Methods and compositions for cell therapy

Abstract

Provided herein is a synthetic construct (synHLA) comprising one or more human leukocyte antigens, a targeting moiety, and a linker region, the synthetic construct not eliciting an immune response. Also provided are a nucleic acid molecule encoding the construct, an immunocompromised stem cell comprising the construct or the nucleic acid molecule, and a method of treating a disease or disorder comprising administering the construct or the nucleic acid molecule.

Description

[Technical field]

[0001] (cross reference) This application claims the benefit of U.S. Patent Application No. 2205771.5, filed April 20, 2022, which is incorporated by reference in its entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated by reference in its entirety. The XML copy created on April 18, 2023 is named 62600-718_601_SL.xml and is 352,190 bytes in size. [Background technology]

[0003] Cellular therapy holds great promise for combating previously intractable diseases. While the use of autologous cells is generally optimal, this approach can be cost-prohibitive and cumbersome. Allogeneic pluripotent stem cells (PSCs) offer greater scalability and cost savings, but their utility is limited by the need to match human leukocyte antigen (HLA) class 1 alleles, the most genetically polymorphic region in the human genome. Mismatches in HLA class 1 haplotypes result in a "self versus non-self" immune response that can result in the body's rejection of transplanted therapeutic cells. The general utility of recent efforts to engineer HLA constructs that block the elicitation of T cell-activating immune responses has been limited by the inability of these constructs to successfully engage killer cell immunoglobulin-like receptors (KIRs), resulting in a "missing self" immune response. As a result, there remains an unmet need for the development of engineered pluripotent stem cells that circumvent both T cell- and KIR-mediated immune responses. Summary of the Invention

[0004] One aspect of the disclosure provides a construct comprising one or more targeting moieties: one or more major histocompatibility complex (MHC) regions, at least one of the one or more MHC regions comprising a cluster of differentiation 8 (CD8) binding site and one or more linker regions. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the construct further comprises one or more disulfide staple pairs. In some embodiments, the one or more MHC regions are inhibited from eliciting a T cell or NK response when the construct is interrogated by one or more T cells. In some embodiments, the one or more MHC regions comprise one or more mutated residues compared to a wild-type version of the one or more MHC regions, the one or more mutated residues being located at a position corresponding to the tyrosine 84 (Y84) residue of human leukocyte antigen (HLA) protein HLA-C. In some embodiments, the one or more mutated residues comprise an alanine. In some embodiments, the one or more mutated residues comprise a cysteine. In some embodiments, the CD8 binding site comprises a mutation to a residue corresponding to the Q226 residue of HLA-C. In some embodiments, the CD8 binding site comprises a mutation to a residue corresponding to the D227K residue of HLA-C. In some embodiments, the T225 residue of the CD8 binding site is deleted. In some embodiments, the Q226 residue of the CD8 binding site is deleted. In some embodiments, the T225 residue of the CD8 binding site is deleted. In some embodiments, the E232 residue of the CD8 binding site is deleted. In some embodiments, the one or more MHC regions further comprise one or more mutations to a residue corresponding to the C1 residue of HLA-C. In some embodiments, the residue is glycine. In some embodiments, a disulfide staple pair is formed between a residue of the one or more MHC regions corresponding to the Y84 residue of HLA-C and a residue in one or more linker regions. In some embodiments, a disulfide staple pair is formed between a residue of the one or more MHC regions corresponding to the R69 residue of HLA-C and a residue in one or more targeting moieties.In some embodiments, disulfide staple pairs are formed between residues in one or more MHC regions corresponding to the A150 residue of HLA-C and residues in one or more targeting moieties. In some embodiments, disulfide staple pairs are formed between residues in one or more MHC regions corresponding to the A73 residue of HLA-C and residues in one or more targeting moieties. In some embodiments, the construct is soluble. In some embodiments, the construct is insoluble. In some embodiments, the construct comprises a beta-2 microglobulin (B2M) leader sequence. In some embodiments, the construct further comprises an N-terminal signal sequence. In some embodiments, the construct further comprises a C-terminal signal sequence. In some embodiments, the construct is a single chain trimer (SCT). In some embodiments, the construct is a single chain dimer (SCD). In some embodiments, the one or more MHC regions comprise one or more human HLA class 1 heavy chain sequences. In some embodiments, the one or more human HLA class 1 heavy chain sequences are derived from HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, or some combination thereof. In some embodiments, the one or more targeting moieties comprise a peptide. In some embodiments, the peptide comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, a linker region of the one or more linker regions comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the construct further comprises a first linker region and a second linker region, wherein the first linker region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186, and the second linker region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186.In some embodiments, the construct further comprises a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 30-129 and 142-157. In some embodiments, the construct comprises, in order from N-terminus to C-terminus, a targeting moiety of the one or more targeting moieties; a first linker of the one or more linkers; and an MHC region of the one or more MHC regions; and a disulfide stable pair configured to associate the targeting moiety with the MHC region or configured to associate the first linker with the MHC region. In some embodiments, the targeting moiety of the one or more targeting moieties comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, the first linker comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the MHC region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 130-141. In some embodiments, the construct further comprises a B2M leader sequence between the first linker and the MHC region. In some embodiments, the construct further comprises a second linker between the B2M leader sequence and the MHC region. In some embodiments, the second linker comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186.

