novel chimeric receptors {e.g., " HCRs") comprising a portion of a MHC molecule (e.g., class I, class II, non-classical IvIHC) and a portion of the ICR. In some embodiments, the fVIHCR comprises a portion of an antigen peptide. The present invention also features ceils, such as T cells, expressing said Sv HCRs (cells expressing a MHCR are herein referred to as "redirected cells"). The MHCRs are adapted to recognize and bind to appropriate (specific) TCRs. Redirected cells (e.g., redirected T celis) expressing a IvlHCR would mimic antigen presenting eels (APCs), the cells that normally express MHC molecules. In some cases, binding of a TCR of a target T cell to the fV!HCR of the redirected cell may then result in destruction of the target T cell; thus, in this case, the redirected celis may function as "anti-T ceil" T cells. The present invention is not limited to redirected cells functioning to destroy a target. For example, in some embodiments, the redirected ceil is adapted to help reprogram a target cell, e.g., the redirected cei! may deliver instructions to the target cell. jOOOS] The present invention also features engineered cells expressing both an IV1HCR and an SCR. It was surprisingly discovered that engineered ceiis co-expressing an SV!HCR and an SCR had enhanced effects (e.g., increased SL~2 expression, see FIG. 5) as compared to engineered cells expressing a !V!HCR without co-expression of an SCR. Without wishing to limit the present invention to any theory or mechanism, it is believed that the use of an SCR in combination with a MHCR enhances signaling and/or other downstream effects. Without wishing to limit the present invention to any theory or mechanism, it is believed that the combination of the MHCR and SCR may provide a synergistic effect, e.g., effects of the combination of the MHCR and SCR may provide effects greater than those of the MHCR and SCR individually. 00 9j Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skii! in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

SUMMARY OF THE INVENTION

fee 10] The present invention features novel chimeric receptors for engineering redirected cells. For example, the present invention features an engineered ceil co- expressing on its surface a chimeric receptor (MHCR) comprising a major histocompatibility complex ( HC) portion (derived from a MHC protein) directly or indirectly fused to a T cell receptor (TCR) portion (derived from a TCR protein); and a surrogate co-receptor (SCR) comprising a celi surface receptor Iigand portion directly or indirectly fused to a signaling molecule portion, in some embodiments, 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 ceil. In some embodiments, binding of the MHCR to the TCR of the target cell and binding of the SCR to the ceil surface receptor of the target celi (i) initiates a signaling cascade effective for eliminating the target cell or (ii) instructs the target cell to differentiate to a specific effector function. In some embodiments, the cell (e.g., genetically engineered cell) is a T cell (e.g., CD4+ CD8+); however, the present invention is not limited to T cells. fee 11] In some embodiments, the TCR portion comprises a transmembrane domain of the TCR protein and the MHC portion comprises an extracellular domain of the MHC protein, in some embodiments, the TCR portion comprises at least a portion of a transmembrane domain of the TCR protein and the MHC portion comprises at least a portion of an extracellular domain of the MHC protein. In some embodiments, 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.

[mil] In some embodiments, the MHC portion of the MHCR is N-terminal to the ICR portion of the MHCR. In some embodiments, the MHC portion is directly fused to the TCR portion, !n some embodiments, the MHC portion is indirectly fused to the TCR portion via a linker. In some embodiments, the MHCR further comprises a peptide antigen integrated into the MHC portion, or directly or indirectly fused to the MHC portion. In some embodiments, the peptide antigen is linked to the MHC portion via a linker. In some embodiments, the linker comprises a glycine-rich peptide, in some embodiments, the SCR further comprises a transmembrane domain positioned in between the cell surface receptor Isgand portion and the signaling molecule portion. In some embodiments, the MHC protein, the TCR protein, or both the MHC protein and the TCR protein are mammalian proteins (e.g., human, mouse, cat, dog, etc. in some embodiments, the signaling molecule portion has kinase or phosphatase activity. In some embodiments, the signaling molecule portion comprises a Src kinase.

10013] In some embodiments, the WHC protein comprises 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-E8 beta, H2-EK alpha, H2-EK beta, a fragment thereof, or a combination thereof. In some embodiments, the MHC molecule comprises 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, 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. In some embodiments, the TCR molecule comprises TRAC, TRBC1 , TRBC2, TRDC, TRGC1 , TRGC2, TCRA, TCB1 , TCB2, TCC1 , TCC2, TCC3, TCC4, a fragment thereof, or a combination thereof, in some embodiments, 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. In some embodiments, the eel! surface receptor Iigand portion of the SCR comprises a CD28 iigand, a CTLA-4 Iigand, an ICOS Iigand, an 0X40 Iigand, a PD-1 iigand, or a CD2 iigand. in some embodiments, the CD28 Iigand comprises CD80, CD86, or both CD80 and CD88. in some embodiments, the MHCR is adapted to complex with a C03 subunit. In some embodiments, the engineered ceil further co-expresses a second SCR. f¾ei4] The present invention also features a chimeric receptor (MHCR) as described above. For example, the MHCR may comprise a major histocompatibility complex (MHC) portion derived from a HC protein directly or indirectiy fused to a T ceil receptor (TCR) portion derived from a ICR protein, wherein the MHCR is adapted to bind to a TOR of a target ceil.

ΪΘ015] The present invention also features a method of eliminating a target ceii or reprogramming a target ceil (the target cell comprising a TCR). in some embodiments, the method comprises introducing a genetically engineered cell that expresses on its surface a chimeric receptor (MHCR) according to the present invention to the target cell, wherein the MHCR is specific for the TCR of the target cell, wherein upon binding of the MHCR to the TCR the genetically engineered cell (a) initiates a signaling cascade that eliminates the target cell, or (b) instructs the target cell to differentiate to a specific effector function. In some embodiments, the method is for immunotherapy, in some embodiments, the target ceii is an autoreactive T ceil.

10816] The present invention also features vectors encoding MHCRs of the present invention. The present invention also features vectors encoding SCRs of the present invention, foe 17] Then present invention also features an engineered cell co-expressing on its surface a chimeric receptor (MHCR) comprising a major histocompatibility complex (MHC) portion derived from an extracellular domain of a mammalian MHC protein directly or indirectly linked to a transmembrane domain of a T cell receptor (TCR) portion derived from a mammalian TCR protein, wherein the MHC portion is N-termina! to the TCR portion; and a surrogate coreceptor (SCR) comprising a ceil surface receptor Itgand portion indirectly linked to a signaling molecule portion by a transmembrane domain, wherein the signaling molecule portion has kinase or phosphatase activity. The MHCR may be adapted to bind to a TOR of a target cell and the SCR may be adapted to bind to a ceii surface receptor of the target cell.

|00t8j The present invention also features an engineered T-ceil co-expressing on its surface: a chimeric receptor (MHCR) comprising a major histocompatibility complex { HC) portion derived from an extracellular domain of a mammalian MHC protein directly or indirectly linked to a transmembrane domain of a T cell receptor (TCR) portion derived from a mammalian TCR protein, the MHC portion being N-terroinal to the TCR portion, the MHC portion being selected from HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1 , HLA-DGA1 , HLA-DQB1 , HLA-ORA, HLA-DRB, H2-Aa, H2-B1 , H2-K1 , H2- EB beta, H2-EK alpha, and H2-EK beta, the TCR portion being selected from TRAC, TRBC1 , TRBC2, TRDC, TRGC1 , TRGC2, TCRA, TCB1 , TCB2, TCC1 , TCC2, TCC3, TCC4; and a surrogate coreceptor {SCR} comprising a cell surface receptor !igand portion indirectly linked to a signaiing molecule portion by a transmembrane domain, the signaling molecule portion having kinase or phosphatase activity. The MHCR may be adapted to bind to a TCR of a target ceil and the SCR may be adapted to bind to a ceil surface receptor of the target cell.

