CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of U S. Provisional Application No. 62/821 ,222 filed March 20, 2019, the specification (s) of which is/are incorporated herein in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No R01 AH 01053 awarded by

National institutes of Heaith. The government has certain rights in the invention

REFERENCE TO A SEQUENCE L!STING

[0003] Applicant asserts that the information recorded in the form of an Annex C/ST.25 text file submitted under Rule 13ter.1(a), entitled UNIA _{_} _19_03_PCT _Sequence Listing_ST25.txt , is identical to that forming part of the international app!ication as filed. The content of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0004] The present invention relates to T ceils and T ceil receptors, more particularly to co-receptors for engineered T cells such as T cells expressing components of a modular chimeric receptor system.

BACKGROUND OF THE INVENTION

[0005] T cells normally recognize and respond to peptide antigens embedded within major histocompatibility complex molecules (pMHCs) of antigen presenting ceiis (APCs) via their TCR-CD3 complex. This TCR-CD3 complex is composed of the TCR, which is the receptor module that binds the pMHC, and the CD3ys, CD3bs, and 003zz signaling modules that connect the TCR to the intracellular signaling machinery. The intracellular domains of the CD3 subunits contain immunoreceptor tyrosine- based activation motifs (ITAMs) that are phosphoryiated by the Src kinases, e.g., Lck, Fyn. CD3y, CD36, and CD3E each contain one ITAM while 003z contains three ITAMs for a totai of ten in a single complex. The 4-module TCR-CD3 complex does not have any intrinsic Src kinase activity. In fact, coreceptor modules {e.g., CD4, CDS) appear to sequester Lck away from the TCR-CD3 complex until both a coreceptor and a TCR bind a pMHC, The Lck associated with the coreceptor is then brought into dose proximity to the CDS ITAMs to phosphorylate tyrosines within these motifs and initiate signaiing.

[0006] Ectopic T cell receptors (ectopic ICRs) have been introduced into T cells in an effort to reprogram or alter T cel! specificity However, in some cases, the introduction of ectopic TCRs has been found to lead to cross-pairing events with endogenous TCRs, resulting in novel TCRs with autoimmune specificities. This led to the use of chimeric antigen receptors (referred to herein as one-module CARs or ^1M 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 CD3x intracellular signaling domain as well as signaling domains from a variety of other signaling molecules (e.g., CD28, CD27, ICOS, 4-1 BB, 0X40). However, ^1M CARs bear little resemblance to naturally evolved receptors. Signal initiation moat likely occurs via random receptor clustering and recruitment of kinases to their signaling motifs as ^1M CARs lack the more complex mechanisms that natural receptors have evolved to initiate signaling. Indeed, ^1M CARs are orders of magnitude less sensitive than their natural counterparts, treatment requires the infusion of supra-physiological numbers of ^1M CAR-T cells, and cytokine release syndrome (CRS) is a common side effect The success of ^1M CAR-T cel therapy Is largely limited to CD19* hematopoietic tumors.

BRIEF SUMMARY OF THE INVENTION

[0007] The present invention describes biomimetic five module chimeric receptors (hereinafter referred to as ( ^5M CARs), which feature a chimeric receptor module (CRM) which, In one example, comprises a portion of the TCR and a portion of a MHC molecule such as a Class I, Class II, or non-classical MHC (also referred to herein as ^* CRM ^mhc "), etc., three CD3 signaling modules (e.g., CD3ye, CD3dc, and CD3xx which connect the TCR to the intracellular signaling machinery), and one surrogate co-receptor module (e.g., the a surrogate co-receptor would mirror the function of CD4 and CD8). In some embodiments, the CRM comprises a portion of a peptide antigen presented in the MHC and is referred to as CRM ^pMHC (aka "pMHCR"). For reference, FIG. 1A shows an example of the natural signaling machinery. FIG. IB shows a model of a CAR comprising a CRM ^,MHC and three CDS molecules (CDS Is not shown). The CRMs (e.g., CRM ^pmhc ) are adapted to recognize and bind to appropriate (specific) TCRs.