[0005] Another aspect of the present disclosure provides hypoimmunogenic pluripotent stem cells comprising a construct comprising one or more targeting moieties; one or more major histocompatibility complex (MHC) regions, at least one of the one or more MHC regions comprising a cluster of differentiation 8 (CD8) binding site; and one or more linker regions. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the hypoimmunogenic pluripotent stem cells further comprise one or more disulfide staple pairs. In some embodiments, when the complex is probed by one or more T cells, the one or more MHC regions are inhibited from eliciting a T cell response. In some embodiments, the one or more MHC regions comprise one or more mutated residues compared to a wild-type version of the one or more MHC regions, the one or more mutated residues being located at a position corresponding to the tyrosine 84 (Y84) residue of human leukocyte antigen (HLA) protein HLA-C. In some embodiments, the one or more mutated residues comprise an alanine. In some embodiments, the one or more mutated residues comprise a cysteine. In some embodiments, the CD8 binding site comprises a mutation to a residue corresponding to the Q226 residue of HLA-C. In some embodiments, the CD8 binding site comprises a mutation to a residue corresponding to the D227K residue of HLA-C. In some embodiments, the T225 residue of the CD8 binding site is deleted. In some embodiments, the Q226 residue of the CD8 binding site is deleted. In some embodiments, the T22D2275 residue of the CD8 binding site is deleted. In some embodiments, the E232 residue of the CD8 binding site is deleted. In some embodiments, the one or more MHC regions further comprise one or more mutations to a residue corresponding to the C1 residue of HLA-C. In some embodiments, the residue is glycine. In some embodiments, disulfide staple pairs are formed between a residue of one or more MHC regions corresponding to the Y84 residue of HLA-C and a residue in one or more linker regions. In some embodiments, disulfide staple pairs are formed between a residue of one or more MHC regions corresponding to the R69 residue of HLA-C and a residue in one or more targeting moieties.In some embodiments, disulfide staple pairs are formed between residues in one or more MHC regions corresponding to the A150 residue of HLA-C and residues in one or more targeting moieties. In some embodiments, disulfide staple pairs are formed between residues in one or more MHC regions corresponding to the A73 residue of HLA-C and residues in one or more targeting moieties. In some embodiments, the construct is soluble. In some embodiments, the construct is insoluble. In some embodiments, the construct comprises a beta-2 microglobulin (B2M) leader sequence. In some embodiments, the construct further comprises an N-terminal signal sequence. In some embodiments, the construct further comprises a C-terminal signal sequence. In some embodiments, the construct is a single chain trimer (SCT). In some embodiments, the construct is a single chain dimer (SCD). In some embodiments, the one or more MHC regions comprise one or more human HLA class 1 heavy chain sequences. In some embodiments, the one or more human HLA class 1 heavy chain sequences are derived from HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, or some combination thereof. In some embodiments, the one or more targeting moieties comprise a peptide. In some embodiments, the peptide comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, a linker region of the one or more linker regions comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the hypoimmunogenic pluripotent stem cells further comprise a first linker region and a second linker region, wherein the first linker region comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186, and the second linker region comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186.In some embodiments, the hypoimmunogenic pluripotent stem cells further comprise a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 30-129 and 142-157. In some embodiments, the construct comprises, in order from N-terminus to C-terminus, a targeting moiety of the one or more targeting moieties; a first linker of the one or more linkers; and an MHC region of the one or more MHC regions; and a disulfide stable pair configured to associate the targeting moiety with the MHC region or configured to associate the first linker with the MHC region. In some embodiments, the targeting moiety of the one or more targeting moieties comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, the first linker comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the MHC region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 130-141. In some embodiments, the hypoimmunogenic pluripotent stem cells further comprise a B2M leader sequence between the first linker and the MHC region. In some embodiments, the hypoimmunogenic pluripotent stem cells further comprise a second linker between the B2M leader sequence and the MHC region. In some embodiments, the second linker comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. 1. A method of generating hypoimmunogenic pluripotent stem cells, comprising: generating a construct comprising one or more targeting moieties, one or more linker regions, and one or more major histocompatibility complex (MHC) regions, at least one of which comprises a cluster of differentiation 8 (CD8) binding site; providing the construct in pluripotent stem cells (PSCs); and expressing the construct in the (PSCs). In some embodiments, the CD8 binding site comprises one or more mutations.In some embodiments, when the complex is interrogated by one or more T cells, the one or more MHC regions are inhibited from eliciting a T cell response. In some embodiments, the one or more MHC regions comprise one or more mutated residues compared to a wild type version of the one or more MHC regions, the one or more mutated residues being located at a position corresponding to the tyrosine 84 (Y84) residue of human leukocyte antigen (HLA) protein HLA-C. In some embodiments, the one or more mutated residues comprise an alanine. In some embodiments, the one or more mutated residues comprise a cysteine. In some embodiments, the CD8 binding site comprises a mutation to a residue corresponding to the Q226 residue of HLA-C. In some embodiments, the CD8 binding site comprises a mutation to a residue corresponding to the D227K residue of HLA-C. In some embodiments, the T225 residue of the CD8 binding site is deleted. In some embodiments, the Q226 residue of the CD8 binding site is deleted. In some embodiments, the T22D2275 residue of the CD8 binding site is deleted. In some embodiments, the E232 residue of the CD8 binding site is deleted. In some embodiments, the one or more MHC regions further comprise one or more mutations to a residue corresponding to the C1 residue of HLA-C. In some embodiments, the residue is glycine. In some embodiments, a disulfide staple pair is formed between a residue in one or more MHC regions corresponding to the Y84 residue of HLA-C and a residue in one or more linker regions. In some embodiments, a disulfide staple pair is formed between a residue in one or more MHC regions corresponding to the R69 residue of HLA-C and a residue in one or more targeting moieties. In some embodiments, a disulfide staple pair is formed between a residue in one or more MHC regions corresponding to the A150 residue of HLA-C and a residue in one or more targeting moieties. In some embodiments, a disulfide staple pair is formed between a residue in one or more MHC regions corresponding to the A73 residue of HLA-C and a residue in one or more targeting moieties. In some embodiments, the construct is soluble. In some embodiments, the construct is insoluble. In some embodiments, the construct comprises a beta-2 microglobulin (B2M) leader sequence.In some embodiments, the construct further comprises an N-terminal signal sequence. In some embodiments, the construct further comprises a C-terminal signal sequence. In some embodiments, the construct is a single chain trimer (SCT). In some embodiments, the construct is a single chain dimer (SCD). In some embodiments, the one or more MHC regions comprise one or more human HLA class 1 heavy chain sequences. In some embodiments, the one or more human HLA class 1 heavy chain sequences are derived from HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, or some combination thereof. In some embodiments, the one or more targeting moieties comprise a peptide. In some embodiments, the peptide comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, a linker region of the one or more linker regions comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the method further comprises a first linker region and a second linker region, wherein the first linker region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186, and the second linker region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the method further comprises a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 30-129 and 142-157. In some embodiments, the construct comprises, in order from N-terminus to C-terminus, a targeting moiety of the one or more targeting moieties; a first linker of the one or more linkers; and an MHC region of the one or more MHC regions; and a disulfide stable pair configured to associate the targeting moiety with the MHC region or configured to associate the first linker with the MHC region.In some embodiments, the targeting moiety of the one or more targeting moieties comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, the first linker comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the MHC region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 130-141. In some embodiments, the method further comprises a B2M leader sequence between the first linker and the MHC region. In some embodiments, the method further comprises a B2M leader sequence and an MHC. In some embodiments, the second linker further comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175 to 186.

[0006] Another aspect of the present disclosure is a construct comprising: a targeting moiety; a major histocompatibility complex (MHC) region; and a linker region disposed between the targeting moiety and the MHC region; one of the targeting moiety, the MHC region, and the linker region comprises a first cysteine ​​residue and another of the targeting moiety, the MHC region, and the linker region comprises a second cysteine ​​residue, wherein the first cysteine ​​residue and the second cysteine ​​residue are configured to form a disulfide bond with each other when the construct is expressed on the surface of a cell.

[0007] In some embodiments, the MHC region comprises an MHC class I heavy chain. In some embodiments, the MHC region is derived from an HLA-A, HLA-B, or HLA-C sequence. In some embodiments, the MHC class I heavy chain comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 130-141 and 279-288. In some embodiments, the MHC class I heavy chain comprises a mutation corresponding to the C1 residue of HLA-C (e.g., SEQ ID NO: 194). In some embodiments, the mutation comprises a glycine residue. In some embodiments, the construct further comprises a second MHC region. In some embodiments, the second MHC region is derived from an HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G sequence. In some embodiments, the construct further comprises a beta-2 microglobulin (B2M) region. In some embodiments, the B2M region is disposed between the targeting moiety and the MHC region. In some embodiments, the B2M region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 195. In some embodiments, the construct further comprises a second linker region disposed between the B2M region and the targeting moiety or the MHC region. In some embodiments, the linker region is disposed between the targeting moiety and the B2M region, and the second linker region is disposed between the B2M region and the MHC region. In some embodiments, the linker region is less than 15 amino acid residues in length. In some embodiments, the linker region is less than 14 amino acid residues in length. In some embodiments, the linker region is less than 13 amino acid residues in length. In some embodiments, the linker region is at least 8 amino acid residues in length. In some embodiments, the linker region comprises at least one cysteine ​​residue. In some embodiments, the linker region comprises an amino acid selected from the group consisting of glycine, serine, and cysteine. In some embodiments, the linker region comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs:175-186.In some embodiments, the linker region comprises a sequence selected from SEQ ID NOs: 175-186 and 198. In some embodiments, the second linker region comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the second linker region comprises a sequence selected from SEQ ID NOs: 175-186 and 198. In some embodiments, the linker region and the second linker region are independently selected from SEQ ID NOs: 175-186 and 198. In some embodiments, the linker region comprises SEQ ID NO: 175 or 176 and the second linker region comprises SEQ ID NO: 183. In some embodiments, the targeting moiety comprises a first cysteine ​​residue and the MHC region comprises a second cysteine ​​residue. In some embodiments, the first cysteine ​​residue is located at any one of positions 19 of the targeting moiety. In some embodiments, the first cysteine ​​residue is the C5, C7, or C8 residue of the targeting moiety. In some embodiments, the second cysteine ​​residue corresponds to the Y84 residue of the HLA-C heavy chain. In some embodiments, the second cysteine ​​residue corresponds to the R69 residue of the HLA-C heavy chain. In some embodiments, the second cysteine ​​residue corresponds to the A73 residue of the HLA-C heavy chain. In some embodiments, the second cysteine ​​residue corresponds to the A150 residue of the HLA-C heavy chain. In some embodiments, the linker region comprises the first cysteine ​​and the MHC region comprises the second cysteine ​​residue. In some embodiments, the first cysteine ​​residue is C2 of the linker. In some embodiments, the targeting moiety comprises a peptide configured to form a complex with the MHC region. In some embodiments, the peptide comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, the peptide comprises a second amino acid residue selected from L, M, S, I, F, T, V, and Y. In some embodiments, the second amino acid residue is selected from T, V, and Y. In some embodiments, the peptide comprises a final amino acid residue selected from V, I, F, W, Y, L, R, and K.In some embodiments, the last amino acid residue is selected from Y, L, R, and K. In some embodiments, the peptide comprises a second amino acid residue selected from E, P, L, Q, A, R, H, S, T, V, M, D, and K. In some embodiments, the second amino acid residue is selected from E, P, L, Q, A, R, and H. In some embodiments, the peptide comprises a last amino acid residue selected from V, L, F, A, I, Y, M, W, P, and R. In some embodiments, the last amino acid residue is selected from V, L, and F. In some embodiments, the peptide comprises a second amino acid residue selected from A, Y, S, T, V, I, L, F, Q, R, N, and W. In some embodiments, the second amino acid residue is selected from A and Y. In some embodiments, the peptide comprises a last amino acid residue selected from L, V, M, F, Y, and I. In some embodiments, the last amino acid residue is L. In some embodiments, the last amino acid residue is the 9th, 10th, 11th or 12th residue of the peptide. In some embodiments, the construct comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 30-129, 142-157, and 203-278. In some embodiments, the construct is inhibited from eliciting an NK cell response when the construct is probed by one or more NK cells.