ΙΘΘ19) In some embodiments, the MHC molecule comprises at feast a portion of an extracellular domain of a MHC protein, in some embodiments, the TCR molecule comprises at least a portion of a cytoplasmic domain of a TCR protein, at least a portion of a transmembrane domain of a TCR protein, at least a portion of an extracellular domain of a TCR protein, or a combination thereof, in some embodiments, the chimeric receptor is adapted to bind to a TCR. in some embodiments, the chimeric receptor is adapted to complex with at least one CD3 subunit.

ΪΘ020] The present invention also features a surrogate co-receptor (SCR) comprising a cell surface receptor Sigand portion directly or indirectly fused to a signaling molecule portion via a transmembrane domain, wherein the SCR is adapted to bind to a ceil surface receptor of a target ceii. In some embodiments, the cell surface receptor ligand portion is indirectly fused to the signaling molecule portion via a linker.

}0i2l| The present invention also features genetically engineered cells (e.g., redirected ceils) that express on their surfaces a chimeric receptor according to the present invention. In some embodiments, the ceil is a T ceii (e.g., CD8+ T ceil, CD4+ T cell, etc.). In some embodiments, the ceii co-expresses one or more SCRs according to the present invention. In some embodiments, the chimeric receptor is complexed with at least one CD3 subunit.

ΪΘ022] The present invention also features method of eliminating a target celi or reprogramming a target celi (said target ceii comprising a ICR), in some embodiments, the method comprises introducing a genetically engineered celi that expresses on its surface a chimeric receptor to the target celi, wherein the chimeric receptor is specific for the ICR of the target ceil, in some embodiments, binding of the chimeric receptor on the genetically engineered ceii to the TCR of the target cei! initiates a signaling cascade that eliminates the target cell, in some embodiments, binding of the chimeric receptor of the genetically engineered ceil to the TCR of the target celi instructs the target ceil to differentiate to a specific effector function (e.g. Th1 , Tft2, Th17, Tfh _f Treg or cytotoxic T ceil), in some embodiments, the chimeric receptor (e.g., IvIHCR) is expressed on a Treg and binding of the chimeric receptor to the TCR of a target celi inhibits the target cell's function (e.g., redirect the Treg function against an autoimmune cell), in some embodiments, the geneticaiiy engineered ceil co-expresses a SCR, In some embodiments, the SCR comprises a ceii surface receptor ligand specific for a cell surface receptor on the target cell. In some embodiments, binding of the chimeric receptor to the TCR and binding of the ceii surface receptor ligand of the SCR to the cell surface receptor of the target ceii initiates a signaling cascade that eliminates the target cell, or instructs the target celi to differentiate to a specific effector function. f0 23| in some embodiments, the method Is for immunotherapy, in some embodiments, the genetically engineered ceii is surgically introduced to a host (e.g., a mammal), in some embodiments, the target ceil is an autoreactive T ceii.

ΙΘΘ24) The present invention aiso features nucieotide sequences encoding the chimeric receptors of the present invention. The present invention aiso features vectors encoding the chimeric receptors of the present invention. The present invention also features nucleotide sequences encoding the SCRs of the present invention. The present invention also features vectors encoding the SCRs of the present invention. jO0 5 Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

|0 26] 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:

ΪΘ027] FIG. 1 A shows molecules involved in T cell activation. Engagement of the TCR with pfvlHC ( HC with a peptide antigen) initiates T ceil activation.

ΙΘΘ28] FIG, 1 B shows the molecular components of the Oipha-beta-TCR-CDS complex.

The TCR transfers pMHC-specific information to the CDS subunits and inside the T ceil.

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 a!., 2006, Immunit 24:133-139).

|0029J FiG. 2A shows a redirected T celi expressing a MHCR (pMHCR with peptide antigen) of the present invention. The MHCR in complex with CDS subunits is bound to a target T cell's TCR.

| 30| 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- terminaS to the TCR portion (see orientation of sequences below).

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

10032] FIG. 38 shows a redirected T cell expressing a MHCR (pMHCR) and two surrogate coreceptors (SCRs). The MHCR, bound to a target T cell's ICR, is oompiexed 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 ceil may help initiate CD3 signaling similar io that seen in norma! T eel! activation.

|0d33] FIG. 4 shows expression of pMHCR-CD3 complexes on T cell fiybridomas. 58α ^{" "} cells that lack endogenous TCRs were transduced with a pMHCR composed of MCG:I- E ^k . The proportional expression (diagonal) of !-E ^k and CD3 subunits suggests surface co-dependent expression of the epitopes.

10034] FIG. 5 shows TCR~specific IL-2 production by pMHGR~CD3 expressing T ce!! hyfandomas. 58α ^" β ^' cells that lack endogenous TCRs were transduced with a pMHC composed of CC.i-E as well as a CD80~Lck surrogate coreceptor (SCR). The ceils were co-cultured with parental 12 B cells, or M12 cells stably transduced to express the MCC:!-E ^k -specif!c 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.

lOft j FIG, 6 shows TCR-specific killing of CD4 T ceils by redirected CTLs. Purified CD8 T cells from B10.A mice were activated in vitro and transduced with a MCC:l~E ^k pMHCR (agonist) or an HB:I-E* pMHCR (null) as well as a CD80-Lck surrogate coreceptor. The redirected CTLs were then co-cu!tured at the indicated ratios with naive 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 (MHGRs).

«36] The present invention features chimeric receptors (e.g., "MHGRs") comprising at least a MHC portion (e.g., class I, class II, non-classical, a combination thereof, etc.) and a TCR portion (e.g., αβ, γδ TCR, etc.) (see FIG. 28(i)). For example, the HCR may comprise a MHC portion and a TC portion, a MHC and a TCR portion optionally separated by a linker (see FIG. 2B (iii) and (iv)). A linker may be any appropriate Sinker 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, 28. 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 am antigen portion, wherein the TCR portion and MHC portion are separated by a Sinker; 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 Sinker and the MHC portion and the antigen portion are separate by a linker; etc.

|0#37j The MHC portion may comprise one or more MHC proteins {e.g., HLA«A, HLA-8, HLA-C, HLA-DPA1 , H.LA-DP81, HLA-DQA1 , HLA-DQ81 , HLA-DRA, HLA-DRBi), one or more fragments thereof, o combinations thereof. For reference, non-Nmiting MHC sequences (human, mouse) are listed be!ow in Tabie 1.1 and Table 1.2. Note that MHC genes are highiy 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. ίθ038] Table 1,1. Examples of Human MHC Protein Sequences

SEQ ID NO, Description Amino Acid Sequence

1 Uniprot P01891 MAV APRTLV LLLSGALAIT QTWAGSHSMR

HLA-A gene YFYTSVSRPG RGEPRFIAVG YVDDTQFVRF

(MHC !} DSDAASQRME PRAPWIEQEG PEYWDR TRN

VKAQSQTDRV DLGTLRGYY QSEAGSHTIQ MMYGCDVGSD GRFLRGYRQD AYDGKDYIAL KEDLRSWTAA OMAAQTTKHK WEAAHVAEQW RAYLEGTCVE WLRRYLENGK ETLQRTDAPK THMTHHAVSD HEATLRCWAL SFYPAEITLT WQRDGEDQTQ DTELVETRPA GDGTFQKWVA VVVPSGQEGR YTCHVQHEGL PKPLTLRWEP SSQPTIPiVG iSAGLVLFGA ViTGAWAAV