[0008] More specifically, the present Invention features surrogate co-receptors (SCoRs), which may be featured as part of a ^5M CAR. In some embodiments, the SCoRs comprise a portion of CD8. For example, the SCoRs of the present invention may comprise an Fv antibody portion linked to a portion of CD8. The present invention Is not limited to CD8 and also includes SCoRs comprising other co-receptor molecules such as a portion of CD4 linked to a single chain Fv fragment The present invention is not limited to the aforementioned SCoR components.

[0009] The present invention also features co-receptor fusions, wherein the SCoR is fused directly or indirectly to a Sro kinase such as Lck.

[0010] The present invention also features cells, expressing the co-receptors (SCoRs) herein. The present invention also features cells expressing ^5M CAR that feature the co-receptors (SCoRs) herein. Cells expressing a ^5M CAR (e.g., with a CRM) may herein be referred to as "redirected cells.· Redirected cells, e.g., redirected T cells, expressing a CRM ^pmhc would mimic antigen presenting cells (APCs). In some cases, binding of a TCR of a target T cell to the CRM ^pMHC of the redirected cel may then result in destruction of the target T cell by the redirected T cell; thus, in this case, the redirected cells may function as "anti-T cel" T cells. The present invention is not limited to redirected cels functioning to destroy a target For example, in some embodiments, the redirected cell is adapted to help reprogram a target cel, e.g., the redirected cell may deliver instructions to the target cell.

[0011] The present invention features surrogate co-receptors (SCoRs). In some embodiments, the surrogate co-receptor (SCoR) comprises a CD8a chain and a CD8b chain. The CD8a drain comprises a first binding portion and a CD8a portion, wherein the first binding portion is an immunoglobulin (Ig) domain (e.g., an antibody fragment for a particular target antigen or the receptor binding region of CD80) and the CD8a portion is at least a portion of CD8a. The CD8b chain comprises a second chain comprising a second binding portion and a CD8b portion, wherein the second binding portion is an Ig domain (e.g., an antibody fragment for a particular target antigen or the receptor binding region of CD80) and the CD8b portion is at least a portion of CD8b.

[0012] In some embodiments, the antibody fragments are antibody Fv fragments. For example, the combination of the heavy and fight chains of the Fv antibody fragments creates specificity for an epitope of a TCR. It is to be understood that the present invention is not limited to TCR, and that the invention can include Fv from any antibody specific for any antigen. In some embodiments, the CD8 portions are linked C-terminal to the binding portions. In some embodiments, the CD8 portions are indirectly linked to the binding portions. In some embodiments, the CD8 portions are directly linked to the binding portions.

[0013] In some embodiments, the antibody fragments are wild type fragments. In some embodiments, the antibody fragments are from a particular standard antibody. In some embodiments, the antibody fragments have at least one mutation compared to their wild type sequences (or standard antibody sequences). In some embodiments, the antibody fragments with at least one mutation have a lower biding affinity than their wild type counterparts. In some embodiments, the antibody fragments have a genuine sequence. The antibody fragments may be derived from a tuning process to modify or select particular binding kinetics via mutagenesis.

[0014] The present invention features surrogate co-receptor (SCoR) fusions (fusion molecules). In some embodiments, the fusion molecule comprises a SCoR of the present invention linked directly or indirectly to Lck. In some embodiments, the CD4 intracellular domain (ICD) (e.g., at least a portion of, or al) is used to replace at least a portion of the native CD8 ICD (e.g., CD8a ICD) in a CD8-based SCoR. In some embodiments, at least a portion of the CD4 ICD and at least a portion of the transmembrane domain (TM) are used to replace at least a portion of the native CD8 ICD and CD8 TM domain (respectively) in a CD8- based SCoR.

[0015] In some embodiments, the present invention features engineered cells expressing on their surface a SCoR of the present invention. In other embodiments, the present invention features engineered cells expressing on their surface a SCoR and a fusion molecule of the present invention.

[0016] The present invention also features engineered cells co-expressing on their surface: a chimeric receptor (CRM) comprising a major histocompatibility complex (MHC) portion derived from an MHC protein directly or indirectly fused to a T ceil receptor (TCR) portion derived from a TCR protein, wherein the CRM contains a peptide antigen in the MHC (CRM ^pMHC ) that confers upon it specificity for a first epitope of a TCR; and a surrogate coreceptor according to the present invention, wherein the SCoR is specific for a second epitope of the TCR. In some embodiments, the engineered cells also express a fusion molecule of the present invention.