[0008] A construct comprising: a targeting moiety; a major histocompatibility complex (MHC) region; and a linker region disposed between the targeting moiety and the MHC region; the linker region comprising fewer than 15 amino acid residues.

[0009] In some embodiments, the MHC region comprises an MHC class 1 heavy chain. In some embodiments, the MHC class I heavy chain is derived from an HLA-A, HLA-B, or HLA-C sequence. In some embodiments, the MCH region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 130-141 and 279-288. In some embodiments, the MHC class I heavy chain comprises a mutation corresponding to the C1 residue of HLA-C (e.g., SEQ ID NO: 194). In some embodiments, the mutation comprises a glycine residue. In some embodiments, the construct further comprises a second MHC region. In some embodiments, the second MHC region is derived from an HLA-A, BLA-B, HLA-C, HLAE, HLA-F, or HLA-G sequence. In some embodiments, the construct further comprises a beta-2 microglobulin (B2M) region. In some embodiments, the B2M region is disposed between the targeting moiety and the MHC region. In some embodiments, the B2M region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to SEQ ID NO: 195. In some embodiments, the construct further comprises a second linker region disposed between the B2M region and the targeting moiety or the MHC region. In some embodiments, the linker region is disposed between the targeting moiety and the B2M region, and the second linker region is disposed between the B2M region and the MHC region. In some embodiments, the linker region is less than 14 amino acid residues in length. In some embodiments, the linker region is less than 13 amino acid residues in length. In some embodiments, the linker region is at least 8 amino acid residues in length. In some embodiments, the linker comprises a first cysteine ​​residue configured to form a disulfide staple pair with a second cysteine ​​residue of the MHC region. In some embodiments, the first cysteine ​​residue is C2 of the linker. In some embodiments, the linker comprises an amino acid selected from the group consisting of glycine, serine, and cysteine.In some embodiments, the linker comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186 and 198. In some embodiments, the linker comprises a sequence selected from SEQ ID NOs: 175-186 and 198. In some embodiments, the second linker region comprises a sequence that is at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 175-186. In some embodiments, the second linker region comprises a sequence selected from SEQ ID NOs: 175-186 and 198. In some embodiments, the linker region and the second linker region are independently selected from SEQ ID NOs: 175-186 and 198. In some embodiments, the linker region comprises SEQ ID NO: 175 or 176 and the second linker region comprises SEQ ID NO: 183. In some embodiments, the targeting moiety comprises a peptide configured to form a complex with an MHC region. In some embodiments, the peptide comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99% identical to any one of SEQ ID NOs: 158-174. In some embodiments, the peptide comprises a second amino acid residue selected from L, M, S, I, F, T, V, and Y. In some embodiments, the second amino acid residue is selected from T, V, and Y. In some embodiments, the peptide comprises a final amino acid residue selected from V, I, F, W, Y, L, R, and K. In some embodiments, the final amino acid residue is selected from Y, L, R, and K. In some embodiments, the peptide comprises a second amino acid residue selected from E, P, L, Q, A, R, H, S, T, V, M, D, and K. In some embodiments, the second amino acid residue is selected from E, P, L, Q, A, R, and H. In some embodiments, the peptide comprises a final amino acid residue selected from V, L, F, A, I, Y, M, W, P and R. In some embodiments, the final amino acid residue is selected from V, L and F. In some embodiments, the peptide comprises a second amino acid residue selected from A, Y, S, T, V, I, L, F, Q, R, N and W.In some embodiments, the second amino acid residue is selected from A and Y. In some embodiments, the peptide comprises a final amino acid residue selected from L, V, M, F, Y and I. In some embodiments, the final amino acid residue is L. In some embodiments, the final amino acid residue is the 9th, 10th, 11th or 12th residue of the peptide.

[0010] Another aspect of the disclosure provides a nucleic acid encoding any of the constructs disclosed herein.

[0011] Another aspect of the disclosure provides engineered vectors encoding any of the nucleic acids disclosed herein.

[0012] In some embodiments, the vector is a plasmid, a minicircle, a CELiD, an adeno-associated virus (AAV) derived virion, a lentivirus, an adenovirus, or a herpes simplex virus (HSV).

[0013] Another aspect of the disclosure provides a method of generating a hypoimmunogenic cell, comprising administering to a cell any of the vectors disclosed herein.

[0014] Another aspect of the present disclosure provides a hypoimmunogenic cell comprising any of the constructs disclosed herein.

[0015] In some embodiments, the cells are stem cells, hi some embodiments, the stem cells are embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), or hematopoietic stem cells (HSCs).

[0016] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification and in the appendices attached hereto are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over such conflicting material. [Brief description of the drawings]