WRRKSSDRK GGSYSQAASS DSAQGSDVSL TACKV

2 Uniprot P18464 MRVTAPRTVL LLLWGAVALT ETWAGSHSMR

HLA-B gene YFYTA SRPG RGEPRFIAVG YVDDTQFVRF

(MHC I) DSDAASPRTE PRAPWIEQEG PEYWDRNTQS

FKTNTQTYRE NLRIALRYYN GSEAGSHTWG TMYGCDVGPD GRLLRGHNQY AYDGKDYIAL

NEDLSS TAA DTAAQITQRK WEAAREAEQL

RAYLEGLCVE WLRRHLENGK ETLGRADPPK THVTHHPVSD HEATLRCWAL GFYPAE!TLT

WQRDGEDQTQ DTELVETRPA GDRTFQKWAA WVPSGEEQR YTCHVQHEGL PKPLTLRWEP SSQSTIPfVG IVAGLAVLAV WIGAWATV

MCRRKSSGGK GGSYSQAASS DSAQGSDVSL TA

Uniproi Q29963 MRV APRTLi LLLSGALALT ETWACSHSWR

HLA-C gene YFDTAVSRPG RGEPRF!SVG YVDDTQFVRF

( HC S) DSDAASPRGE PRAPWVEGEG PEYWDRETQ

YKRQAQADRV NLRKLR6YYN QSEDGSHTLQ

WMYGCDLGPD GRLLRGYDQS AYDGKDYIAL NEDLRSWTAA DTAAQITQRK WEAAREAEQW RAYLEGTCVE WLRRYLENGK ETLQRAEHP THVTHHPVSD HEATLRCWAL GFYPAEiTLT

WQRDGEDQTQ DTELVETRPA GDGTFQKWAA WVPSGEEQR YTCHVQHEGL PEPLTLRWEP SSQPTIP!VG IVAGLAVLAV LAVLGAVMAV

V CRRKSSGG KGGSCSQAAS SNSAQGSDES UACKA

Uniproi P20036 MRPEDR FHI RAV!LRALSL AFLLSLRGAG

HLA DPA1 AiKADHVSTY AAFVQTHRPT GEF FEFDED

(M C !l) EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG

LANIAILNN LNTLIQRS H TGATNDPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHW6 LDQPLLKHWE

AQEPIQMPET TETVLCALGL VLGLVGiiVG TVUIKSLRS GHDPRAQGTL

Uniproi P04440 MMVLQVSAAP RTVALTALLM VLLTSVVQGR

HLA DP81 ATPENYLFQG RQECYAF GT QRFLERYIYN

(MHC II) REEFARFDSD VGEFRAVTEL GRPAAEYWNS

QKDiLEEKRA VPDRMCRHNY ELGGPWTLGR RVQPRVNVSP SKKGPLQHHN LLVCHVTDFY

PGS!QVRWFL NGGEETAGVV STNLIRNGDW

TFQiLV LE TPQGGDVYTC QVEHTSLDSP

VTVEWKAQSD SARSKTLTGA GGFVLGLiiC

GVGIFyHRRS KKVQRGSA

Uniprot PQ1909 MILNKALMLG ALALTTVMSP CGGED!VADH

HLA DGAi VASYGVNLYQ SYGPSGGYTH EFDGDEQFYV

(MHC SI) DLGRKETVWC LPVLRQFRFD PQFALTNIAV

LKHNLNSLIK RSNSTAAT E VPEVTVFSKS PVTLGQPNIL iCLVDNIFPP WfSilTWLSNG

HSVTEGVSET SFLSKSDHSF FKiSYLTLLP

SAEESYDCKV EHWGLDKPLL KHWEPEiPAP

MSELTETVVC ALGLSVGLVG iWGTVFHR

GLRSVGASRH QGPL

Uniproi P01 2G MSWKKALRIP GGLRAATVTL MLAMLSTPVA

HLA DQB1 EGRDSPEDFV YQFKAMGYFT NGTERVRYVT

( HC IS) RYiYNREEYA RFDSDVEVYR AVTPLGPPDA

EYWNSQKEVL ERTRAELDTV CRHNYQLELR TTLQRRVEPT VTSSPSRTEA LNHHNLLVGS VTDFYPAQIK VRWFRNDQEE TTGWSTPLi

RNGDWTFQIL V LE TPQHG DVYTCHVEHP SLQNPITVEW RAQSESAQSK MLSGiGGFVL

GLIFLGLGLI iHHRSQKGLL H

Uniprot P01903 MAiSGVPVLG FF!!AVLMSA QESWAiKEEH VHQAEFYLN HLA DRA gene PDQSGEFMFD FDGDEFHVD MAKKETVWRL

(MHC Si) EEFGRFASFE AQGALANIAV DKANLEIMTK

RSNYTPITNV PPEVTVLTNS PVELREPNVL ICFIDKFTPP WNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLP STEDVYDCRV EHWGLDEPLL KHWEFDAPSP LPETTENWC ALGLTVGLVG liiGTFiiK GVRKSNAAER RGPL

Uniprot Q30187 VCLRLPGGS C AVLTVTLM VLSSPLALAG

HLA DRB1 gene DTRPRFLEEV KFEGHFFNGT ERVRLLERRV (MHC SI) HNQEEYARYD SDVGEYRAVT ELGRPDAEYVV

NSQKDLLERR RAAVDTYCRH NYGVGESFFV

QRRVQPKVTV YPSKTQPLQH HNLLVCSVNG

FYPGSiEVRW FRNGQEEKTG VVSTGLIQNG

DWTFQTLVML ETVPQSGEVY TCQVEHPSVM

SPLTVEWRAR SESAQSKMLS GVGGFVLGLL

FLGAGLFIYF RNQKGHSGLP PTGFLS ίθ039] Tabie 1 ,2, Examples of Mouse MHO Protein Sequences

MHC ! H2-K gene RRSGSHRAPP PGPHSLSQAD NPRFEPRAP (Hap!otype d) (H2- fvlEGEGPEYWE EGTGRAKSDE QVVFRVSLRTA K1) GRYYNGSKGG SHTFQRMFGC DVGSDWRLLR

GYQQFAYDGR DYIALNEDLK TWTAADTAAL STRRKWEQAG DAEYYRAYLE GECVEVVLRRY LELGNETLLR TDSPKAHVTY HPRSQVDVTL RCWALGFYPA DITLTVVQLNG EDLTQDMELV ETRPAGDGTF Q WAAWVPL GKEQNYTCHV HHKGLPEPLT LRWKLPPPTV SNTVIIAVLV VLGAAiVTGA WAFVMK RR NTGGKGVNYA LAPGSQTSDL SLPDGKVMVH

Uniprot P04230 VWLPRVPCV AAVILLLTVL SPP ALVRDS H2 Class IS RPWFLEYC S ECHFYNGTQR VRLLERYFYN histocompatibility LEENLRFDSD VGEFHAVTEL GRPDAENWNS antigen E-8 beta QPEFLEGKRA EVDTVCRHNY ESSDKFLVRR chain RVEPTVTVYP TKTQPLEHHN LLVCSVSDFY

PG IEVRWFR NGKEEKTGIV STGLVRNGDW TFQTLV tET VPGSGEVYTC GVEHPSLTDP VTVEWKAGST SAQN IVILSGV GGFVLGLLFL GAGLFSYFR Q GQSGLQPT GLLS

Uniprot P04224 WATIGALVLR FFFIAVLMSS G SWASKEEH MHC li E-K alpha TIIQAEFYLL PD RGEFMFD FDGDEIFHVD chain lEKSETSWRL EEFAKFASFE AGGALANIAV

(underlined portion is DKANLDVMKE RSNNTPDANV APEVTVLSRS portion used in SEQ PVNLGEPNIL iCFIDKFSPP VV VTWLRNG ID NO: 30) RPVTEGVSET VFLPRDDHLF R FHYLTFLP

