REDIRECTED CELLS WITH MHC CHIMERIC RECEPTORS AND METHODS OF USE IN IMMUNOTHERAPY
Chimeric receptors featuring major histocompatibility molecules grafted onto T cell receptor molecules and surrogate co-receptors featuring cell surface receptor ligands fused with signaling molecule domains. The chimeric receptors can be used to redirect cells, altering their specificity. T cells expressing chimeric receptors may bind to TCRs of target T cells for which their chimeric receptors are specific. Surrogate co-receptors may be used to help enhance TCR-CD3 signaling as part of this modular receptor system. The chimeric receptors and surrogate coreceptors may be used to help eliminate autoreactive T cells or program T cells to desired effector functions.
This application is a Divisional and claims benefit of U.S. patent application Ser. No. 15/738,467 filed Dec. 20, 2017 which is a 371 application and claims benefit of International Patent Application No. PCT/US16/40177 filed Jun. 29, 2016, which claims benefit of U.S. Provisional Patent Application No. 62/186,865 filed Jun. 30, 2015, the specification(s) of which is/are incorporated herein in their entirety by reference.
GOVERNMENT SUPPORTThis invention was made with government support under Grant No. R01 AI101053 awarded by NIH. The government has certain rights in the invention.
REFERENCE TO SEQUENCE LISTINGApplicant asserts that the paper copy of the Sequence Listing is identical to the Sequence Listing in computer readable form found on the accompanying computer file, entitled UNIA_15_04_PCT_US_DIV_Sequence_Listing_ST25, and 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 INVENTIONThe present invention relates 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.
BACKGROUND OF THE INVENTIONT cells normally recognize and respond to peptide antigens embedded within major histocompatibility complex molecules (pMHCs) of antigen presenting cells (APCs) via their TCR-CD3 complex (see
Ectopic T cell receptors (TCRs) have been introduced into T cells in an effort to reprogram or alter T cell 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 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 CD3ζ intracellular signaling domain as well as signaling domains from a variety of other signaling molecules (e.g., CD28, CD27, ICOS, 4-1BB, OX40). Without wishing to limit 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 cell activation and effector functions. CARs are likely to be delivering incomplete signals that could have unintended consequences or side effects.
The present invention features novel chimeric receptors (e.g., “MHCRs”) comprising a portion of a MHC molecule (e.g., class I, class II, non-classical MHC) and a portion of the TCR. In some embodiments, the MHCR comprises a portion of an antigen peptide. The present invention also features cells, such as T cells, expressing said MHCRs (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 cells) expressing a MHCR would mimic antigen presenting cells (APCs), the cells that normally express MHC molecules. In some cases, binding of a TCR of a target T cell to the MHCR of the redirected cell may then result in destruction of the target T cell; thus, in this case, the redirected cells may function as “anti-T cell” T cells. The present invention is not limited to redirected cells functioning to destroy a target. For example, in some embodiments, the redirected cell is adapted to help reprogram a target cell, e.g., the redirected cell may deliver instructions to the target cell.
The present invention also features engineered cells expressing both an MHCR and an SCR. It was surprisingly discovered that engineered cells co-expressing an MHCR and an SCR had enhanced effects (e.g., increased IL-2 expression, see
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.
SUMMARY OF THE INVENTIONThe present invention features novel chimeric receptors for engineering redirected cells. For example, the present invention features an engineered cell co-expressing on its surface a chimeric receptor (MHCR) comprising a major histocompatibility complex (MHC) portion (derived from a MHC protein) directly or indirectly fused to a T cell receptor (TCR) portion (derived from a TCR protein); and a surrogate co-receptor (SCR) comprising a cell surface receptor ligand 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 cell. In some embodiments, binding of the MHCR to the TCR of the target cell and binding of the SCR to the cell surface receptor of the target cell (i) initiates a signaling cascade effective for eliminating the target cell or (ii) instructs the target cell to differentiate to a specific effector function. 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.
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.