[0017] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figure" and "FIG"), in which: [Figure 1] FIG. 1 shows a domain diagram of the synthetic human leukocyte antigen (synHLA) constructs provided herein. [Diagram 2] FIG. 1 shows a three-dimensional representation of the construct structure of the synthetic human leukocyte antigen (synHLA) constructs provided herein. [Diagram 3] 1 shows HLA-bound immunogenic peptides that engage the T cell receptor and result in T cell activation. [Figure 4] We demonstrate that the synthetic human leukocyte antigen (synHLA) constructs provided herein engage T cell receptors and result in the failure of T cell activation. [Diagram 5] We present single-chain trimers (SCT) in constructs with killer cell immunoglobulin-like receptors (KIR), which block KIR interactions and generate a "missing self" immune signal. [Figure 6] We demonstrate that synthetic human leukocyte antigen (synHLA) constructs provided herein in constructs with killer cell immunoglobulin-like receptors (KIRs) result in successful KIR interactions and no "missing self" immune signals. [Figure 7]1 shows an overlay of a single chain trimer (SCT) in a construct with a killer cell immunoglobulin-like receptor (KIR) and a synthetic human leukocyte antigen (synHLA) construct provided herein in a construct with a killer cell immunoglobulin-like receptor (KIR). [Figure 8] shows a synthetic human leukocyte antigen (synHLA) construct provided herein that engages CD8. [Figure 9] 1 shows an immunocompromised cell provided herein. [Figure 10] 1 shows an SDS-PAGE gel of the synHLA constructs described herein recombinantly expressed in bacteria. [Figure 11] 1 shows an SDS-PAGE of synHLA constructs described herein recombinantly expressed in bacteria. [Figure 12A] Raw thermal melting curves of SYNC4-1 and SYNC4-1+KIR2DL2 are shown. [Figure 12B] The first derivatives of the thermal melting curves of SYNC4-1 and SYNC4-1+KIR2DL2 are shown. [Figure 13A] Raw thermal melting curves of SYNC4-1, SYNC4-1+KIR2DL2, and KIR2DL2 are shown. [Figure 13B] The first derivatives of the thermal melting curves of SYNC4-1, SYNC4-1+KIR2DL2, and KIR2DL2 are shown. [Figure 14A] Raw thermal melting curves of SYNA1-1, SYNA1-1+KIR2DL2, and KIR2DL2 are shown. [Figure 14B] The first derivatives of the thermal melting curves of SYNA1-1, SYNA1-1+KIR2DL2, and KIR2DL2 are shown. [Figure 15] FIG. 1 shows a domain diagram of the synthetic human leukocyte antigen (synHLA) constructs provided herein designed for expression in bacteria. [Figure 16] 1 shows an SDS-PAGE gel of the synHLA constructs described herein recombinantly expressed in bacteria. [Figure 17]1 shows an SDS-PAGE gel of the synHLA constructs described herein recombinantly expressed in bacteria. [Figure 18] 1 shows the mass spectrum of the synHLA constructs described herein measured by TOF-MS. [Figure 19A] 1 shows the results of dynamic light scattering (DLS) experiments performed on constructs described herein. [Figure 19B] 1 shows the results of dynamic light scattering (DLS) experiments performed on constructs described herein. [Figure 19C] 1 shows the results of dynamic light scattering (DLS) experiments performed on constructs described herein. [Figure 19D] 1 shows the results of dynamic light scattering (DLS) experiments performed on constructs described herein. [Figure 19E] 1 shows the results of dynamic light scattering (DLS) experiments performed on constructs described herein. [Figure 19F] 1 shows the results of dynamic light scattering (DLS) experiments performed on constructs described herein. [Figure 20] 1 shows an SDS-PAGE gel of the synHLA constructs described herein recombinantly expressed in bacteria. [Figure 21] FIG. 1 shows a domain diagram of the synHLA constructs provided herein, which are designed for expression in bacteria through multiple transcription units. [Figure 22] 1 shows an SDS-PAGE gel of the synHLA constructs described herein recombinantly expressed in bacteria. [Figure 23] 1 shows representative surface plasmon resonance (SPR) sensorgrams for binding of representative HLA proteins disclosed herein to immobilized killer cell immunoglobulin-like receptors (KIRs). [Figure 24] 1 shows the relative binding of the HLA proteins disclosed herein to KIRs as determined by SPR. [Diagram 25]Representative flow cytometry data showing the generation of beta-2 microglobulin (B2M) deficient EBV cell lines are shown. [Figure 26] Representative histograms from flow cytometry experiments showing expression of synthetic HLA proteins described herein on the surface of B2M-deficient EBV cells are shown. Expression of HLA-A2 on the surface of β2M-null EBV 9031 cells was examined by staining with an antibody specific for HLA-A2 (BB7.2). Plots show the staining profile of negative control samples transfected with an irrelevant protein or not transfected (indicated by arrows). HLA-A2 (indicated by arrows) expressed on the surface of cells after transfection with SCTA2-M1 or SCDA1-M1 constructs is shown. All transfected cells were grown in the presence of puromycin for at least 7 days to select for transfected cells. MFI, mean fluorescence intensity. [Figure 27] Schematic diagram of multigene expression vector product. HLA proteins were fused to a GFP reporter in the multigene expression vector. Schematic showing predicted predominant products assuming furin cleavage of the T2A peptide and ribosomal skipping at the 2A "cleavage" site. Furin cleavage site, VRAKR (SEQ ID NO: 196); T2A peptide, EGRGSLLTCGDVEENPGP (SEQ ID NO: 197); SGSG linker (SEQ ID NO: 198); GFP, green fluorescent protein. [Figure 28] shows a table summarizing the synthetic HLA constructs and the resulting HLA-C expression. The percentage of HLA-C positive cells within the GFP+ population is reported for two experiments. HLA-C staining is reported after subtraction of staining from the isotype control. [Figure 29]Histograms measuring surface expression of HLA-C after transfection of B2M-deficient EBV cells with different synthetic HLA constructs are shown. The B2M-deficient EBV-transformed B cell line 9031 was transfected with a derivative of the pCE plasmid encoding the expression of an HLA-C single-chain trimer variant fused to green fluorescent protein (GFP) via the T2A peptide. For analysis, the GFP+ population was gated ("start population" in the leftmost plot) and from this population the % of positive cells was determined after staining with an isotype control (clone MPC-11 AlexaFluor 647) and then the % of HLA-C expressing cells (after staining with mAb DT-9 AlexaFluor 647). Numbers refer to the % of cells falling within the black box from the entire plot. Unstained GFP-ve cells fall in the lower left quadrant. [Diagram 30] A graph summarizing the percentage of cells expressing GFP and presenting synthetic HLA proteins on the cell surface is shown. The B2M-deficient EBV-transformed B cell line 9031 was transfected with derivatives of the pCE plasmid encoding the expression of synthetic HLA variants fused to green fluorescent protein (GFP) via the T2A peptide. For analysis, the GFP+ population was gated ("start population" in the leftmost plot) and from this population the % of positive cells was determined after staining with an isotype control (clone MPC-11 AlexaFluor 647) and then the % of HLA-C expressing cells (after staining with mAb DT-9 AlexaFluor 647) was determined. [Diagram 31]Kinetics of surface expression of HLA-C after transfection of B2M-deficient EBV cells with different constructs. The B2M-deficient EBV-transformed B cell line 9031 was transfected with derivatives of the pCE plasmid encoding the expression of the HLA-C single-chain trimer variant fused to green fluorescent protein (GFP) via the T2A peptide. For analysis, the percentage of GFP+HLA-C+ detected by staining with mAb DT-9 AlexaFluor 647 minus the % of positive cells after staining with an isotype control (clone MPC-11 AlexaFluor 647) was determined for each construct 1, 2, 5, 9 and 13 days after transfection. [Diagram 32] A gel showing expression of GFP in transfected cells is shown. Cells (2.0 x 105) were lysed in RIPA buffer (Sigma, P0278-50ML) containing complete protease inhibitor cocktail (Sigma, P8340-1ML) and run on a reducing 4-12% SDS PAGE gel (Thermo Fisher Scientific, NW 04127BOX). Proteins were transferred to an Amersham Protran 0.45mm NC nitrocellulose membrane (Sigma, GE10600008) and probed with mouse anti-GFP mAb (clone B34, Biolegend catalog number 902601), which was detected using a goat anti-mouse IgG (H+L) antibody conjugated to DyLight 800 (Thermo Fisher Scientific, SA5-35521). Membranes were imaged using an Odyssey CLx imaging system. The highest molecular weight band corresponds to GFP linked to HLA-C, and the approximately 62 kDa band corresponds to human immunoglobulin (Hu-Ig). GFP (approximately 27 kDa) is observed primarily in the free form. [Diagram 33]Flow cytometry results showing NK cell expression are shown. NK cells were purified from peripheral blood mononuclear cells (PBMCs) of healthy volunteer donors using the Miltenyi NK Cell Isolation Kit (Cat. No. 130-092-657). Purified NK cells were expanded using bead-coupled antibodies from the Miltenyi NK Activation / Proliferation Kit (Cat. No. 130-094-483) in Miltenyi's NK medium supplemented with 5% pooled human serum and 500 U / mL IL-2. Expanded NK cells were used for functional assays at least 7-10 days after initiation of culture. [Diagram 34] 1 shows thermostability data for the constructs described herein. [Diagram 35] 1 shows representative surface plasmon resonance (SPR) sensorgrams characterizing the interaction between killer cell immunoglobulin-like receptors (KIRs) and constructs described herein. [Diagram 36] 4 shows the results of a chromium release assay demonstrating the protection against NK cell cytotoxicity provided by the constructs described herein. [Figure 37A] FIG. 1 shows a schematic diagram of synergistic inhibition of NK cells by co-expression of HLA-E and the constructs described herein. [Figure 37B] FIG. 1 shows a schematic diagram of the interaction between HLA-E and the NKG2A receptor. [Figure 38] 4 shows the results of a chromium release assay demonstrating the synergistic protection against NK cell cytotoxicity provided by the constructs described herein in combination with HLA-E. [Figure 39] 1 shows a portion of the crystal structure of the complex between the KIR2DL2 receptor and a construct described herein. [Diagram 40] 1 shows the results of a luciferase-based assay for cytotoxicity described herein.

[0018] Brief Description of the Sequence Listing The Sequence Listing submitted herewith provides exemplary polynucleotide and polypeptide sequences for use in the methods, compositions, and systems according to the present disclosure. Below are representative descriptions of the sequences.

[0019] SEQ ID NOs: 1-15 show representative amino acid sequences of single-chain trimer (SCT) constructs described herein.

[0020] SEQ ID NOs: 16-29 and 199-202 show representative amino acid sequences of single-chain dimer (SCD) constructs described herein.

[0021] SEQ ID NOs: 30-129, 142-157, and 203-278 show full-length amino acid sequences of representative synthetic HLA (synHLA) constructs described herein.

[0022] SEQ ID NOs: 130-141 and 279-288 show representative amino acid sequences of the HLA heavy chain (HHC) constructs described herein.

[0023] SEQ ID NOs: 158-174 show the amino acid sequences of representative peptides configured to attenuate or inhibit HLA activity as described herein.

[0024] SEQ ID NOs: 175-186 and 198 show the amino acid sequences of representative linkers between inhibitory amino acids and HLA sequences or between the B2M region and HLA sequences described herein.

[0025] SEQ ID NOs: 187-189 show the amino acid sequences of representative CD8-α chains expressed and described herein.

[0026] SEQ ID NOs: 190-192 show the amino acid sequences of the Killer Ig-like Receptor (KIR) domains expressed and described herein.