STDDFYDCEV DHWGLEEPLR KHWEFEEKTL

LPET ENVVC ALGLFVGLVG IVVGI!LIMK

GIK RNVVER RQGAL

GenBank iD: IViWLPRVPCVAAVILLLTVLSPPVALVRDSRPWFLE M36939.1 YCKSECHFYNGTQRVRLLVRYFY LEE LRFDSD MHC II E-K beta VGEFRAVTELGRPDAENWNSGPEFLEGKRAEVD chain TVCRHNYEIFD FLVPRRVEPTVTVYPT TQPLEH (underlined portion is HNLLVCSVSDFYPGNiEVRWF G EEKTGlVSTG used in SEQ !D NO: LVRNGDWTFQTLVMLETVPGSGE TCQVEHPSL 31 , 32) TDPVnVEWKAQSTSAQNKMLSGVGGFVLGLLFLG

AGLFtYFRNQKGQSGLQPTGLLS f0040] Referring to Tab!e 1.1 , the HLA-A (M C 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 beiieved 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 H) 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 beiieved to make up the transmembrane region, and amino acids 248-260 are believed to make up the cytoplasmic region. The HLA DPB1 (MHC IS) 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 228-248 are beiieved to make up the transmembrane region, and amino adds 247-258 are beiieved to make u the cytoplasmic region. The HLA DGA1 (MHC li) sequence (SEQ ID NO: 8) includes the signal peptide (amino acids 1-23); amino acids 24-216 are beiieved 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 beiieved to make up the extracellular region, amino acids 231-251 are believed to make up the transmembrane region, and amino acids 252-281 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 beiieved 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 u the transmembrane region, and amino acids 251-268 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 26-2 8), the transmembrane domain (aa 217-24), and a cytoplasmic portion (aa 243-255). *4i] As previously discussed, the MHCR of the present invention comprises at ieast a MHC portion and a TCR portion, in some embodiments, a MHC portion comprises one or more MHC proteins (e.g., HLA-A, HLA-8, HLA-C, HLA-DPA1, HLA-DPB1 , HLA- DQA 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.

|0f 2| In some embodiments, the MHC portion comprises a pepiide 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 Ieast 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 pepiide that is at least 99% identical to a MHC protein or a fragment thereof.

|0f 3| 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 aa in length, in some embodiments, a fragment of a MHC protein is from 10 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 10 to 300 aa in length, !n some embodiments, a fragment of a MHC protein is from 10 to 350 aa in length, in some embodiments, a fragment of a MHC protein is from 25 to 50 aa In length. In some embodiments, a fragment of a MHC protein is from 25 to 100 aa in !ength. in some embed merits a fragment of a iVIHC protein i from 25 to 150 aa in length. in some embod merits a fragment of a MHC protein is from 25 to 200 aa in iength. in some embod ments a fragment of a WHO protein is from 25 to 250 aa in iength. in some embod menfs a fragment of a MHC protein is from 25 to 300 aa in iength. In some embod ments a fragment of a MHC protein is from 25 to 350 aa in iength. in some embod ments a fragment of a MHC protein is from 50 to 100 aa in iength. in some embod ments a fragment of a MHC protein is from 50 to 150 aa in length. in some embod ments a fragment of a MHC protein is from 50 to 200 aa in Iength. in some embod ments a fragment of a MHC protein is from 50 to 250 aa in length. in some embod ments a fragment of a MHC protein is from 50 to 300 aa in iength. In some embod ments a fragment of a MHC protein is from 50 to 350 aa in iength. in some embod ments a fragment of a MHC prote n is from 100 to 150 aa in iength, in some embod ments a fragment of a MHC prote in is from 100 to 200 aa in length, in some embod ments a fragment of a MHC prote n is from 100 to 250 aa in iength, in some embod ment a fragment of a MHC prote in is from 100 to 300 aa in iength. in some embod ments a fragment of a MHC prote rt is from 100 to 350 aa in iength. in some embod ments a fragment of a MHC prote n is from 150 to 200 aa in Iength. in some embod ments a fragment of a MHC prote n is from 150 to 250 aa in iength, in som embod ments a fragment of a MHC prote n is from 150 to 300 aa in length, In some embod ments, a fragment of a MHC prote n is from 150 to 350 aa in iength. In some embod ments, a fragment of a MHC prote n is from 200 to 250 aa in iength, in some embod ments, a fragment of a iVIHC prote in is from 200 to 300 aa in length, in some embod ments, a fragment of a MHC prote o is from 200 to 350 aa in iength. in some embod ments, a fragment of a MHC prote in is from 250 to 300 aa in iength. in some embod ments, a fragment of a MHC prote n is from 250 to 350 aa in iength. in sortie embod ments, a fragment of a !HC protein is more than 350 aa in iength.

10044] A TCR portion may comprise one or more TCR proteins (e.g., TC A, TCRB), one or more fragments thereof, or combinations thereof. For reference, non-iimiting ICR sequences (human and mouse) are listed beiow in Table 2.1 and Tabie 2,2, The present invention is not limited to the TCR sequences in Tabie 2.1 and Tabie 2.2. f0&45] Tabie 2.1. Examples of Human TCR Protein Sequences SEQ

ID MO. Description Amino Acid Sequence

18 Uniprot P01848 PNIQNPDPAV YQLRDSKSSD KSVCLFTDFD

T ceii receptor SQTNVSQSKD SDVYiTD TV LDMRSMDFKS alpha chain NSAVAWSNKS DFACANAFNN SiiPEDTFFP constant region SPESSCDVKL VEKSFETDT LNFQNLSViG (TRAC, TCRA) FRiLLLKVAG FNLL TLRLW SS

17 Uniprot P01850 EDLNKVFPPE VAVFEPSEAE iSHTQKATLV

T ceii receptor CLATGFFPDH VELSWWV GK EVHSGVSTDP beta-1 chain QPLKEQPALN DSRYCLSSRL RVSATFWQNP constant region RNHFRCQVQF YGLSENDEWT QDRAKPVTQ) (TRBC1 ) VSAEAWGRAD CGFTSVSYQQ G VIS AT! LYE

ILLGKATLYA VLVSALVLMA MVKRKDF

18 Uniprot A0A5B9 DLKNVFPPEV AVFEPSEAEI SHTQKATLVC

T ceii receptor LATGFYPDHV ELSW VNGKE

beta-2 chain VHSGVSTDPQ PLKEQPALND SRYCLSSRLR constant region VSATFWQNPR HFRCQVQFY

(TRBC2, GLSENDEWTQ DRAKPVTQIV SAEAWGRADC TCRBC2) GFTSESYQQG VLSAT!LYEi

LLGKATLYAV LVSALVLMAM V RKDSRG

19 Uniprot B7Z8 6 SQPHTKPSVF V KNGTNVAC LVKEFYPKDi

T ceii receptor RiNLVSSKKI TEFDPAiVIS PSGKYNAVKL delta chain GKYEDSNSVT CSVQHDNKTV HSTDFEVKTD constant region STDHVKPKET ENTKQPSKSC HKPKAiVHTE (TRDC) KVN SLTVL GLR LFAKTV AVNFLLTAKL FFL 0 Uniprot P0CF51 DKQLDADVSP KPTiFLPSfA ETKLQKAGTY

T ceil receptor LCLLEKFFPD VIKIHWQEKK SNTILGSQEG gamma-1 chain NT KTNDTYM KFSWLTVPEK SLDKEHRCIV constant region RHENNKNGVD QEilFPPIKT DVITMDPKDN (TRGC1 ) CSKDANDTLL LQLTNTSAYY MYLLLLLKSV