In some embodiments, the MHC portion of the MHCR is N-terminal to the TCR portion of the MHCR. In some embodiments, the MHC portion is directly fused to the TCR portion. In 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 ligand 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.
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. In some embodiments, the cell surface receptor ligand portion of the SCR comprises a CD28 ligand, a CTLA-4 ligand, an ICOS ligand, an OX40 ligand, a PD-1 ligand, or a CD2 ligand. In some embodiments, the CD28 ligand comprises CD80, CD86, or both CD80 and CD86. In some embodiments, the MHCR is adapted to complex with a CD3 subunit. In some embodiments, the engineered cell further co-expresses a second SCR.
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 MHC protein directly or indirectly fused to a T cell receptor (TCR) portion derived from a TCR protein, wherein the MHCR is adapted to bind to a TCR of a target cell.
The present invention also features a method of eliminating a target cell or reprogramming a target cell (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 cell is an autoreactive T cell.
The present invention also features vectors encoding MHCRs of the present invention. The present invention also features vectors encoding SCRs of the present invention.
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-terminal to the TCR portion; and a surrogate coreceptor (SCR) comprising a cell surface receptor ligand 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 TCR of a target cell and the SCR may be adapted to bind to a cell surface receptor of the target cell.
The present invention also features an engineered T-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, the MHC portion being N-terminal to the TCR portion, the MHC portion being selected from 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, 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 ligand portion indirectly linked to a signaling 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 cell and the SCR may be adapted to bind to a cell surface receptor of the target cell.
In some embodiments, the MHC molecule comprises at least 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.
The present invention also features a surrogate co-receptor (SCR) comprising a cell surface receptor ligand portion directly or indirectly fused to a signaling molecule portion via a transmembrane domain, wherein the SCR is adapted to bind to a cell surface receptor of a target cell. In some embodiments, the cell surface receptor ligand portion is indirectly fused to the signaling molecule portion via a linker.
The present invention also features genetically engineered cells (e.g., redirected cells) that express on their surfaces a chimeric receptor according to the present invention. In some embodiments, the cell is a T cell (e.g., CD8+ T cell, CD4+ T cell, etc.). In some embodiments, the cell 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.
The present invention also features a method of eliminating a target cell or reprogramming a target cell (said target cell comprising a TCR). In some embodiments, the method comprises introducing a genetically engineered cell that expresses on its surface a chimeric receptor to the target cell, wherein the chimeric receptor is specific for the TCR of the target cell. In some embodiments, binding of the chimeric receptor on the genetically engineered cell to the TCR of the target cell initiates a signaling cascade that eliminates the target cell. In some embodiments, binding of the chimeric receptor of the genetically engineered cell to the TCR of the target cell instructs the target cell to differentiate to a specific effector function (e.g. Th1, Th2, Th17, Tfh, Treg or cytotoxic T cell). In some embodiments, the chimeric receptor (e.g., MHCR) is expressed on a Treg and binding of the chimeric receptor to the TCR of a target cell inhibits the target cell's function (e.g., redirect the Treg function against an autoimmune cell). In some embodiments, the genetically engineered cell co-expresses a SCR. In some embodiments, the SCR comprises a cell 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 cell surface receptor ligand of the SCR to the cell surface receptor of the target cell initiates a signaling cascade that eliminates the target cell, or instructs the target cell to differentiate to a specific effector function.
In some embodiments, the method is for immunotherapy. In some embodiments, the genetically engineered cell is surgically introduced to a host (e.g., a mammal). In some embodiments, the target cell is an autoreactive T cell.
The present invention also features nucleotide sequences encoding the chimeric receptors of the present invention. The present invention also 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.
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.
The patent application or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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:
Chimeric MHC Receptors (MHCRs)
The present invention features chimeric receptors (e.g., “MHCRs”) comprising at least a MHC portion (e.g., class I, class II, non-classical, a combination thereof, etc.) and a TCR portion (e.g., αβ, γδ TCR, etc.) (see
The MHC portion may comprise one or more MHC proteins (e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1), one or more fragments thereof, or combinations thereof. For reference, non-limiting MHC sequences (human, mouse) are listed below in Table 1.1 and Table 1.2. Note that MHC genes are highly polymorphic, and thus the present invention is not limited to the sequences in Table 1.1 And Table 1.2. The present invention includes MHC polymorphisms and any other appropriate variant of MHC proteins.