[0027] SEQ ID NO: 193 shows the amino acid sequence of the leukocyte immunoglobulin-like receptor (LILR) domain expressed and described herein.

[0028] SEQ ID NO: 194 shows the amino acid sequence of a representative HLA-C allele.

[0029] SEQ ID NO: 195 shows the amino acid sequence of a representative beta-2 microglobulin (B2M) sequence described herein.

[0030] SEQ ID NO: 196 shows the amino acid sequence of a representative furin cleavage site.

[0031] SEQ ID NO: 197 shows the amino acid sequence of a representative T2A peptide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] definition Whenever the terms "at least," "greater," or "greater than or equal to" precede the first number in a series of two or more numerical values, the terms "at least," "greater," or "greater than or equal to" apply to each and every number in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0033] Whenever the terms "no more than," "less than," or "less than or equal to" precede the first number in a series of two or more numbers, the terms "no more than," "less than," or "less than or equal to" apply to each of the numbers in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0034] As used herein, "T cell" generally refers to a cell that contains a T cell receptor.

[0035] As used herein, a "peptide" is a chain of 2 to 50 amino acid residues.

[0036] "Pharmaceutically acceptable carrier" generally refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives, such as those known in the art, for example, those described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0037] As used herein, "treatment" or "treating" is an approach to obtain beneficial or desired results, preferably including clinical results. For example, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing symptoms caused by the disease, improving the quality of life of a person suffering from the disease, reducing the dose of other drugs required to treat the disease, slowing the progression of the disease, and / or prolonging the survival of an individual.

[0038] As used herein, an "effective dosage" or "effective amount" of a construct, a nucleic acid molecule, an immunocompromised cell, or a pharmaceutical composition thereof generally refers to an amount sufficient to produce a beneficial or desired result. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, reducing the severity, or delaying the onset of a disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes manifested during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms resulting from the disease, increasing the quality of life of a person suffering from the disease, reducing the dose of another drug required to treat the disease, enhancing the effect of another drug through targeting, etc., delaying the progression of the disease, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may be effective in reducing the number of cancer cells; reducing tumor size; inhibiting (e.g., slowing to some extent, preferably stopping) cancer cell invasion into peripheral organs; inhibiting (e.g., slowing to some extent, preferably stopping) tumor metastasis; inhibiting to some extent tumor growth; and / or alleviating to some extent one or more symptoms associated with the disorder. An effective dosage may be administered in one or more administrations. For purposes of the present invention, an effective dosage of a construct, nucleic acid molecule, immunocompromised cell, or pharmaceutical composition thereof is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As will be understood in a clinical context, an effective dosage of a construct, nucleic acid molecule, immunocompromised cell, or pharmaceutical composition thereof may or may not be achieved in conjunction with another construct, nucleic acid molecule, immunocompromised cell, or pharmaceutical composition thereof. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given at an effective dose if, in conjunction with one or more other agents, a desired result can be or is achieved.

[0039] As defined herein, the terms "inhibit," "inhibit," "inhibiting," and the like, with respect to protein-inhibitor interactions, generally refer to adversely affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. Inhibition may refer to a reduction in a disease or disease symptoms. Inhibition may refer to a decrease in the activity of a particular protein or nucleic acid target. The protein may be deoxycytidine kinase. Thus, inhibition includes, at least in part, partially or completely blocking a stimulus, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating the amount of a signal transduction or enzyme activity or protein.

[0040] The term "modulator" refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule or the physical state of a target of the molecule.

[0041] The term "modulate" is used according to its plain and ordinary meaning and generally refers to the act of changing or varying one or more properties. "Regulation" refers to the process of changing or varying one or more properties. For example, a modulator of a target protein changes the property or function of the target molecule by increasing or decreasing the amount of the target molecule. A modulator of a disease reduces the symptoms, cause, or characteristics of the target disease.

[0042] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" generally refer to substances that aid in the administration and absorption of an active agent to a subject and may be included in the compositions of the present invention without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solution, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, saline (such as Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents. Such preparations are sterilized and may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring agents, and / or aromatic substances that do not adversely react with the constructs, nucleic acid molecules, or immunologically ineligible cells of the present invention, as appropriate. Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0043] As used herein, the term "administering" includes oral administration, administration as a suppository, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained release device, such as a mini-osmotic pump, to a subject. Administration may be by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0044] "Patient", "subject", "patient in need thereof" and "subject in need thereof" refer to an organism suffering from or susceptible to a disease or condition that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human. A "cancer patient" is a patient suffering from or susceptible to developing cancer.

[0045] Unless expressly indicated otherwise, the term "individual" as used herein generally refers to a mammal, including but not limited to a cow, horse, cat, rabbit, dog, rodent, or primate (e.g., human). In some embodiments, the individual is a human. In some embodiments, the individual is a non-human primate, such as chimpanzees and other ape and monkey species. In some embodiments, the individual is a farm animal, such as cows, horses, sheep, goats, and pigs; pets, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, guinea pigs, and the like. In some embodiments, the invention finds use in both human and veterinary medicine.

[0046] "Disease" or "condition" generally refers to an existing or health state of a patient or subject that may be treated using the constructs, nucleic acid molecules, immunocompetent cells, or methods provided herein. In some embodiments, disease as used herein refers to cancer.

[0047] As used herein, "immune checkpoint modulator" generally refers to an agent that results in the activation or inhibition of one or more immune checkpoint proteins. For example, immune checkpoint modulators can include, but are not limited to, CD47, PD-L1, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TIM-3, VISTA, and SIGLEC7.

[0048] As used herein, a "mutation" generally refers to a change in the sequence of a nucleic acid molecule. Mutations include, but are not limited to, insertions, deletions, and substitutions.

[0049] As used herein, abbreviations for amino acids are conventional and may be as follows: alanine (A, Ala); arginine (R, Arg); asparagine (N, Asn); aspartic acid (D, Asp); cysteine ​​(C, Cys); glutamic acid (E, Glu); glutamine (Q, Gln); glycine (G, Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K, Lys); methionine (M, Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T, Thr); tryptophan (W, Trp); tyrosine (Y, Tyr); valine (V, Val). Other amino acids include citrulline (Cit); homocysteine ​​(Hey); hydroxyproline (Hyp); ornithine (Orn); and thyroxine (Thx). Examples of amino acids that are uncharged at physiological pH include, but are not limited to, alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0050] As used herein, an "anchor residue" of a peptide generally refers to a conserved amino acid residue that plays a role in binding of the peptide to the groove of a given HLA allele.

[0051] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] It is understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0053] Synthetic human leukocyte antigen (synHLA) constructs Provided herein, in one aspect, is a construct comprising one or more human leukocyte antigens (HLA). In some embodiments, the one or more HLA are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the one or more HLA are inhibited from eliciting a natural killer (NK) cell response when the construct is interrogated by one or more NK cells. In some embodiments, the one or more HLA are inhibited from eliciting a T cell response when interrogated by one or more T cells and inhibited from eliciting a NK cell response when interrogated by one or more NK cells.

[0054] In some embodiments, the construct comprises, from N-terminus to C-terminus, a segment comprising a peptide and a segment comprising a beta-2 microglobulin (B2M) sequence. In some embodiments, the construct comprises, from N-terminus to C-terminus, a segment comprising a peptide and a segment comprising a human HLA class 1 heavy chain sequence. In some embodiments, the construct comprises, from N-terminus to C-terminus, a segment comprising a peptide, a segment comprising a B2M sequence, and a sequence comprising a human HLA class 1 heavy chain sequence.

[0055] In some embodiments, the human HLA class 1 heavy chain sequence comprises one or more class 1 HLA. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-A, HLA-B, HLA-C, or any combination thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-A. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-B. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-A and HLA-B. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-A and HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-B and HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-A, HLA-B, and HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-A, HLA-B, HLA-C, or any combination thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-A. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-B. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-A and HLA-B. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-A and HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-B and HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises multiple versions of HLA-A, HLA-B, and HLA-C. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-A, which is positioned between HLA-B and HLA-C.