[0017] In some embodiments, the MHC portion of the CRM is N-terminal to the TCR portion of the CRM. In some embodiments, the MHC portion is indirectly fused to the TCR portion via a Inker. 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 extraoeluiar 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 extracelular domain of the MHC protein. In some embodiments, the CRM further comprises a peptide antigen integrated into the MHC portion, or directly or indirectly fused to the MHC portion. In some embodiments, the MHC 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-EB 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.

[0018] In some embodiments, the CRM can complex with a CD3 subunit In some embodiments, the engineered cell further co-expresses a SCoR. In some embodiments, the engineered ceB further co- expresses a second fusion molecule.

[0019] The present invention also features vectors encoding surrogate co-receptors according to the present invention. The present invention also features DMA sequences encoding surrogate co-receptors according to the present invention. The present invention also features protein sequences encoding surrogate co-receptors according to the present invention. The present invention also features vectors encoding fusion molecules according to the present invention. The present invention also features DMA sequences encoding fusion molecules according to the present invention. The present invention also features protein sequences encoding fusion molecules according to the present invention.

[0020] The present invention also features methods for eliminating or redirecting a target cell. In some embodiments, the method comprises introducing a genetically engineered cel according to the present invention, wherein the CRM of the genetically engineered cel is specific for a TCR of the target cel, wherein upon binding of the CRM of the genetically engineered cel to the TCR of the target cel, the genetically engineered cel (a) inmates a signaling cascade that eliminates the target cel, or (b) Instructs the target cell to differentiate to a specific effector function.

[0021] 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 SEVERAL VIEWS OF THE DRAWING(S)

[0022] 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:

[0023] FIG· 1A shows an «(ample of the natural signaling machinery.

[0024] FIG. 1B shows an example of a CAR comprising a CRM ^pMHC (aka pMHCR) and three CDS molecules (CD8 is not shown).

[0025] FIG. 2 shows an example of a surrogate co-receptor (SCoR) featuring a portion of CD8. More specifically, the SCoR comprises the Fv regions of the anti-mTCRb mAb H57-597 (H57) fused to the stalks of CD8«p. The CD8a Ig domain was replaced with the H57 heavy chain (HC), and the CD8b Ig domain was replaced with the light chain (LC).

[0026] FIG. 3 shows IL-2 expression in cells expressing a surrogate co-receptor with a F32A mutant on

T ^CR

the H57 light chain (LC) (SCoR ) with the MCC:I-E* pMHCIIR on 58a b ^" cells. The pMHCIIR* SCoR™** cells made more IL-2 than pMHCIIR* cells when co-incubated with M12 B cels expressing the 2B4 TCR, CD4, and truncated CDS subunits (COST) that lack ITAMs. The TCR-CDT complexes cannot signal, and M12 cels do not make IL-2, therefore the pMHCIIR-CD3 complexes signal more with this SCoR than without.

[0027] FIG. 4 shows IL-2 expression from 58a ^" b" cells (58a"b" T cel hybridomas) expressing the 5c.c7 TCR and indicated CD4-Lck fusions alter 16 hours of co-culture with peptide-pulsed APCs.

[0028] FIG. 5 shows IL-2 expression from 58a ^' b ^" cells (58a ^" b ^* T cel hybridomas) expressing the 5c.c7 TCR and indicated CD4-Lck fusions after 16 hours of co-culture with APCs expressing high density single-species agonist, weak agonist, antagonist, shaved or null tethered pMHC.

DETAILED DESCRIPTION OF THE INVENTION

[0029] It is to be noted that CRM ^MHC and MHCR are interchangeable and both refer to a chimeric receptor that is comprised of a portion of the TCR and a portion of an MHC molecule. Additionaly, CRM ^pMHC and pMHCR are also Interchangeable, both referring to a CRM ^MHC with a portion of a peptide antigen presented within the context of the MHC portion.