Referring to Table 1.1, the HLA-A (MHC 1) sequence (SEQ ID NO: 1) includes the signal peptide (amino acids 1-24); amino acids 25-308 are believed to make up the extracellular region, amino acids 309-332 are believed to make up the transmembrane region, and amino acids 333-365 are believed to make up the cytoplasmic region. The HLA-B (MHC 1) 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 1) sequence (SEQ ID NO: 3) includes the signal peptide (amino acids 1-24); amino acids 25-308 are believed to make up the extracellular region, amino acids 309-333 are believed to make up the transmembrane region, and amino acids 334-366 are believed to make up the cytoplasmic region. The HLA DPA1 (MHC II) sequence (SEQ ID NO: 4) includes the signal peptide (amino acids 1-28); amino acids 29-222 are believed to make up the extracellular region, amino acids 223-245 are believed to make up the transmembrane region, and amino acids 246-260 are believed to make up the cytoplasmic region. The HLA DPB1 (MHC II) sequence (SEQ ID NO: 5) includes the signal peptide (amino acids 1-29); amino acids 30-225 are believed to make up the extracellular region, amino acids 226-246 are believed to make up the transmembrane region, and amino acids 247-258 are believed to make up the cytoplasmic region. The HLA DQA1 (MHC 11) sequence (SEQ ID NO: 6) includes the signal peptide (amino acids 1-23); amino acids 24-216 are believed to make up the extracellular region, amino acids 217-239 are believed to make up the transmembrane region, and amino acids 240-254 are believed to make up the cytoplasmic region. The HLA DQB1 (MHC II) sequence (SEQ ID NO: 7) includes the signal peptide (amino acids 1-32); amino acids 33-230 are believed to make up the extracellular region, amino acids 231-251 are believed to make up the transmembrane region, and amino acids 252-261 are believed to make up the cytoplasmic region. The HLA DRA (MHC II) sequence (SEQ ID NO: 8) includes the signal peptide (amino acids 1-25); amino acids 26-216 are believed to make up the extracellular region, amino acids 217-239 are believed to make up the transmembrane region, and amino acids 240-254 are believed to make up the cytoplasmic region. The HLA DRB1 (MHC II) sequence (SEQ ID NO: 9) includes the signal peptide (amino acids 1-29); amino acids 30-227 are believed to make up the extracellular region, amino acids 228-250 are believed to make up the transmembrane region, and amino acids 251-266 are believed to make up the cytoplasmic region. The MHC E-K alpha chain (SEQ ID NO: 14) includes the signal peptide (aa 1-25), the extracellular domain (aa 26-216), the transmembrane domain (aa 217-24), and a cytoplasmic portion (aa 243-255).
As previously discussed, the MHCR of the present invention comprises at least a MHC portion and a TCR portion. In some embodiments, a MHC portion comprises one or more MHC proteins (e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, MHC E-K alpha, MHC E-K beta, etc.), fragments thereof, or combinations thereof. For example, in some embodiments, the MHC portion comprises a fragment of any of SEQ ID NO: 1-15.
In some embodiments, the MHC portion comprises a peptide that is at least 80% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 85% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 90% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 95% identical to a MHC protein or a fragment thereof. In some embodiments, the MHC portion comprises a peptide that is at least 99% identical to a MHC protein or a fragment thereof.