[0056] In some embodiments, the construct further comprises one or more immune checkpoint modulators. In some embodiments, the one or more immune checkpoint modulators comprise CD47, PD-L1, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, or any combination thereof. In some embodiments, the construct comprises CD47. In some embodiments, the construct comprises PD-L1. In some embodiments, the construct comprises A2AR. In some embodiments, the construct comprises B7-H3. In some embodiments, the construct comprises B7-H4. In some embodiments, the construct comprises BTLA. In some embodiments, the construct comprises CTLA-4. In some embodiments, the construct comprises IDO. In some embodiments, the construct comprises KIR. In some embodiments, the construct comprises LAG3. In some embodiments, the construct comprises NOX2. In some embodiments, the construct comprises PD-1. In some embodiments, the construct comprises TIM-3. In some embodiments, the construct comprises VISTA. In some embodiments, the construct comprises SIGLEC7.

[0057] In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof, HLA-F or a fragment thereof, HLA-G or a fragment thereof, or any combination thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-F or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-G or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof and HLA-F or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof and HLA-G or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-F or a fragment thereof and HLA-G or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof, HLA-F or a fragment thereof, and HLA-G or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof, HLA-F or a fragment thereof, and HLA-G or a fragment thereof. In some embodiments, the human HLA class 1 heavy chain sequence comprises HLA-E or a fragment thereof, HLA-F or a fragment thereof, and HLA-G or a fragment thereof. In some embodiments, at least one of HLA-E or a fragment thereof, HLA-F or a fragment thereof, HLA-G or a fragment thereof, or any combination thereof, is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, HLA-E or a fragment thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, HLA-F or a fragment thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, HLA-G or a fragment thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, HLA-E or a fragment thereof and HLA-F or a fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, HLA-E or a fragment thereof and HLA-G or a fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, HLA-F or a fragment thereof and HLA-G or a fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells.In some embodiments, HLA-E or a fragment thereof, HLA-F or a fragment thereof, and HLA-G or a fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells.

[0058] In some embodiments, the construct further comprises an epitope configured to allow detection of the construct, hi some embodiments, the epitope comprises 3,5-dinitrosalicylic acid.

[0059] In some embodiments, the construct comprises a human beta-2 microglobulin (B2M) sequence. In some embodiments, the human B2M sequence is a wild-type human B2M sequence. In some embodiments, the B2M sequence comprises a sequence that is at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to SEQ ID NO:195.

[0060] In some embodiments, the construct comprises, in order from N-terminus to C-terminus: A peptide; b. a first linker of the one or more linkers; c. human B2M sequence; d. a second linker of the one or more linkers; and e. Human HLA class 1 heavy chain sequence Includes.

[0061] Provided herein, in another aspect, is a construct comprising one or more human leukocyte antigens (HLA). In some embodiments, the construct comprises, in order from N-terminus to C-terminus: a. a peptide that is unable to activate one or more T cells; b. a first linker; and C. a segment containing a human HLA class 1 heavy chain sequence Including, The first linker comprises a conformation configured to not block one or more killer cell immunoglobulin-like receptor (KIR) binding sites on the human HLA class 1 heavy chain sequence, in some embodiments, the conformation is further configured to resist proteolytic cleavage.

[0062] In some embodiments, the one or more HLAs are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the one or more HLAs are inhibited from eliciting a NK cell response when the construct is interrogated by one or more NK cells. In some embodiments, the construct comprises one or more targeting moieties (e.g., a peptide); one or more linker regions; one or more major histocompatibility complex (MHC) regions (e.g., an HLA class 1 heavy chain sequence), at least one of the one or more MHC regions comprises a cluster of differentiation 8 (CD8) binding site, the CD8 binding site comprising one or more mutations; and one or more disulfide staple pairs.

[0063] The one or more targeting moieties can include peptides that, when associated with an MHC region (e.g., an HLA class I heavy chain sequence), are incapable of activating one or more T cells as described elsewhere herein (e.g., as listed in Table 2). The targeting moieties can include peptides that, when associated with an HLA class I heavy chain sequence, are incapable of activating one or more NK cells as described elsewhere herein (e.g., as listed in Table 2).

[0064] The one or more MHC regions may comprise one or more human HLA class I heavy chain sequences. In some cases, the human HLA class I heavy chain sequences are derived from HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, or any combination thereof. In some cases, the human HLA class I heavy chain sequences comprise a sequence that is at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 130-141 and 279-288.

[0065] The one or more MHC regions may be inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. The one or more MHC regions may comprise one or more mutated residues compared to a wild-type version of the one or more MHC regions. In some embodiments, the mutation is at a site corresponding to the tyrosine 84 (Y84) residue of the human leukocyte antigen (HLA) protein HLA-C (e.g., SEQ ID NO: 194). The mutation may be to any suitable amino acid. In some embodiments, the mutation comprises an alanine residue (e.g., a Y84A mutation). In some embodiments, the mutation comprises a cysteine ​​residue (e.g., a Y84C mutation).

[0066] The CD8 binding site may include a mutation at any residue to any other amino acid or a deletion at any residue. In some embodiments, the mutation may include an insertion before or after the residue. In some embodiments, the CD8 binding site may include a mutation at a position corresponding to glutamine 226 (Q226) of HLA-C (e.g., SEQ ID NO: 194). In some embodiments, the CD8 binding site may include a mutation to a residue corresponding to aspartic acid 227 (D227) of HLA-C. In some embodiments, the mutation at D227 of HLA-C is to a lysine residue (e.g., a D227K mutation). In some embodiments, the CD8 binding site may include a deletion of a residue corresponding to threonine 225 (T225) of HLA-C. In some embodiments, the CD8 binding site may include a deletion of the Q226 residue. In some embodiments, the CD8 binding site may include a deletion of the D227 residue. In some embodiments, the CD8 binding site may comprise a deletion of a residue corresponding to glutamic acid 232 (E232) of HLA-C.

[0067] In some embodiments, the construct may further comprise a mutation to a residue corresponding to the cysteine ​​1 (C1) residue of HLA-C (e.g., SEQ ID NO: 194). In some embodiments, the mutation is to a glycine (e.g., a C1G mutation).

[0068] In some embodiments, the construct comprises a single chain trimer (SCT). The single chain trimer may comprise a targeting peptide (e.g., a moiety), a beta-2 microglobulin (B2M), and an HLA heavy chain, which are optionally connected by one or more linkers. In some embodiments, the SCT construct comprises a peptide comprising a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 30-129, 142-157, and 203-278. In some embodiments, the SCT construct comprises a B2M region comprising a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to SEQ ID NO:195. In some embodiments, the SCT construct comprises an HLA heavy chain comprising a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 130-141 and 279-288. In some embodiments, the SCT construct comprises a linker between the peptide and the B2M region that comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 175-186 and 198.In some embodiments, the SCT construct comprises a linker between the B2M region and an HLA heavy chain that comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 175-186 and 198. In some embodiments, the SCT construct comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs:1-15.

[0069] In some embodiments, the construct comprises a single chain dimer (SCD). The single chain dimer may comprise a targeting moiety (e.g., a peptide) and a beta-2 microglobulin (B2M). The single chain dimer may comprise a targeting moiety (e.g., a peptide) and a B2M region connected by a linker. In some embodiments, the SCD construct comprises a peptide comprising a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 16-29 and 199-202. In some embodiments, the SCD construct comprises a B2M region comprising a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to SEQ ID NO:195. In some embodiments, the SCD construct can be combined with an HLA heavy chain that comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 130-141 and 279-288. In some embodiments, the SCD construct comprises a linker between the peptide and the B2M region that comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 175-186 and 198.In some embodiments, the SCD construct comprises a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 16-29 and 199-202.

[0070] In some embodiments, the constructs of the present disclosure include an SCD construct combined with an HLA heavy chain construct described herein. In one example, the SCD construct includes a targeting moiety (e.g., a peptide) described herein, optionally connected by a linker to a B2M sequence described herein. The SCD construct can then be combined with an HLA heavy chain to provide a complex including a targeting moiety, a B2M sequence, and one or more HLA heavy chain sequences. In some embodiments, the SCD construct and the HLA heavy chain construct each include one portion of a disulfide staple pair described herein that is configured to form a disulfide bond. In some embodiments, the SCD comprises a sequence that is at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 16-29 and 199-202. In some embodiments, the HLA heavy chain comprises a sequence that is at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to any one of SEQ ID NOs: 130-141 and 279-288.

[0071] In some embodiments, the construct comprises a sequence that is at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to a sequence listed in Table 1 below.