VYFAIiTCCL LRRTAFCCNG EKS

1 Uniprot P03986 DKQLDADVSP KPTiFLPSf ETKLQKAGTY

T ceii receptor LCLLEKFFPD IIKiHWGEKK SNTiLGSQEG gamma-2 chain NT KTNDTYM KFSWLTVPEE SLDKEHRCiV constant region RHENNKNGID QEIiFPPIKT DVTTVDPKDS

(TRGC2, YSKDANDViT MDPKDNWSKD A DTLLLQLT

TCRGC2) NTSAYYMYLL LLLKSWYFA tiTCCLLGRT

AFCCNGEKS

| ( 6j Table 2.2. Examples of Mouse TC Protein Sequ

DVLQFQFTST SAYYTYLLLL L SViYLAif

SFSLLRRTSV CGNEKKS

28 Uniprot P0398S DKKLDADI8P KPTiFLPSVA ETNLH TGTY

T celi receptor gamma LCVLEKFFPD VIRVYWKEKK GNTILDSQEG chain constant region DMLKTNDTY KFSWLTVPER S GKEHRCtV C7.S (TCC2-mouse) KHENNKGGAD QEiFFPTIKK VAVSTKPTTC

WQDKNDVLQL QFT!TSAYYT YLLLLLKSVI

YLAHSFSLL RRTSVCCNEK KS

27 Uniprot P06334 PSDKRLDADS SPKPTIFLPS VAETNLHKTG

T ceii receptor gamma TYLCiLE FF PDViRVYWKD KNGNT!LDSQ chain constant region EGDTLKTKGT Y KFSWLTVP ERSMGKEHRC DFL12 (TCC3-mouse) IVKHENNKGG ADQEFFPSi KKVATTCWQD

KNDVLQLQF STSAYYTYLL LLLKSVIYLA

iiSFSLLRRT SVCC EKRS

28 Uniprot P06335 DKRTDSDFSP KPTIFLPSAA ETNLHKAGTY

T ceil receptor gamma LCLLEKFFPK ViRVYWKEKD GE iLESQEG chain constant region NTIKTNDRY KFSWLTVTED S AKEHSCIV 5/10-13 (TCC4-mouse) KHENNKRGVD QEILFPPIGK AFTTINVNPR

DSVLRHENVN MATDLEDCMK GRKD LQLQV TTTYAFYTYL ILFFKSMVHL AFVVFCLFRR AA SCDDQRS ίθΦ 7| Referring to the TRAC protein (SEQ !D NO: 18) in Table 2, amino acids 1 18-137 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 !D NO: 17) in Table 2, amino acids 151 -171 are beiieved 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 Tabie 2, amino acids 130-152 are beiieved to make up the transmembrane domain. Referring to the TRGC1 protein (SEQ ID NO; 20) in Table 2, amino acids 139-161 are beiieved to make up the transmembrane domain. Referring to the TRGC2 protein (SEQ !D NO: 21 ) in Tabie 2, amino acids 157-177 are beiieved to make up the transmembrane domatn, and amino acids 178-189 are beiieved to make up the cytoplasmic domain.

J0i4s;| As previously discussed, the MHC of the present invention comprises at ieast a MHC portion and a ICR 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: 18-28. (in some embodiments, the fragment is from 5 to 10 aa in iength. In some embodiments, the fragment is from 10 to 20 aa in Iength, in some embodiments, the fragment is from 10 to 30 aa in iength. IN some embodiments, the fragment is from 10 to 40 aa in Iength. In some embodiments, the fragment is from 10 to 50 aa in length, etc.

I004.9] In some embodiments, the TCR portion comprises a peptide that is at least 80% identical to a TCR protein (e.g., any of SEQ SO NO: 16-28}, or a fragment thereof. In some embodiments, the TCR portion comprises a peptide that is at ieast 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 Ieast 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 ieast 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 ieast 99% identical to a TCR protein (e.g., any of SEQ ID HO: 16-28), or a fragment thereof.

{ΘΘ50] in some embodiments, a fragment of a TCR protein is from 10 to 25 aa in length. In some embodiments, a fragment of a TCR protein is from 10 to 50 aa in Iength. in some embodiments, a fragment of a TCR protein is from 10 to 100 aa in Iength. 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 aa in Iength. In some embodiments, a fragment of a TCR protein is from 25 to 100 aa in Iength. In some embodiments, a fragment of a TCR protein is from 25 to 150 aa in length, in some embodiments, a fragment of a TCR protein is from 50 to 100 aa in length. In some embodiments, a fragment of a TCR protein is from 50 to 150 aa in iength. In some embodiments, a fragment of a TCR protein is from 100 to 150 aa in length. In some embodiments, a fragment of a TC protein is more than 150 aa in iength. }Θ051| 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 ceils with TCRs that are specific for that peptide antigen/p HCR. A non-limiting example of a peptide antigen that may be used with the MHCR is moth cytochrome c peptide {aa 88-103, ANERADLIAYLKQATK {SEQ ID NO: 29)). The peptide antigens used in the Examples (see below) are peptides commonly used as model antigens in mous 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 IS 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.

Ji)i52] in some embodiments, the MHCR comprises at ieast a portion of a MHC molecule that aliows 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 moiecu!e that allows for appropriate signaling and/or compiexing subunits such as CD3 subunits). In some embodiments, the MHCR comprises a transmembrane domain that is at Ieast 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 ail) of the transmembrane domain is not derived from a MHC molecule or a TCR mo!ecu!e. In some embodiments, the MHCR comprises an extracellular domain that is at Ieast 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.

J0053J As an example, in some embodiments, the MHCR comprises at least a portion of the extraceliular 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 moiecuie. As another example, in some embodiments, the MHCR comprises at least a portion of the extracellular domain of a TCR moiecuie. 605 j The present invention also features redirected ceils, such as redirected T ee!is, expressing MHCRs of the present Invention, e.g., as described above. Without wishing to Iimit the present invention to any theory or mechanism, the MHCRs are generaly 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 _c cells, CTLs), In some embodiments, the MHCR is expressed in a CD4+ T ceii (e.g., a T helper ceil, TH cell or a regulatory T ceii (Treg ceil)). The present invention is not limited to the expression of MHCRs in T ceils, 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, γδΤ celis, N ceils, NK T ce!is, etc. In some embodiments, the MHCR of the redirected T ceil complexes or is adapted to complex with CDS subunits (e.g., forming a MHCR-CD3 complex).

|Θ055{ 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 Sinker, in some embodiments, the linker comprises a giycine-rich linker.

{0 6] 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 ceii surface receptor ligands described herein, e.g., the present invention may be applicable to a wide range of MHC molecules, TCR molecules, antigens, signaling moieucles cell surface receptor ligands, etc.

Surrogate Corecepio (SCRs)

{0057] 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 th HCR and/or GD3 subunits. The present invention also features cells expressing a SCR. In some embodiments, redirected cells, e.g., redirected T cells, express both a ivlHCR and a SCR. In some embodiments, ceils 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 p HCR~CD3 complex. i ^' 0058] In some embodiments, the SCR comprises a eel! 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 ceii surface receptor ligand and the kinase are separated by a Sinker, e.g. , a peptide linker or any other appropriate iinker. The signaling moSecule is not limited to a kinase or a phosphatase.