[0030] Referring now to FIG. 1-5, the present invention features surrogate co-receptors (SCoRs), such as SCoRs comprising at least a portion of CD8. For example, the SCoRs of the present invention comprise an Fv antibody portion linked to a portion of CD6. The present invention is not limited to CD8 and also includes SCoRs comprising at least a portion of CD4, e.g., a single chain Fv antibody fragment linked to a portion of CD4. In a non-limiting embodiment, the light chain and/or heavy chain Fv regions are placed separately on the CD8 alpha or beta chains so that they form a heterodimer. In some embodiments, the SCoRs of the present invention may be featured as part of the ^5M CARs.

[0031] The present invention also features co-receptor fusions, wherein a SCoR is fused to a Src kinase (such as Lck) or to portions of the CD4 intracellular domain that would increase association with Lck.

[0032] The present invention also features cells, such as T cels, expressing SCoRs of the present invention. The present invention also features cells, such as T cells, expressing ^5M CARs featuring SCoRs of the present invention.

I. ^5M CARs with Chimeric MHC Receptors (CRMs)

[0033] As previously discussed, the surrogate co-receptors (SCoRs) of the present invention may be featured as part of ^5M CARs (e.g., as part of ^5M CARs with chimeric MHC receptors, e.g., CRMs, CRM ^{, pMHC} etc.). The CRMs described herein comprise an MHC portion (e.g., class I, class II, non-classical, a combination thereof, etc.) and a TCR portion (e.g., ab, gd TCR, etc.). In some embodiments, the MHC portion and TCR portion are separated by a linker, e.g., peptide linker. In some embodiments, the CRM further comprises a peptide antigen (a CRM comprising a peptide antigen may herein be referred to as a pMHCR or CRM ^pMHC 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. a. MHC Portion of CRMs

[0034] The MHC portion may comprise one or more MHC peptides (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.

[0035] Table 1.1. Examples of Human MHC Protein Sequences

[0037] Referring to Table 1.1, the HLA-A (MHC I) sequence (SEQ ID NO: 1) includes the signal peptide (amino adds 1-24); amino adds 25-308 are believed to make up the extracellular region, amino adds 309-332 are believed to make up the transmembrane region, and amino adds 333-365 are believed to make up the cytoplasmic region. The HLA-B (MHC I) sequence (SEQ ID NO: 2) indudes the signal peptide (amino adds 1-24); amino adds 25-308 are believed to make up the extracellular region, amino adds 309-332 are believed to make up the transmembrane region, and amino adds 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 adds 1-24); amino adds 25-308 are believed to make up the extracellular region, amino adds 309-333 are believed to make up the transmembrane region, and amino adds 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 adds 1-28); amino adds 29-222 are believed to make up the extracellular region, amino adds 223-245 are believed to make up the transmembrane region, and amino adds 246-260 are believed to make up the cytoplasmic region. The HLA DPB1 (MHC II) sequence (SEQ ID NO: 5) Indudes the signal peptide (amino adds 1-29); amino adds 30-225 are believed to make up the extracellular region, amino acids 226-246 are believed to make up the transmembrane region, and amino adds 247- 258 are believed to make up the cytoplasmic region. The HLA DQA1 (MHC II) sequence (SEQ ID NO: 6) indudes the signal peptide (amino adds 1-23); amino adds 24-216 are believed to make up the extracellular region, amino adds 217-239 are believed to make up the transmembrane region, and amino adds 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 adds 1-32); amino adds 33-230 are believed to make up the extracellular region, amino adds 231-251 are believed to make up the transmembrane region, and amino adds 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 adds 1-25); amino adds 26-216 are believed to make up the extracellular region, amino adds 217-239 are believed to make up the transmembrane region, and amino adds 240-254 are believed to make up the cytoplasmic region. The HLA DRB1 (MHC II) sequence (SKI ID NO: 9) includes the signal peptide (amino adds 1-29); amino adds 30-227 are believed to make up the extracellular region, amino adds 228-250 are believed to make up the transmembrane region, and amino adds 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).

[0038] As previously discussed, the CRM of the present invention comprises an MHC portion and a TCR portion. In some embodiments, the MHC portion comprises one or more MHC proteins (e.g., HLA-A, HLA- B, HLA-C, HLArDPAI, 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.