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. In 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 as in length. In some embodiments, a fragment of a MHC protein is from 25 to 100 aa in length. In some embodiments, a fragment of a MHC protein is from 25 to 150 aa in length. In some embodiments, a fragment of a MHC protein is from 25 to 200 aa in length. In some embodiments, a fragment of a MHC protein is from 25 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 25 to 300 aa in length. In some embodiments, a fragment of a MHC protein is from 25 to 350 aa in length. In some embodiments, a fragment of a MHC protein is from 50 to 100 aa in length. In some embodiments, a fragment of a MHC protein is from 50 to 150 aa in length. In some embodiments, a fragment of a MHC protein is from 50 to 200 aa in length. In some embodiments, a fragment of a MHC protein is from 50 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 50 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 50 to 350 aa in length. In some embodiments, a fragment of a MHC protein is from 100 to 150 aa in length. In some embodiments, a fragment of a MHC protein is from 100 to 200 aa in length. In some embodiments, a fragment of a MHC protein is from 100 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 100 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 100 to 350 aa in length. In some embodiments, a fragment of a MHC protein is from 150 to 200 aa in length. In some embodiments, a fragment of a MHC protein is from 150 to 250 aa in length. In some embodiments, a fragment of a MHC protein is from 150 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 150 to 350 aa in length. In some embodiments, a fragment of a MHC protein is from 200 to 250 as in length. In some embodiments, a fragment of a MHC protein is from 200 to 300 as in length. In some embodiments, a fragment of a MHC protein is from 200 to 350 as in length. In some embodiments, a fragment of a MHC protein is from 250 to 300 aa in length. In some embodiments, a fragment of a MHC protein is from 250 to 350 as in length. In some embodiments, a fragment of a MHC protein is more than 350 aa in length.
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.
Referring to the TRAC protein (SEQ ID NO: 16) in Table 2, amino acids 118-137 are believed to make up the transmembrane domain, and amino acids 138-142 are believed to make up the cytoplasmic domain. Referring to the TRBC1 protein (SEQ ID NO: 17) in Table 2, amino acids 151-171 are believed to make up the transmembrane domain. Referring to the TRBC2 protein (SEQ ID NO: 18) in Table 2, amino acids 145-167 are believed to make up the transmembrane domain. Referring to the TRDC protein (SEQ ID NO: 19) in Table 2, amino acids 130-152 are believed to make up the transmembrane domain. Referring to the TRGC1 protein (SEQ ID NO: 20) in Table 2, amino acids 139-161 are believed to make up the transmembrane domain. Referring to the TRGC2 protein (SEQ ID NO: 21) in Table 2, amino acids 157-177 are believed to make up the transmembrane domain, and amino acids 178-189 are believed to make up the cytoplasmic domain.
As previously discussed, the MHCR of the present invention comprises at least a MHC portion and a TCR portion. In some embodiments, a TCR portion comprises one or more TCR proteins (e.g., TRAC, TRBC1, TRBC2, TRDC, TRCG1, TRCG2, TCRA-mouse, TCB1-mouse, TCB2-mouse, TCC1-mouse, TCC2-mouse, TCC3 mouse, TCC4 mouse, etc.), fragments thereof, or combinations thereof. For example, in some embodiments, the TCR portion comprises a fragment of any of SEQ ID NO: 16-28. (In some embodiments, the fragment is from 5 to 10 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.
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.
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 as in length. In some embodiments, a fragment of a TCR protein is from 10 to 100 aa in length. In some embodiments, a fragment of a TCR protein is from 10 to 150 aa in length. In some embodiments, a fragment of a TCR protein is from 25 to 50 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 as in length. In some embodiments, a fragment of a TCR protein is more than 150 aa in length.
In some embodiments, the MHCR comprises a peptide antigen. Any appropriate peptide antigen may be used. The peptide antigen in the pMHCR complex directs the specificity of the pMHCR molecule, therefore the pMHCR molecule will be specific for T cells with TCRs that are specific for that peptide antigen/pMHCR. A non-limiting example of a peptide antigen that may be used with the MHCR is moth cytochrome c peptide (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 mouse models. Any appropriate peptide antigen may be used, and the present invention is not limited to the peptide antigens disclosed herein. For example, in some embodiments, the peptide antigen comprises any immunodominant peptide antigen identified to bind a class I or class 11 MHC. In some embodiments, the peptide antigen comprises any immunodominant peptide antigen identified to bind a class I or class II MHC and elicit a response. A response may include but is not limited to an autoimmune response, an allergic response, an asthma response, or an inappropriate Treg response. The peptide antigen may be any appropriate length.