ΪΘ059] 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 ceii surface receptor ligand (or a fragment thereof) may be used. For example, in some embodiments, the ceil surface receptor ligand comprises a CD28 ligand, a CTLA-4 iigand, an iCOS ligand, an 0X40 ligand, a PD-1 ligand (e.g., PD-1 L), a CD2 ligand, etc.

|Θ06Ο| 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 CDS subunits, forming a piV1HCR-CD3 complex. If the cell is also expressing a CD80-Lck SCR, then when the plVIHCR 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 pfvlHCR~CD3 complex to phosphorylate the pMHCR~GD3 STAMs for robust signaling.

| ( 1] 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 ceils to make autoantibodies in autoimmune diseases such as Lupus. The ligand for PD-1 is PD-1 L, so a SCR comprising PD-1 L and Lck may be co-expressed with a pMHCR recognized by the ICR of the T follicular helper cell. This may allow for targeting of this specific T follicular heiper cell population, 0062] The present invention also features methods of use of said SViHCRs, SCRs, and/or said redirected cells, for example for immunotherapy, in some embodiments, the redirected ceils may eliminate autoreactive T cells, regulatory T oeiis (Tregs) that protect tumor cells by suppressing anti-tumor T cell responses, or any othe appropriate T cell. For example, in some embodiments, the MHCR is an auto-antigen MHCR, and the MHCR's target is an autoreactive T ceii,

EXAMPLES

ft) 3| Example 1: Redirected T cells targeting CD4 T Helper Cells. Example 1 describes a non-limiting experimental approach to target CD4 T oeiis. A prototype pfV HCR was engineered with a peptide antigen: the moth cytochrome c peptide (SEQ ID NO: 29) was fused to the mouse class II MHC !-E ^k (MCCJ-E*; e.g., see SEQ ID NO: 31 ). This pMHCR was expressed (e.g., retroviraiiy expressed) in T ceil hybridomas. It was determined that this p!VIHCR (e.g., pMHCR~GD3 complex) was expressed on the surface of T ceil hybridomas (see FIG. 4). iL-2 production was induced after interactions with cognate TCRs (e.g., 5c.c7, 2B4), yet an irrelevant peptide (control peptide antigen) in the pMHCR~CD3 complex rendered it non-stimulatory (data not shown).

|0i64] Lck fusions were generated with known !igands for T cell surface receptors. For example, all T cells express CD28, Lck fusions with CD28 figands (e.g., CD80, C086) were engineered to generate surrogate coreceptors (SCRs), e.g., CD80-Lck (see SEQ ID NO: 33, SEQ !D NO: 38), e.g., CD86-Lck (see SEQ ID NO: 34, SEQ ID NO: 39). When the pWHCR~CD3 complex was co-expressed with SCR CD8Q-Lck in hybridomas, these cells produced significantly more IL-2 in response to cells expressing the 2B4 ICR !igand + CD28 than they did in response to celis expressing only the 2B4 TCR ligand (see FiG. 5). This suggested that signaling through the pMHCR~CD3 complex could be augmented through the use of a SCR.

ΙΘ065Ι MCC;IE ^k p HCR-CD3 and the SCR CD8u-Lck or HB;IE ^k p HCR-CD3 (e.g., see SEQ ID NO; 32) and the SCR CD80-Lck were expressed in in vitro differentiated CDS cytotoxic T celis (CTLs) and their ability to kill 5c,c7 TCR transgenic CD4 T ceils expressing the TCR specific for the MCC:iE ^k p HCR was evaluated. Surface expression of the pMHCRs on the redirected CTLs was observed, suggesting that these chimeric receptor modules compete with th endogenous ICR for assembly with the endogenous CD3 subunits (data not shown). CTLs expressing the MGC:IE ^k pMHCR robustly killed the target CD4 T ceils while those expressing the null HB:IE ^k p HCR did not (see FIG. 6). This suggests that CD8 T cells can be redirected to target and eliminate antigen-specific CD4 T ceils. 0#66j Example 2: Redirected T cells targeting CD4 T Helper Ceils in Allergic Asthma. Example 2 describes a non-limiting experimental approach to target CD4 T helper ceils involved in allergic asthma, e.g., to help eliminate naive Der p 1 -specific CD4 T ceils 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 ceils from the repertoire may help prevent the onset of T _H 2 immunity upon HDM: sensitization.

|Θ067] Α pMHCR {pMHCR-CD3 compiex) will be refrovirally expressed in in vitro activated CTLs. The pMHCR will bear a pi IHCR comprising either the immunodominant HDM-derived Der p 1 epitope (aa1 17-127) in the context of i~A ^b {Derp1:IA ^b ) or the immunodominant West Nile Virus peptide from the envelope protein (aaS41~655) in the context of l-A (E641 :IA ^b ). The E641 :IA ^b pMHCR ceils will serve as a non-specific control population.

ΙΘ 68] 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 C57BS/6 mice to target and eliminate Derp1 :!A ^t:i - or E641:IA ^b -specific naive CD4 T cells from the endogenous repertoire. After a certain length of time, e.g., 1 week, the elimination of antigen-specific CD4 T ceils will be evaluated. This will be performed via tetramer enrichment experiments using a Derp1 :IA ^b tetramer and a E641 :IA ^b tetramer. The presence of the redirected CDS T cells will also be assessed by flow cytometry by gating on CD3 ^* CD8 !A ^b T cells since mouse T cells do not express class II SvlHC. Θ069| 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 ceils specific for Derp1 ;IA are detected, but oniy if redirected T cells are still present in the mice. This may help to determine if activation of the CD4 T ceiis made them more susceptible to targeting by the redirected CTLs. 0070] Example 3: Redirected T cells targeting CD4 T Heiper Ceiis in Lungs After Sensitization. Example 3 describes a non-limiting experimental approach to target CD4 T helper ceils in iungs 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 Iungs of HDM-sensitized mice may help attenuate T _H 2 immunity.

[Θ#71] 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 ΌΜ according to the protocol described above. They will then receive redirected Derp1 ;IA ^b or E641 :IA ^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 TI :-4-Lck SCR (since the Tllv -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 ceiis on the lung cytokine milieu and ceSiuiarity. The status of the redirected CTLs will also be evaluated.

|0i72] Example 4: Attenuation of Der p 1 '-specific CD4 T ce!i function in situ.

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 _H 2 Immunity.

|0S?3| to 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 a in Example 3. Evaluation of the lung cytokine milieu and ceSiuiarity will then be performed as described above.

[0074] 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 !D NO: 23, and a portion is derived from SEQ ID NO: 29 (and other residues may correspond to a g!ycine-rich linking region). In SEQ !D NO: 32, a portion is derived from SEQ !D NO: 15 and a portion is derived from SEQ ID NO: 23 (and other residues may correspond to a glycine-rich Sinking region). fe#7Sj Table 3. Peptide sequences for reagents in Exampies.

SEQ ID HQ. Description Amino Acid Sequence

30 !-E ^k a-TCRa yATIGALLLRFFFIAVLSVlSSQKSWAiKEEHTi!QAEFY

Note: underlined LLPDKRGEFMFDFDGDEiFHVDIEKSETIWRLEEFA portion is from SEQ KFASFEAQGALANIAVDKANLDVMKERSNNTPDAN ID NO: 14 ( HC VAPEVTVLSRSPVNLGEPNJLICFIDKFSPPWNVTW portion), bold portion FRNGRPVTEGVSETVFLPRDDHLFRKFHYLTFLPS is from SEQ ID NO: TDDFYDCEVDHWGLEEPLRKHWEFEEKTLLPETK 22 (TCR portion) ECDATLTEKSFETDM L FQNLSV!VIGLRiLLL VA

GFNLLMTLRLWSS

31 CC:l-E ^k p-TCRp MNArVlPRVPGVAAVILLLTVLSPPVALVRDSGSAWE

(note: italic portion RflDLM V/J QATKEFRSGGGGSLVPRGSGGGGSV shows peptide DRPWFLEYC SECHFYNGTQRVRLLVRYFYNLEE antigen sequence, NLRFDSDVGEFRAVTELGRPDAENWNSQPEFLEQ underlined portion is KRAEVDTVCRHNYEiFDNFLVPRRVEPTVTVYPTKT from SEQ ID NO: 15 QPLEHHNLLVCSVSDFYPGNIEVRWFRNGKEEKTG (fv HC portion), and SVSTGLVRNGDWTFQTLVMLETVPQSGEVYTCQVE bold portion is from HPSLTDPVTVEWKAQSTSAQNKCGITSASYHQGV SEQ ID NO: 24 (TCR LSATfLYEILLGKATLYAVLVSGLVLMAMV K NS portion) AAA