[0039] In some embodiments, the MHC portion comprises a peptide that is at least 80% identical to an MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 85% identical to an MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 90% identical to an MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 95% identical to an MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 99% identical to an MHC protein or a fragment thereof.

[0040] In some embodiments, a fragment of an MHC protein is from 10 to 25 aa in length. In some embodiments, a fragment of an MHC protein is from 10 to 50 aa in length. In some embodiments, a fragment of an MHC protein is from 10 to 100 aa in length. In some embodiments, a fragment of an MHC protein is from 10 to 150 aa in length. In some embodiments, a fragment of an MHC protein is from 10 to 200 aa in length. In some embodiments, a fragment of an MHC protein is from 10 to 250 aa in length. In some embodiments, a fragment of an MHC protein Is from 10 to 300 aa in length. In some embodiments, a fragment of an MHC protein is from 10 to 350 aa in length. In some embodiments, a fragment of an MHC protein Is from 25 to 50 aa in length. In some embodiments, a fragment of an MHC protein is from 25 to 100 aa in length. In some embodiments, a fragment of an MHC protein is from 25 to 150 aa in length. In some embodiments, a fragment of an MHC protein is from 25 to 200 aa In length, in some embodiments, a fragment of an MHC protein is from 25 to 250 aa in length. In some embodiments, a fragment of an MHC protein Is from 25 to 300 aa in length. In some embodiments, a fragment of an MHC protein is from 25 to 350 aa in length. In some embodiments, a fragment of an MHC protein is from 50 to 100 aa in length. In some embodiments, a fragment of an MHC protein is from 50 to 150 aa in length. In some embodiments, a fragment of an MHC protein is from 50 to 200 aa in length. In some embodiments, a fragment of an MHC protein is from 50 to 250 aa in length. In some embodiments, a fragment of an MHC protein is from 50 to 300 aa in length. In some embodiments, a fragment of an MHC protein is from 50 to 350 aa in length. In some embodiments, a fragment of an MHC protein is from 100 to 150 aa in length. In some embodiments, a fragment of an MHC protein is from 100 to 200 aa in length. In some embodiments, a fragment of an MHC protein Is from 100 to 250 aa in length. In some embodiments, a fragment of an MHC protein is from 100 to 300 aa In length. In some embodiments, a fragment of an MHC protein is from 100 to 350 aa in length. In some embodiments, a fragment of an MHC protein is from 150 to 200 aa in length. In some embodiments, a fragment of an MHC protein is from 150 to 250 aa in length. In some embodiments, a fragment of an MHC protein Is from 150 to 300 aa in length. In some embodiments, a fragment of an MHC protein is from 150 to 350 aa in length. In some embodiments, a fragment of an MHC protein is from 200 to 250 aa in length. In some embodiments, a fragment of an MHC protein is from 200 to 300 aa in length. In some embodiments, a fragment of an MHC protein is from 200 to 350 aa In length. In some embodiments, a fragment of an MHC protein is from 250 to 300 aa in length. In some embodiments, a fragment of an MHC protein is from 250 to 350 aa in length. In some embodiments, a fragment of an MHC protein is more than 350 aa in length. b. TCR Portion of CRMs

[0041] 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.

[0042] Table 2.1. Examples of Human TCR Protein Sequences

[0043] Table 2.2. Examples of Mouse TCR Protein Sequences

[0044] Referring to the TRAC protein (SEQ ID NO: 16) in Table 2.1, amino adds 118-137 are believed to make up the transmembrane domain, and amino adds 138-142 are believed to make up the cytoplasmic domain. Referring to the TRBC1 protein (SEQ ID NO: 17), amino adds 151-171 are believed to make up the transmembrane domain. Referring to the TRBC2 protein (SEQ ID NO: 18), amino adds 145-167 are believed to make up the transmembrane domain. Referring to the TRDC protein (SEQ ID NO: 19), amino adds 130-152 are believed to make up the transmembrane domain. Referring to the TRGC1 protein (SEQ ID NO: 20), amino adds 139-161 are believed to make up the transmembrane domain. Referring to the TRGC2 protein (SEQ ID NO: 21), amino acids 157-177 are believed to make up the transmembrane domain, and amino adds 178-189 are believed to make up the cytoplasmic domain.