[0072] [Table 1-1]

[0073] [Table 1-2]

[0074] [Table 1-3]

[0075] [Table 1-4]

[0076] [Table 1-5]

[0077] [Table 1-6]

[0078] [Table 1-7]

[0079] [Table 1-8]

[0080] [Table 1-9]

[0081]

Table 1-10

[0082]

Table 1-11

[0083]

Table 1-12

[0084]

Table 1-13

[0085]

Table 1-14

[0086]

Table 1-15

[0087]

Table 1-16

[0088]

Table 1-17

[0089]

Table 1-18

[0090]

Table 1-19

[0091]

Table 1-20

[0092]

Table 1-21

[0093]

Table 1-22

[0094]

Table 1-23

[0095]

Table 1-24

[0096]

Table 1-25

[0097]

Table 1-26

[0098]

Table 1-27

[0099]

Table 1-28

[0100]

Table 1-29

[0101]

Table 1-30

[0102]

Table 1-31

[0103]

Table 1-32

[0104]

Table 1-33

[0105]

Table 1-34

[0106]

Table 1-35

[0107]

Table 1-36

[0108]

Table 1-37

[0109]

Table 1-38

[0110]

Table 1-39

[0111] [Table 1-40]

[0112] [Table 1-41]

[0113] [Table 1-42]

[0114] [Table 1-43]

[0115] [Table 1-44]

[0116] [Table 1-45]

[0117] targeting part Provided herein, in one aspect, is a targeting moiety. In some embodiments, the targeting moiety comprises a peptide. In some embodiments, the peptide is configured to attenuate or inhibit HLA activity. In some embodiments, the peptide is configured to attenuate or inhibit T cell activity. In some embodiments, the peptide is configured to attenuate or inhibit NK cell activity.

[0118] In some embodiments, the peptide does not elicit a T cell response when the peptide (or a complex comprising the peptide) is interrogated by one or more T cells. In some embodiments, the peptide is unable to activate one or more T cells. In some embodiments, the peptide is capable of binding to a receptor on one or more T cells, and the binding is insufficient to activate one or more T cells. In some embodiments, the peptide does not elicit an NK cell response when the peptide (or a complex comprising the peptide) is interrogated by one or more NK cells. In some embodiments, the peptide is unable to activate one or more NK cells. In some embodiments, the peptide is capable of binding to a receptor on one or more NK cells, and the binding is insufficient to activate one or more NK cells.

[0119] In some embodiments, the peptide is configured to covalently bind to HLA. In some embodiments, the peptide is configured to bind to the N-terminus of the beta chain of HLA class II or the N-terminus of the beta-2 microglobulin (B2M) chain of HLA class 1. In some embodiments, the peptide is configured to be specific for the MHC binding groove, but does not contain the precise TCR-facing / solvent-exposed amino acids required for recognition by the T-cell receptor (TCR). The presence of certain "anchor residues" in the MHC binding groove of HLA acts to anchor the bound peptide.

[0120] In some embodiments, residues in the peptide can be modified to configure the peptide so that the TCR does not recognize and / or bind peptide-bound MHC (pMHC) (e.g., a peptide bound to a human HLA class 1 heavy chain). While the TCR can interact with residues in the MHC, one to three TCR-facing, solvent-exposed residues from the peptide also directly contribute to TCR interaction. In some embodiments, the TCR-facing residues of the peptide are configured to antagonize TCR interaction.

[0121] In some embodiments, the peptide binds to one or more HLA binding groove domain residues of a human HLA class 1 heavy chain sequence. In some embodiments, the peptide modulates the conformation of the human HLA class 1 heavy chain sequence. In some embodiments, the conformation prevents one or more T cells from binding to the human HLA class 1 heavy chain sequence.

[0122] In some embodiments, the sequence of the bound peptide may affect the inherent flexibility of the pMHC. In some embodiments, the conformational flexibility of the pMHC facilitates TCR interaction. In some embodiments, the peptide configured to bind to HLA is further configured to increase the conformational variability of the pMHC and prevent TCR engagement.

[0123] In some embodiments, the peptide is about 8 to about 15 amino acids in length. In some embodiments, the peptide is about 8 to about 9 amino acids in length, about 8 to about 10 amino acids in length, about 8 to about 11 amino acids in length, about 8 to about 12 amino acids in length, about 8 to about 13 amino acids in length, about 8 to about 14 amino acids in length, about 8 to about 15 amino acids in length, about 9 to about 10 amino acids in length, about 9 to about 11 amino acids in length, about 9 to about 12 amino acids in length, about 9 to about 13 amino acids in length, about 9 to about 14 amino acids in length, about 9 to about 15 amino acids in length, about 10 to about 11 amino acids in length, The peptide is about 10 to about 12 amino acids in length, about 10 to about 13 amino acids in length, about 10 to about 14 amino acids in length, about 10 to about 15 amino acids in length, about 11 to about 12 amino acids in length, about 11 to about 13 amino acids in length, about 11 to about 14 amino acids in length, about 11 to about 15 amino acids in length, about 12 to about 13 amino acids in length, about 12 to about 14 amino acids in length, about 12 to about 15 amino acids in length, about 13 to about 14 amino acids in length, about 13 to about 15 amino acids in length, or about 14 to about 15 amino acids in length. In some embodiments, the peptide is about 8 amino acids in length, about 9 amino acids in length, about 10 amino acids in length, about 11 amino acids in length, about 12 amino acids in length, about 13 amino acids in length, about 14 amino acids in length, or about 15 amino acids in length. In some embodiments, the peptide is at least about 8, 9, 10, 11, 12, 13, or 14 amino acids in length. In some embodiments, the peptide is up to about 9, 10, 11, 12, 13, 14, or 15 amino acids in length. In some embodiments, the peptide comprises more than 14 amino acids.

[0124] In some embodiments, the use of unusually long peptides to bind to the MHC-binding groove of HLA inhibits HLA activity. MHC-I can bind peptides 8-10 amino acids long, but can also bind non-canonical, longer peptides (e.g., 13 amino acids). The terminus of such long peptides binds to the MHC-binding groove at an anchor residue, creating a "bulge" in the center of the peptide-binding site. In such pMHC-TCR constructs, the TCR makes relatively few contacts with the MHC (typically, in canonical short peptides in such constructs, the MHC heavy chain dominates the interface with the TCR), and instead, interactions with the TCR are directly dominated by the peptide. Bulged peptides also represent a steric burden for TCR engagement. Given the dominance of peptide-TCR interactions in such systems, it is possible to prevent TCR binding by selecting peptide sequences in the bulge. In some embodiments, the peptide is configured to block and / or silence amino acids of the HLA required for molecular contact with the TCR and / or the peptide does not contain sufficient amino acid residues for TCR binding and / or activity. In some embodiments, the peptide is configured to increase the conformational heterogeneity of the HLA in this region to render the HLA incapable of TCR binding and / or activity. In some embodiments, the peptide is configured to perform any combination of the above functions.

[0125] In some embodiments, the peptide is coupled to the construct by a disulfide bond. The peptide can be further covalently linked to the remainder of the construct. The disulfide bond can connect the disulfide staple pairs. The disulfide staple pairs can be located or distributed over the appropriate portion(s) of the construct, such as on the peptide and on the human HLA class I heavy chain, or on the peptide and on the linker.

[0126] In some embodiments, the peptide comprises or is otherwise derived from an influenza A virus M1 peptide (e.g., SEQ ID NO: 158) or a mutant thereof. In some embodiments, the peptide comprises or is otherwise derived from a histone H3 peptide (e.g., SEQ ID NO: 159) or a mutant thereof.

[0127] In some embodiments, the construct further comprises a regulatory peptide. In some embodiments, the regulatory peptide is an apoptosis-inducing peptide. In some embodiments, the apoptosis-inducing peptide acts as a "kill switch" for the construct.

[0128] In some embodiments, the targeting moiety comprises a peptide comprising a sequence at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to a sequence listed in Table 2 below.

[0129] [Table 2]

[0130] Linker In some embodiments, the construct comprises one or more linkers between the targeting moiety (e.g., a peptide) and the MHC region (e.g., a human HLA class 1 heavy chain sequence). In some embodiments, the one or more linkers are configured to resist proteolytic cleavage. In some embodiments, the one or more linkers comprise a conformation configured to not block one or more killer cell immunoglobulin-like receptor (KIR) binding sites on the human HLA class 1 heavy chain sequence. In some embodiments, the one or more linkers are structurally stable. In some embodiments, the one or more linkers are rigid. In some embodiments, the one or more linkers have limited flexibility. In some embodiments, the structural stability, rigidity, and limited flexibility of the one or more linkers increase resistance to proteolysis. In some embodiments, the one or more linkers comprise a cysteine ​​that is part of a disulfide staple pair as described herein.