In some embodiments, the MHCR comprises at least a portion of a MHC molecule that allows for binding to an appropriate TCR. In some embodiments, the MHCR comprises at least a portion of a MHC molecule that allows for binding to an appropriate TCR and at least a portion of a TCR molecule (e.g., a portion of a TCR molecule that allows for appropriate signaling and/or complexing subunits such as CD3 subunits). In some embodiments, the MHCR comprises a transmembrane domain that is at least partially derived from (i) a MHC molecule, (ii) a TCR molecule, or (iii) both the MHC molecule and TCR molecule. In some embodiments, the MHCR comprises a transmembrane domain, wherein a portion (or all) of the transmembrane domain is not derived from a MHC molecule or a TCR molecule. In some embodiments, the MHCR comprises an extracellular domain that is at least partially derived from (i) a MHC molecule, (ii) a TCR molecule, or (iii) both the MHC molecule and TCR molecule. In some embodiments, the MHCR comprises an extracellular domain, wherein a portion of the extracellular domain is not derived from a MHC molecule or a TCR molecule.
As an example, in some embodiments, the MHCR comprises at least a portion of the extracellular domain of a MHC molecule (e.g., the extracellular domain of HLA-DRA) and at least a portion of the transmembrane domain of a TCR molecule and at least a portion of the cytoplasmic domain of a TCR molecule. As another example, in some embodiments, the MHCR comprises at least a portion of the extracellular domain of a TCR molecule.
The present invention also features redirected cells, such as redirected T cells, expressing MHCRs of the present invention, e.g., as described above. Without wishing to limit the present invention to any theory or mechanism, the MHCRs are generally adapted to recognize and bind to appropriate (specific) TCRs. In some embodiments, the MHCR is expressed in a CD8+ T cell (e.g., a cytotoxic T cell, TC cells, CTLs). In some embodiments, the MHCR is expressed in a CD4+ T cell (e.g., a T helper cell, TH cell or a regulatory T cell (Treg cell)). The present invention is not limited to the expression of MHCRs in T cells, nor is the present invention limited to expression of MHCRs in CD8+ or CD4+ T cells, e.g., the MHCRs may be expressed in CD8+/CD4+ thymocytes, γδT cells, NK cells, NK T cells, etc. In some embodiments, the MHCR of the redirected T cell complexes or is adapted to complex with CD3 subunits (e.g., forming a MHCR-CD3 complex).
In some embodiments, the MHCR comprises a MHC portion derived from an extracellular portion of a MHC protein and a TCR portion derived from a transmembrane domain of a TCR protein. In some embodiments, the MHC portion and TCR portion are directly linked. In some embodiments, the MHC portion and TCR portion are separated by a linker. In some embodiments, the linker comprises a glycine-rich linker.
The present invention is not limited to the MHC portions and TCR portions described herein. For example, the MHC portion may comprise any MHC peptide, e.g., an extracellular domain (or a portion thereof) of any MHC peptide. The TCR portion may comprise any TCR peptide, e.g., a transmembrane domain (or portion thereof) of any TCR peptide. Further, the present invention is not limited to antigens, signaling molecules, and cell surface receptor ligands described herein, e.g., the present invention may be applicable to a wide range of MHC molecules, TCR molecules, antigens, signaling molecules cell surface receptor ligands, etc.
Surrogate Coreceptors (SCRs)The present invention also features chimeric surrogate coreceptors (SCR), e.g., receptors that recruit signaling molecules (e.g., kinases such as but not limited to Src kinases (e.g., Lck), phosphatases, etc.). In some embodiments, the SRCs recruit signaling molecules (e.g., kinases) to the MHCR and/or CD3 subunits. The present invention also features cells expressing a SCR. In some embodiments, redirected cells, e.g., redirected T cells, express both a MHCR and a SCR. In some embodiments, cells express more than one type of SCR. Without wishing to limit the present invention to any theory or mechanism, it is believed that certain SCRs may enhance signaling through the pMHCR-CD3 complex.