[0045] As previously discussed, the CRM of the present invention comprises an 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, the TCR portion may comprise a fragment of any of SEQ ID NO: 16-28. In some embodiments, the fragment is from 5 to 10 aa 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 aa 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 aa in length, etc.

[0046] 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.

[0047] 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 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 150aa in length. In some embodiments, a fragment of a TCR protein is from 25 to 50 aa in length. In some embodiments, a fragment of a TCR protein is from 25 to 100 aa in length. 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 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.

[0048] In some embodiments, the CRM comprises a peptide antigen. Any appropriate peptide antigen may be used. The peptide antigen in CRM ^pMHC complex directs the specificity of the CRM ^pMHC molecule, therefore the CRM ^pMHC molecule will be specific for T cells with TCRs that are specific for that peptide antigen CRM ^pMHC A non-limiting example of a peptide antigen that may be used with the CRM is moth cytochrome c peptide (aa 88-103, AN ERADLIAYLKQATK (SEQ ID NO: 29)). 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 responee. The peptide antigen may be any appropriate length.

[0049] In some embodiments, the CRM comprises at least a portion of an MHC molecule that allows for binding to an appropriate TCR. In some embodiments, the CRM comprises at least a portion of an 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 CDS subunits). In some embodiments, the CRM comprises a transmembrane domain that is at least partially derived from (i) an MHC molecule, (i) a TCR molecule, or (iii) both the MHC molecule and TCR molecule. In some embodiments, the CRM comprises a transmembrane domain, wherein a portion (or all) of the transmembrane domain is not derived from an MHC molecule or a TCR molecule. In some embodiments, the CRM comprises an extracelular domain that is at least partialy derived from (i) an MHC molecule, (ii) a TCR molecule, or (iii) both the MHC molecule and TCR molecule. In some embodiments, the CRM comprises an extracellular domain, wherein a portion of the extracellular domain is not derived from an MHC molecule or a TCR molecule.

[0050] In one embodiment, the CRM comprises at least a portion of the extraceBular domain of an 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. In another embodiment, the CRM comprises at least a portion of the extracellular domain of a TCR molecule.

//. Surrogate Coreceptors-(SCoR)

[0051] In some embodiments, the present invention features surrogate coreceptors (SCoRs), e.g., receptors that recruit signaling molecules (e.g., kinases such as but not limited to Src kinases (e.g., Lck), phosphatases, etc.). The SCoRs may recruit signaling molecules (e.g., kinases) to the CRM and/or CDS subunite. Without wishing to limit the present invention to any theory or mechanism, it is believed that certain SCoRs may enhance signaling through the CRM ^pMHC -CDS complex. The present invention is not limited to the aforementioned uses of SCoRs. For example, the SCoRs of the present invention may be used in 5M-GARs that have an Fv-based CRM module (CRM ^Fv ) instead of a CRM ^pMHC . The SCoRs of the present invention may be used in any appropriate application. For «cample, the SCoRs of the present invention may be used in combination with conventional single chain CARs (aka 1 module or IM-CARs. Without wishing to be bound to a particular theory or mechanism, the SCoRs may enhance function of the 1M-CARs.

[0052] For example, the present invention provides SCoRs comprising a portion of CD8. Referring to FIG. 2 (left side), CD8 forms a dimer, consisting of a CD8a and CD8p chain. The two chains of CD8 each have an immunoglobulin variable-like extracellular domain (ECD), a transmembrane domain (TMD), and an intracellular domain (ICD). Wild type CDS binds to class I MHCs.

[0053] The SCoRs of the present invention may comprise: (1) an alpha chain with a binding portion (e.g., antibody fragment, e.g., Fv Ig domain, etc.) linked to a CD8a portion (e.g., the portion of CD8a except for the Ig domain); and (2) a beta chain with a binding portion (e.g., antibody fragment, e.g., Fv Ig domain, etc.) linked to a CD8b portion (e.g., the portion of CD8p except for the Ig domain).