32 ΗΒ:Ι~Ε ^κ β~ΤΟΒβ VWLPRVPCVAAVILLLTVLSPPVALVRDSGSGKK

Note: italic portion ViTAFNEGLKEFRSGGGGSLVPRGSGGGGSVDRP shows peptide VVF LEYCKS ECH FYN GTQRVRLLVRYF YN LEEN LRF antigen sequence, DSDVGEFRAVTELGRPDAENWNSQPEFLEQKRAE underlined portion is VDTVCRHNYESFDNFLVPRRVEPTVTVYPT TQPLE from SEQ ID NO: 15 HHNLLVCSVSDFYPGNIEVRWFR GKEEKTG!VST (MHC portion), and GLVRNG DWTFQTLVM LETVPQSGEV YTCQVE H PS bold portion is from LTDPVTVEWKAQSTSAQNKC6ITSASYHCM3VLSAT SEQ ID HO: 24 (ICR SLYESLLGKATLYAVtVSGLVLMAMV K NSAAA portion)

CD80~Lck (mCD80~ ACNCQLMQDTPLLKFPCPRLiLLFVLURLSQVSS mLck fusion) DVDEQLSKSVKDKVLLPCRYNSPHEDESEDRiYWQ

KHDKVVLSVlAG L V PEYK RTLYDNTTYSLliLG

LVLSDRGTYSCWQKKERGTYEVKHtALVKLSIKAD

FSTPNITESGNPSADTKRiTCFASGGFPKPRFSWLE

NGRELPGiNTTiSQDPESELYTISSQLDFNTTRNHU

KCLi YGDAHVSEDFTWE PPEDPPDS NTLVLFG

AGFGAViTWViVVSiKCFCKHRSCFRRNEASRETNN

SLTFGPEEAlAEQTVFLTTSHYPIVPLDSKISLPiRN

GSEVRDPLVTYEGSLPPASPLQDNLV!ALHSYEPSH

DGDLGFEKGEQLR!LEQSGEWWKAQSLTTGQEGF

!PFNFVA ANSLEPEPWFFKNLSRKDAERQLLAPG

NTHGSFLIRESESTAGSFSLSVRDFDQNGGEVVKH

YKIRNLDNGGFYiSPRITFPGLHDLVRHYT ASDGL

CTKLSRPCQTQKPQKPW EDEWEVPRETLKLVER

LGAGQFGEVWMGYYNGHTKVAVKSLKQGSMSPD

AFLAEANLMKQLQHPRLVRLYAWTQEPIYIITEYME

NGSLVDFLKTPSGIKLNV KLLDMAAQtAEGMAFIE

EQNYIHRDLR NILVSDTLSCKIADFGLARLIEDNE

YTAREGA FPI WTAPE AS YGTFT !KSD VWS FG i LL

TEIVTHGRIPYPGMT PEViQNLERGYRMVRPDNC

PEELYHLM LCWKERPEDRFTFDYLRSVLDDFFTA

TEGQYQPQPGT

CDSe-Lck (mCD86- M DPRCT GLA ί L S FVTVLL ί SDAVSVETQAYF NGT Y mlck fusion) LPCPFTKAQNiSLSEtWFWQDQQKLVLYEHYLGT

EKLDSV AKYLGRTSFDRNNWTLRLH VQIKDMG

SYDCFIQKKPPTGSIILQQUTELSVIANFSEPEIKLA

QNVTGNSGINLTCTSKQGHPKPKK YFLITNSTNE YGDNMQISQONVTELFSiSNSLSLSFPDGWVHMTV

VCVLETES Μ ί SSKP LN FTGEFPS PGTYWKE !TAS V

TVALLLVMLLIiVCHKKPNQPSRPSNTASKLERDSN

ADRET! NL E LEPQ IASAKP N AECTSHYP S VPLDSK i

SLPIRNGSEVRDPLVTYEGSLPPASPLQDNLVIALH

SYEPSHDGDLGFEKGEGlRfLEGSGEWWKAQSLT

TGQEGFIPFNFVAKANSLEPEPWFFKNLSRKDAER

QLLAPGNTHGSFLIRESESTAGSFSLSVRDFDQNQ

GEWKHYKiRNLDNGGFYiSPRITFPGLHDLVRHYT

N ASDGLCTKLSRPCQTQ KPQ PWW ED EWEVPRE

TLKLVERLGAGQFGEVW GYYNGHTKVAVKSLKQ

GSMSPDAFLAEA L QLQHPRLVRLYAWTQEPI

YI!TEYMENGSLVDFLKTPSGIKLNVNKLLD AAQIA

EG AFiEEQNYiHRDLRAANiLVSDTLSCKIADFGLA

RL!EDNEYTAREGA FPiKWTAPEAiNYGTFTiKSDV

WSFGILLTEiVTHGRIPYPG TNPEVIQNLERGYRM

VRPDNCPEELYHLMMLCWKERPEDRPTFDYLRSV

LDDFFTATEGQYQPQPGT

10076] Tabie 4, Examples of ONA sequences for encoding the proteins in Tabie 3.

SEQ

ID MO. Description Gene Sequence

35 i-E ^k a-TCRa aataagctictcgagcgccaccATGGCCACAATTGGAGCCCTGCTGTTAAGATTT

fusion TTCTTCATTGCTGTTCTGATGAGCTCCCAGAAGTCATGGGCTATCAAA

GAGGAACACACCATCATCCAGGCGGAGTTCTATCTTTTACCAGACAAA

CGTGGAGAGTTTATGTTTGACTTTGACGGCGATGAGATTTTCCATGTA

GACATTGAAAAGTCAGAGACCATCTGGAGACTTGAAG.AATTTGCAAAG

TTTGCCAGCTTTGAGGCTCAGGGTGCACTGGCTAATATAGCTGTGGAG

AAAGCTAACCTGGATGTCATGAAAGAGCGTTCCAACAACACTCCAGAT

GCCAACGTGGCCCCAGAGGTGACTGTACTCTCCAGAAGCCCTGTGAA

CCTGGGAGAGCCCAACATCCTCATCTGTTTCATT6ACAAGTTCTGCCC

TCCAGTGGTCAATGTCACCTGGTTCCGGAATGGACGGCCTGTCACCG

AAGGCGTGTCAGAGACAGTGTTTCTCCCGAGGGACGATCACCTGTTC

CGCAAATTCCACTATCTGACCTTCCTGCCCTCCACAGATGATTTCTATG

ACTGTGAGGTGGATGACTGGGGTTTGGAGGAGCCTCTGCGGAAGCAC GACCCTGTCACGGTCGAGTGGAAAGCACAGTCCACATCTGCACAGAA

CAAGtgiggaateadagtgcatoctatcaieaggggg

ctggggaaggG accctatatgctgtgciggtcagiggcctagigGigatgGCCATGGTCAAGA AAAAAAATTCCgcggccgcatgaigagatctgagctccatagaggcg