In some embodiments, the SCR comprises a cell surface receptor ligand (e.g., T cell surface receptor ligand) fused to a signaling molecule (e.g., kinase (e.g., Lck or other appropriate kinase), phosphatase, etc.). In some embodiments, the cell surface receptor ligand and the kinase are separated by a linker, e.g., a peptide linker or any other appropriate linker. The signaling molecule is not limited to a kinase or a phosphatase.
In some embodiments, the cell surface receptor ligand (e.g., T cell surface receptor ligand) comprises CD80, CD86, fragments thereof, or combinations thereof. The present invention is not limited to CD80 and CD86; any other appropriate cell surface receptor ligand (or a fragment thereof) may be used. For example, in some embodiments, the cell surface receptor ligand comprises a CD28 ligand, a CTLA-4 ligand, an ICOS ligand, an OX40 ligand, a PD-1 ligand (e.g., PD-1L), a CD2 ligand, etc.
As an example, in some embodiments, when a T cell is expressing a pMHCR (a MHCR with a peptide antigen), the pMHCR may complex with CD3 subunits, forming a pMHCR-CD3 complex. If the cell is also expressing a CD80-Lck SCR, then when the pMHCR binds a TCR on a target T cell, the CD80-Lck may also bind to CD28 on the same target T cell. Without wishing to limit the present invention to any theory or mechanism, it is believed that then the CD80-Lck SCR should recruit Lck to the pMHCR-CD3 complex to phosphorylate the pMHCR-CD3 ITAMs for robust signaling.
In some embodiments, the SCR is engineered (e.g., a particular cell surface receptor ligand of the SCR is selected) to target a specific set of target cells. For example, T follicular helper cells express a molecule called PD-1 and these cells provide help to B cells to make autoantibodies in autoimmune diseases such as Lupus. The ligand for PD-1 is PD-1L, so a SCR comprising PD-1L and Lck may be co-expressed with a pMHCR recognized by the TCR of the T follicular helper cell. This may allow for targeting of this specific T follicular helper cell population.
The present invention also features methods of use of said MHCRs, SCRs, and/or said redirected cells, for example for immunotherapy. In some embodiments, the redirected cells may eliminate autoreactive T cells, regulatory T cells (Tregs) that protect tumor cells by suppressing anti-tumor T cell responses, or any other appropriate T cell. For example, in some embodiments, the MHCR is an auto-antigen MHCR, and the MHCR's target is an autoreactive T cell.
ExamplesExample 1: Redirected T cells targeting CD4 T Helper Cells. Example 1 describes a non-limiting experimental approach to target CD4 T cells. A prototype pMHCR was engineered with a peptide antigen: the moth cytochrome c peptide (SEQ ID NO: 29) was fused to the mouse class II MHC I-Ek (MCC:I-Ek; e.g., see SEQ ID NO: 31). This pMHCR was expressed (e.g., retrovirally expressed) in T cell hybridomas. It was determined that this pMHCR (e.g., pMHCR-CD3 complex) was expressed on the surface of T cell hybridomas (see
Lck fusions were generated with known ligands for T cell surface receptors. For example, all T cells express CD28. Lck fusions with CD28 ligands (e.g., CD80, CD86) were engineered to generate surrogate coreceptors (SCRs), e.g., CD80-Lck (see SEQ ID NO: 33, SEQ ID NO: 38), e.g., CD86-Lck (see SEQ ID NO: 34, SEQ ID NO: 39). When the pMHCR-CD3 complex was co-expressed with SCR CD80-Lck in hybridomas, these cells produced significantly more IL-2 in response to cells expressing the 2B4 TCR ligand+CD28 than they did in response to cells expressing only the 284 TCR ligand (see
MCC:IEk pMHCR-CD3 and the SCR CD80-Lck or HB:IEk pMHCR-CD3 (e.g., see SEQ ID NO: 32) and the SCR CD80-Lck were expressed in in vitro differentiated CD8 cytotoxic T cells (CTLs) and their ability to kill 5c.c7 TCR transgenic CD4 T cells expressing the TCR specific for the MCC:IEk pMHCR was evaluated. Surface expression of the pMHCRs on the redirected CTLs was observed, suggesting that these chimeric receptor modules compete with the endogenous TCR for assembly with the endogenous CD3 subunits (data not shown). CTLs expressing the MCC:IEk pMHCR robustly killed the target CD4 T cells while those expressing the null HB:IEk pMHCR did not (see
Example 2: Redirected T cells targeting CD4 T Helper Cells in Allergic Asthma. Example 2 describes a non-limiting experimental approach to target CD4 T helper cells involved in allergic asthma, e.g., to help eliminate naïve Der p 1-specific CD4 T cells from the repertoire prior to House Dust Mite (HDM) sensitization. Without wishing to limit the present invention to any theory or mechanism, it is believed that eliminating allergen-specific CD4 T cells from the repertoire may help prevent the onset of TH2 immunity upon HDM sensitization.