[0054] In some embodiments, the binding domains of the SCoR are antibody Fv domains, e.g., the Ig domains of both CD8at and CD8b are each replaced with an antibody Fv domain, e.g., an antibody Fv domain specific for a target TCR. Referring to FIG. 2 (right side), a SCoR was constructed to target an epitope on TCRp (the FG-loop) that is ubiquitous to aN mouse TCRs. The SCoR comprises an Fv region of the anti-mTCRb mAb H57-597 (H57) fused to the stalks of CD8a and CD8p (referred to as SCoR™ in FIG.2). More specifically, the CD8a Ig domain was replaced with the H57 heavy chain (HC) Fv Ig domain, and the CD8b Ig domain was replaced with the light chain (LC) Fv Ig domain.

[0055] The present invention also features SCoRs synthesized by tuning the binding kinetics of the particular Fv. Co-receptors do not typically bind their target with high affinity so that they can cycle through various ligands, in some cases, cells expressing the native Fv affinity may bind any T cel, regardless of TCR specificity, and not disengage. Thus, mutations may be introduced to the Fv(s) that affect the binding of the Fv to the TCR of interest. For example, the SCoR described above may be modified by introducing alanine mutations for the large hydrophobic H57 residues that are believed to mediate binding to the TCR (e.g., light chain (LC): L28, F32, Y34; HC: W33, Y35, F98). The present invention is not limited to the aforementioned SCoR nor the specific mutations described herein. Further, the present invention is not limited to SCoRs made based on mouse sequences. The sequences of any of the SCoRs described herein may be tailored to a particular animal or subject (e.g., human, primate, dog, rat, etc.).

[0056] As previously discussed, the present invention features methods and compositions for“tuning" the binding kinetics (e.g., via mutagenesis, etc.) as desired, e.g., to tune the binding to mimic the natural binding kinetics of CD8 for MHCI. For example, one of ordinary skil in the art may start with a high affinity Fv and alter said Fv to weaken the binding by introducing mutations to the sequence or reverting the sequence to that of the germline sequence (or a sequence similar to the germlne sequence). In some embodiments, the methods feature mutating the antigen binding COR loops of the Fv to tune its affinity for the target ligand, e.g., to tune the affinity more closely to natural binding kinetics (e.g., tune to natural binding kinetics of CD8 for MHCI).

[0057] A SCoR was constructed comprising a CD8a chain wherein the CD8a Ig domain was replaced with the H57-597 heavy chain (HC) and a CD8p chain wherein the CD8p Ig domain was replaced with the light chain (LC), wherein the LC comprises a F32A mutation (SCoR™ F32A). Referring to FIG. 3, when expressed with the MCC:l-E ^k pMHCIIR on 58a b ^' cels, the pMHCIIR* SCoR™ F32A* cels made more IL- 2 than pMHCIIR* cels when co-incubated with M12 B cells expressing the 2B4 TCR, CD4, and truncated CD3 subunits (CD3T) that lack ITAMs. The TCR-CD3T complexes cannot signal, and M12 cells do not make IL-2, therefore the pMHCIIR-CD3 comp!exee elgnal more with this SCoR”* F32A than without

[0058] Table 3 lists the sequence for CD8a and CD8p and non-limiting examples of SCoRs featuring H57 heavy chain and H57 light chain as the binding portions of the SCoRs, as wel as examples of SCoRs featuring portions of the 145-2C11 antibody (monoclonal antibody specific for mouse CD3epsilon

[0059] Table 3. Examples of SCoRs

[0060] The present invention is not limited to any particular antibody: the present Invention indudes grafting any appropriate Ig domains onto the CD8 stalks to make a SCoR.

[0061] It is also possible to reconstruct the germline sequence of the non-affinity matured antibody (e.g., H57 mAb). The germline sequence may be useful for considering a mutagenesis strategy.

[0062] Without wishing to Imit the present invention to any theory or mechanism, it is believed that a SCoR that binds the target TCR-CD3 complex with faster kinetics than the CRM ^pMHC (and approximate the spatial relationship between the TCR-CD3 complex and CD8) would be optimal.

[0063] The present invention is not limited to SCoRs comprising a portion of CD8. In some embodiments, the SCoR comprises a portion of CD4. For example, in some embodiments, single-chain Fv antibody fragments are fused to the D2-D4 domains of CD4.

[0064] The present invention also features SCoRs synthesized based on tuning signaling intensity by tuning linkage to Lck.