CD80~Lck acgfcFagatecctogaggcc ccATGGCTTGC

(mCD8G- tGcteaagittccatgtec^aggctc ttct^^

a¾aacaac{giccaagtcagtgaaagataaggiattgctgcctigccg¾acaactctc ctcaigaagai niLck fusion)

gagicigaagaccgaaiciaciggcaaaaacaigacaaagiggtgcigtcigicailgci gggaaaoia aaagigtggcccgagiafaagaaccggaoittatatgaGaacaciaeGiactoicttaic atccigggoGi ggtcclticagaccggggcacatacagctgtgtcgitcaaaagaaggaaagaggaacgia igaagtta aacacttggciitagtaaagttgtccatcaaagcigacttoictacccccaacataactg agtetggaaac ccatctgcagacactaaaaggattacctgctttgcftGcgggggittcGcaaagcctcgc ttdcftggttgg aaaatggaagagaattaccfggcafcaatacgacaaittcccaggaicctgaatcigaal igtacaccai tagtagccaaGtagatttcaatacgactcgcaaccacaccattaagtgtcica acgigteagaggacttcacctgggaaaaacccccagaagaccctcctgatagcaagaaca cacttgt gctcttfggggcaggattcggcgcagiaa aacagtegicgteatcgagtcateaicaaatgctlctgiaa gcacagaagctgiftcagaagaaatgaggcaagcagagaaacaaacaacagcciiaccii cgggcc igaagaagcattagctGAACAGACCGTCTTCCTTaccactag!CACTATCCCATAG

TCccaciggaGagGaagatcicgctgcccaiccggaatggctcigaagtgcgggacc cactggicac ctaigagggaicictcccaccagGatcGCGgctgcaagaGaaccfggitategeGetgGa cagttatgag cccicccatgatggagac tgggciitgagaagggtgaacagctccgaaicclggagcagagcggtga gtggtggaaggcieagtecctgacgactggGcaagaaggGttcaitGcctlcaaGtlcgt ggcgaaagc aaacagcctggagccigaacGttggitcficaagaateigagccgiaaggacgccgagcg gcagcttii ggcgcccgggaacacgcatggatccttcctga!ccgggaaagcgaaagcac!geggggic cttiiccc tgtcggtcagagacttegaccagaaccagggagaagiggigaaacatiacaagaiccgia acctaga caacg tggcttciacatcteeectcg^^

cgectctgafgggdgfgcacaaagtigagcegtcetf^

gggaggacgaatgggaagttcccagggaaacac¾aagttggiggag ggc¾ggagciggccagt tojgggaagigtggaiggggiaefaeaacggacaeacg^

ggagcaigicccccgacgccttcciggctgaggctaaccicatgaagcagctgcageacc cgcggcia gtoGggcitiaigcagtggicacGcaggaacccatctacatGatcaeggaaiacaiggag aaGgggag cdaglagattticicaagactccctcgggcatcaagtigaatgtcaacaaaciliiggac atggcagccc agatigcagagggcaiggcgitcatcgaagaaGagaattacaiccatogggaccigcgcg ccgccaa catcc.tggtgtcigacacgctgagcigcaagatigcagaGi%gGc¾gcgcgcctca igaggacaai gagtacacggcccgggagggggccaaatttecca!taagtggacagcaceagaagccatt aactatg ggacciicaccafcaaglcagac^gtggtc ttcgggatcttgctia

aatocctiacccaggaatgaccaaccc¾aagtcattcagaacctggagagaggctaccg catggtga gacfitgacaaeigtccggaagagctgiaccaectca¾

ccggcGcacgtitgactaccttcggagtgtictggatgacttcticacagccacagaggg cCAGTAC CAGCCCCAGCCTggiacciagtgagaatictacaig 39 CD86~Lck tactetagatacctcgaggccaccATGeACCCCAGATGCACCatgggctiggcaaiccif ai (mCD86- cttigfgacagtettgetgateteagaig^^

tgcGgfgcccatiiaGaaaggcicaaaacataagccigagigagGiggtagtatiiiggG aggaccagc nriLck fusiori)

aaaagitggiicigiacgagcactatttgggcacagagaaacttgatagtgigaa!gcGa agiaeciggg ccgcacgagcttigacaggaacaaciggactctaGgacitcacaatgitcagatcaagga ca^ggctc gtatgaUgiiiiatacaaaaaaagccaccGacaggatcaattatcctocaacagacaita acagaacig tcaglgatcgGcaacttcagigaacctgaaataaaactggctcagaatgiaacaggaaat fctggcaia aatligacclgcacgtciaagcaagg!cacccgaaacctaagaagatgtattitctgata actaaitcaac iaatgagta¾gigataacaigcagaiaicacaagaiaatgtcacagaacigifcagiat ctccaacagc

Gtcictctfteatieceggaiggtgtgtggea^

ttectceaaaceietcaaitteaetcaa agitte^ ^{^} ^

gitaetgfggcccteetcettgtgatgef cteaic^

cccagcaacacagcciciaagitagagcgggaiagtaa gctgacagagagaciatcaacctgaag gaacitgaaccccaaattgctteagcaaaaccaaatgGagagtg!actagtCACTATCCC ATA

GTCccactggacagcaagatetcgctgc<%;atccggaatggGic¾aagigcg ggacccaGiggtca cciaigagggaietctcccaccagcaic-cccgctgcaagacaacctggitatcgccGtg cacagttafga gccctcccaigatggagacitgggci!fgagaagggtgaacagctecgaaicciggagca gagcggig agtgglggaaggctcagiccctgacgactggccaagaaggcttcaiicccitcaacitcg tggcgaaag caaacagcctggagcc gaaccttggttcttcaagaaic gagccgiaaggacgccgagcggcagcttt tggcgcccgggaacacgcatggatcciioctgatccgggaaagcgaa

c¾teggtcagagac{tegaccagaaccagggagaagtggtgaaacaitacaagatcq)t aacciag acaacggtggctictacatctcccctegfatcactfttcccggattgcacgatciagtec gccaiiscacca acgcctc{gatgggctgtgcacaaagt¾agccgicctigccagacccagaagccccaga aacca¾3 gigggaggacgaaigggaagttGCGagggaaacaetgaagtiggiggagcggctgggagc iggeGa gticggggaagtgtggatggggtaciacaacggacacaegaaggiggeggigaagagtet gaaaca agggagcatgicccccgacgccticctggGigaggctaaGctcaigaagcagetgcagGa cccgcgg ctaglccggctttatgcagtggtcac caggaacccatolacatcaicacggaaiacatggagaaoggg agcciagiagattttctcaagactcccicgggcateaagtigaaigicaacaaactitig gacatggcagc ccagattgcagagggcatggcgticatcgaagaacagaatiacaiccaicgggaccigcg cgccgcc aacatectggtgictgacacgctgagc¾^

atgag acacggcccgggagggggccaaa t ccca taagtggacagcaccagaagcca taacta tgggaectieaecateaagtcagacgtgfggteGtte^

gaafcGcSacccaggaatgaccaaccctgaagicaiicagaacciggagagaggctaccg catggtg agaccigacaacigtccggaagagctgtaccacctcatgatgdgtgctggaaggagcgcc cag accggGCGacgt igaciaccticggagigitG^gatgacitGitcacagccacagagggcCAGTAC CAGCCCCAGCCTggtacctagigagaaiictacatg

| ^β β?7 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. 8450112; U.S. Pat No. 7741465; U.S. Pat. No. 6319494; CA 2209300; CA 2104957; EP 0574512; U.S. Pat No. 6407221; U.S. Pat No. $268411 ; U.S. Pat..Application No. 20040258697; EP 1292621 ; EP 2659893; WO 2011101681; WO 2005054292; EP 1379870; U.S. Pat No. 6056952; U.S. Pat. No. 6410319; U.S. Pat No. 8524234; U.S. Pat. No. 7871817.

ΙΘ078) As used herein, the term "about" refers to plus or minus 10% of the referenced number.

[0079] Various modifications of the invention, in addition to those described herein, will be apparent to those skiiied 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. fO08O| 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