A pMHCR (pMHCR-CD3 complex) will be retrovirally expressed in in vitro activated CTLs. The pMHCR will bear a pMHCR comprising either the immunodominant HDM-derived Der p 1 epitope (aa117-127) in the context of I-Ab (Derp1:IAb) or the immunodominant West Nile Virus peptide from the envelope protein (aa641-655) in the context of I-Ab (E641:IAb). The E641:IAb pMHCR cells will serve as a non-specific control population.
The in vitro activated CTLs will also be transduced with a CD80-Lck SCR to enhance signaling. These redirected CTLs will then be transferred intravenously into C57Bl/6 mice to target and eliminate Derp1:IAb- or E641:IAb-specific naïve CD4 T cells from the endogenous repertoire. After a certain length of time, e.g., 1 week, the elimination of antigen-specific CD4 T cells will be evaluated. This will be performed via tetramer enrichment experiments using a Derp1:IAb tetramer and a E641:IAb tetramer. The presence of the redirected CD8 T cells will also be assessed by flow cytometry by gating on CD3+CD8+IAb+ T cells since mouse T cells do not express class II MHC.
After determining if the redirected CTLs eliminate the target population, mice that received redirected CTLs one-week prior will be sensitized with HDM (e.g., intranasally, e.g., with HDM extracts). This will be done even if endogenous CD4 T cells specific for Derp1:IAb are detected, but only if redirected T cells are still present in the mice. This may help to determine if activation of the CD4 T cells made them more susceptible to targeting by the redirected CTLs.
Example 3: Redirected T cells targeting CD4 T Helper Cells in Lungs After Sensitization. Example 3 describes a non-limiting experimental approach to target CD4 T helper cells in lungs of HDM-sensitized mice. Without wishing to limit the present invention to any theory or mechanism, it is believed that eliminating allergen-specific CD4 T cells from the lungs of HDM-sensitized mice may help attenuate TH2 immunity.
Der p 1-specific CD4 T cells will be targeted similarly to Example 2, but only after HDM sensitization. In brief, mice will be sensitized with HDM according to the protocol described above. They will then receive redirected Derp1:IAb or E641:IAb pMHCR-CD3 CTLs on day 14. Various surrogate co-receptors will be employed to explore the efficacy of the technology and approach. For example, the CD80-Lck fusion SCR will be used, as well as others, e.g., a TIM-4-Lck SCR (since the TIM-1 expressed on CD4 T cells is genetically linked with asthma and this combination for targeting might enhance effectiveness). One week after transfer of redirected CTLs, cytokine and cellular analysis will be performed as described above in Example 2 so as to assess the impact of these cells on the lung cytokine milieu and cellularity. The status of the redirected CTLs will also be evaluated.
Example 4: Attenuation of Der p 1-specific CD4 T cell function in situ. 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 TH2 immunity.