III. Lck Fusions

[0065] Other approaches to tuning the SCoR signaling include other modifications to CDS and Lck fusions. For example, the present invention also features a CD8-Lck fusion and a CD4-Lck fusion.

[0066] For example, the present invention provides a fusion molecule wherein Lck is fused to CD4 after its second intracellular residue (see below). This fusion protein has been shown to increase signaling and allow detection of TCR scanning of MHCII.

[0067] The fusion proteins may be constructed a number of ways, e.g., connecting the CD4 portion to the Lck portion directly, connecting the CD4 portion to the Lck portion indirectly, connecting amino adds 1- 420 of CD4 to amino acids 1-509 of Lck (indirectly or directly), connecting amino adds 1-420 of CD4 to amino adds 4-509 of Lck (indirectly or directly), etc. In some embodiments, the Lck molecule attached to CD4 comprises one or more mutations. In some embodiments, the CD4 attached to Lck comprises one or more mutations. In some embodiments, both the CD4 and the Lck comprise one or more mutations.

[0068] in some embodiments, the CD4 Intracellular domain (ICD) (e g., at least a portion of, or all) will be used to replace at least a portion of the native CD8 ICD (e.g., CD8a ICD) in a CD8-based SCoR. In some embodiments, at least a portion of the CD4 ICD and at least a portion of the transmembrane domain (TM) are used to replace at least a portion of the native CDS ICD and CDS TM domain (respectively) in a CD8- based SCoR.

[0069] Table 4 lists the sequence for wild type CD4, wld type Lck, and non-limiting examples of the Inking legions of Lck Fusions (e.g., where the CD4 links to Lck).

[0070] Table 4. Examples of linkage regions of Lck Fusions

[0071] FIG. 4 shows IL-2 expression from 58a ^" b ^" ceils expressing the 5c.c7 TCR and indicated CD4-Lck fusions alter 16 hours of co-culture with peptide-pulsed APCs.

[0072] The present invention is not limited to Lck fusion molecules disclosed herein, e.g., those made based on mouse sequences. The sequences of any of the Lck fusion molecules described herein may be tailored to a particular animal or subject (e.g., human, primate, dog, rat, etc.).

IV. Methods and Applications

[0073] The present invention also features applications and methods of use of said CRMs, SCoRs, Lck fusions. For example, the present invention also features ceis expressing a SCoR of the present invention. The present invention also features ceils expressing a Lck fusion of the present invention. The present invention also features cells expressing a SCoR and/or Lck fusion as part of a *¾AR f redirected" ceils). In some embodiments, cells express more than one type of SCoR.

[0074] As an example, in some embodiments, redirected cells may be used to help eliminate autoreactive T cels, regulatory T cells (Tregs) that protect tumor cells by suppressing anti-tumor T cell responses, or any other appropriate T cel. For example, in some embodiments, the CRM is an auto-antigen CRM, and the CRM’s target » an autoreactive T cel. The present invention Is not limited to the expression of CRMs in T cells.

[0075] 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 ceil surface receptor figands 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. In some embodiments, the CRM ^MHC comprises an MHC portion derived from an extracellular portion of an 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 Inker. In some embodiments, the linker comprises a glycine-rich Inker.

[0076] Without wishing to limit the present invention to any theory or mechanism, it is beloved that the ^5M CARS of the present invention (e.g., those comprising the SCoRs herein) wll be more sensitive and will be under better control as compared to 1 module CAR _S . Thus, it would be possible to introduce an amount of T cells closer to a physiological level, which may help reduce toxicity and the likelihood of cytokine release syndrome.

[0077] The disclosures of the following patents are incorporated in their entirety by reference herein: U.S. Pat. Application No. 2018/0179260, 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.6268411; U.S. Pat. Application No. 20040258697; EP 1292621; EP 2659893; WO 2011101681; WO 2005054292; EP 1379670; U.S. Pat No. 6056952; U.S. Pat No. 6410319; U.S. Pat No. 8524234; U.S. Pat No. 7871817.

[0078] Although there has been shown and described the preferred embodiment of the present invention, it wil 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 Imited 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 essentialy of or‘consisting of, and as such the written description requirement for claiming one or more embodiments of the present invention using the phrase "consisting essentially of or "consisting of is met