In vitro generated induced Tregs (iTregs) expressing a MHCR will be tested for efficacy in reducing HDM-induced airway hypersensitivity. Induced Tregs (iTregs) will be generated in vitro and transduced with pMHCR and SCRs as described in Examples 2 and 3 above. These cells will then either be transferred prior to HDM sensitization as in Example 2 or after sensitization as in Example 3. Evaluation of the lung cytokine milieu and cellularity will then be performed as described above.
Table 3 shows examples of protein sequences for reagents the above examples. Table 4 shows the nucleotide sequences for the proteins in Table 3. Note that in SEQ ID NO: 30, a portion is derived from SEQ ID NO: 14 and a portion is derived from SEQ ID NO: 22. In SEQ ID NO: 31, a portion is derived from SEQ ID NO: 15, a portion is derived from SEQ ID NO: 23, and a portion is derived from SEQ ID NO: 29 (and other residues may correspond to a glycine-rich linking region). In SEQ ID NO: 32, a portion is derived from SEQ ID NO: 15 and a portion is derived from SEQ ID NO: 23 (and other residues may correspond to a glycine-rich linking region).
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. Nos. 8,450,112; 7,741,465; 6,319,494; CA 2209300; CA 2104957; EP 0574512; U.S. Pat. Nos. 6,407,221; 6,268,411; U.S. Pat. Application No. 20040258697; EP 1292621; EP 2659893; WO 2011101681; WO 2005054292; EP 1379670; U.S. Pat. Nos. 6,056,952; 6,410,319; 8,524,234; 7,871,817.
As used herein, the term “about” refers to plus or minus 10% of the referenced number.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.
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.
Claims
1. An engineered cell, comprising:
- a. A chimeric receptor module (MHCR) that comprises: i) an extracellular domain of a major histocompatibility complex (MHC); ii) a T-cell receptor (TCR) portion comprising a transmembrane domain of a TCR, and a cytoplasmic domain of a TCR; and
- b. A surrogate coreceptor (SCR) that comprises: i) an extracellular region of a cell surface receptor ligand; ii) a transmembrane region; and iii) a kinase.
2. The engineered cell of claim 1, wherein the extracellular domain of the MHC is directly bound to the TCR portion.
3. The engineered cell of claim 1, wherein an antigenic peptide is bound to the extracellular domain of the MHC.
4. The engineered cell of claim 1, wherein the extracellular domain of the MHC is derived from an MHC selected from the group consisting of: HLA-A, HLA-B, HLA-C, Beta2-microglobulin, HLA-DPA, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB, H2-Aa, H2-B1, H2-K1, H2-EB beta, H2-EK alpha, and H2-EK beta.
5. The engineered cell of claim 1, wherein the transmembrane domain and the cytoplasmic domain of the TCR are derived from a TCR selected from the group consisting of: TRAC, TRBC1, TRBC2, TRDC, TRGC1, and TRGC2.
6. The engineered cell of claim 1, wherein the cell surface receptor ligand is a T-cell surface receptor ligand.
7. The engineered cell of claim 1, wherein the T-cell surface receptor ligand is selected from the group consisting of: a CD28 ligand, a CTLA-4 ligand, an ICOS ligand, an OX40 ligand, and a CD2 ligand.
8. The engineered cell of claim 1, wherein the T-cell surface receptor ligand is selected from the group consisting of: CD80 and CD86.
9. The engineered cell of claim 1, wherein the kinase is a Src kinase.
10. The engineered cell of claim 9, wherein said Src kinase is Lck or Fyn.
11. The engineered cell of claim 6, wherein the T-cell surface ligand is CD80, and the kinase is Lck.
12. The engineered cell of claim 6, wherein the T-cell surface ligand is CD86, and the kinase is Lck.
13. The engineered cell of claim 1, wherein said engineered cell is a T cell, NK cell, or NK T cell.
Type: Application
Filed: Jun 11, 2021
Publication Date: Oct 7, 2021
Inventors: Michael S Kuhns (Tucson, AZ), Thomas Serwold (Boston, MA)
Application Number: 17/345,425