[0131] In some embodiments, a linker of the one or more linkers is disposed between the targeting moiety (e.g., a peptide) and a human beta-2 microglobulin (B2M) sequence, between the human B2M sequence and the human HLA class 1 heavy chain sequence, or both. In some embodiments, a linker of the one or more linkers is disposed between the peptide and the human B2M sequence. In some embodiments, a linker of the one or more linkers is disposed between the human B2M sequence and the human HLA class 1 heavy chain sequence. In some embodiments, a first linker of the one or more linkers is disposed between the peptide and the human B2M sequence, and a second linker of the one or more linkers is disposed between the human B2M sequence and the human HLA class 1 heavy chain sequence. In some embodiments, the second linker comprises a conformation configured to resist proteolytic cleavage. In some embodiments, the second linker is further configured not to block one or more killer cell immunoglobulin-like receptor (KIR) binding sites on the human HLA class 1 heavy chain sequence. In some embodiments, the conformation of the second linker allows KIR binding to human HLA class 1 heavy chain sequences and prevents "missing self" immune responses. In some embodiments, the conformation of the second linker prevents attack by one or more natural killer cells.

[0132] The linker may be configured to confer a certain secondary, tertiary, or quaternary structure when the construct sequence is arranged in three-dimensional space (e.g., expressed in a host cell). The secondary, tertiary, or quaternary structure may be determined from experimental structural biology data (e.g., X-ray crystallography data, cryo-electron microscopy data, nuclear magnetic resonance data), biochemical data (e.g., mass spectrometry data, chromatography data, electrophoresis data), or computer simulation or modeling data (e.g., molecular dynamics simulation, de novo or ab initio prediction, homology modeling, fragment assembly, secondary structure prediction). Alternatively or additionally, the linker may be configured to confer a certain functional outcome on the expressed construct. In some embodiments, the linker enhances expression (e.g., cell surface expression) of the imaging agent, enhances stability of the construct, prevents exchange of the peptide, or some combination thereof. In some embodiments, the linker enhances expression of the construct.

[0133] In some embodiments, the linker may be configured to increase expression in a host cell (e.g., compared to wild-type or other constructs lacking the linker). In some embodiments, the expression is cell surface expression. Expression may be measured, for example, by flow cytometry, fluorescence microscopy, mass spectrometry, or any other suitable quantitative method. In some embodiments, the presence of the linker increases expression of the construct in a host cell compared to a reference (e.g., wild-type) construct. In some embodiments, expression is increased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more. In some embodiments, expression is increased by about 1% to about 100%.In some embodiments, expression is from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 1% to about 30%, from about 1% to about 40%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, from about 1% to about 80%, from about 1% to about 90%, from about 1% to about 100%, from about 5% to about 10%, from about 5% to about 20%, from about 5% to about 30%, from about 5% to about 40%, from about 5% to about 50%, from about 5% to about 60%, %, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60 %, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%. In some embodiments, expression is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, expression is increased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, expression is increased by up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0134] In some embodiments, the linker may be configured to increase the stability of the construct (e.g., compared to a construct that does not include a linker, such as a wild-type construct). The presence of a linker may increase stability by limiting the conformational flexibility of the construct to help the construct fold and retain a particular secondary, tertiary, and / or quaternary structure. The stability of the construct may be measured by differential scanning calorimetry (DSC), pulse chase assays (e.g., bleach chase and cycloheximide chase assays), thermal shift assays, circular dichroism (CD) spectroscopy, UV-vis spectroscopy, nuclear magnetic resonance (NMR), gel filtration, isothermal calorimetry, light scattering, or any other suitable assay or instrument. In some embodiments, the stability of the construct is expressed in relative terms (e.g., percent or fold change in stability relative to a reference, such as a wild-type construct). In some embodiments, the stability of the construct is expressed in absolute terms (e.g., with respect to a thermodynamic coordinate, such as melting or other phase transition temperature or free energy of folding).

[0135] In some embodiments, the presence of a linker increases the stability of a construct by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to a reference (e.g., a construct that does not contain a linker, such as a wild-type construct). In some embodiments, the stability is increased by about 1% to about 100%. In some embodiments, the stability is about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5 ...80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, %, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60 %, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%. In some embodiments, the stability is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.In some embodiments, the stability is increased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, the stability is increased by up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0136] In some embodiments, the presence of the linker increases the stability of the construct by about 2-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, about 100-fold, about 1000-fold, or more, compared to the refence (e.g., a construct that does not include a linker, such as a wild-type construct). In some embodiments, the stability is increased by about 1-fold to about 1,000-fold. In some embodiments, the stability is about 1-fold to about 2-fold, about 1-fold to about 3-fold, about 1-fold to about 4-fold, about 1-fold to about 5-fold, about 1-fold to about 10-fold, about 1-fold to about 100-fold, about 1-fold to about 1,000-fold, about 2-fold to about 3-fold, about 2-fold to about 4-fold, about 2-fold to about 5-fold, about 2-fold to about 10-fold, about 2-fold to about 100-fold, about 2-fold to about 1,000-fold, about 3-fold to about 4-fold, about 3-fold to about 5-fold, about 3-fold to about 10-fold, about 3-fold to about 100-fold, about 3-fold to about 1,000-fold, about 4-fold to about 5-fold, about 4-fold to about 10-fold, about 4-fold to about 100-fold, about 4-fold to about 1,000-fold, about 5-fold to about 10-fold, about 5-fold to about 100-fold, about 5-fold to about 1,000-fold, about 10-fold to about 100-fold, about 10-fold to about 1,000-fold, or about 100-fold to about 1,000-fold. In some embodiments, the stability is increased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold. In some embodiments, the stability is increased by at least about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 100-fold. In some embodiments, the stability is increased by up to about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold.

[0137] In some embodiments, the constructs described herein that include a linker have a higher melting temperature (T m) (e.g., as determined by a thermodynamic technique such as DSC). In some embodiments, T m is increased by about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 20° C., about 30° C., or more, as compared to the reference. m In some embodiments, T m is about 1°C to about 2°C, about 1°C to about 3°C, about 1°C to about 4°C, about 1°C to about 5°C, about 1°C to about 6°C, about 1°C to about 7°C, about 1°C to about 8°C, about 1°C to about 9°C, about 1°C to about 10°C, about 1°C to about 20°C, about 1°C to about 30°C, about 2°C to about 3°C, about 2°C to about 4°C, about 2°C to about 5°C, about 2°C to about 6°C, about 2°C to about 7°C, about 2°C to about 8°C, about 2 ... ~ about 8°C, about 2°C to about 9°C, about 2°C to about 10°C, about 2°C to about 20°C, about 2°C to about 30°C, about 3°C ​​to about 4°C, about 3°C ​​to about 5°C, about 3°C ​​to about 6°C, about 3°C ​​to about 7°C, about 3°C ​​to about 8°C, about 3°C ​​to about 9°C, about 3°C ​​to about 10°C, about 3°C ​​to about 20°C, about 3°C ​​to about 30°C, about 4°C to about 5°C, about 4°C to about 6°C, about 4°C to about 7°C °C, about 4°C to about 8°C, about 4°C to about 9°C, about 4°C to about 10°C, about 4°C to about 20°C, about 4°C to about 30°C, about 5°C to about 6°C, about 5°C to about 7°C, about 5°C to about 8°C, about 5°C to about 9°C, about 5°C to about 10°C, about 5°C to about 20°C, about 5°C to about 30°C, about 6°C to about 7°C, about 6°C to about 8°C, about 6°C to about 9°C, about 6°C to about 10°C , about 6°C to about 20°C, about 6°C to about 30°C, about 7°C to about 8°C, about 7°C to about 9°C, about 7°C to about 10°C, about 7°C to about 20°C, about 7°C to about 30°C, about 8°C to about 9°C, about 8°C to about 10°C, about 8°C to about 20°C, about 8°C to about 30°C, about 9°C to about 10°C, about 9°C to about 20°C, about 9°C to about 30°C, about 10°C to about 20°C, about 10°C to about 30°C, or about 20°C to about 30°C. m increases by about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 20° C., or about 30° C. In some embodiments, T m increases by at least about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., or about 20° C. In some embodiments, T mincreases by up to about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 20°C, or about 30°C.

[0138] In some embodiments, the linker may be configured so as not to block killer cell immunoglobulin-like receptor (KIR) receptors. The linker may impart a particular secondary, tertiary, and / or quaternary structure to the construct that does not block (e.g., inhibitory) KIR binding to the construct. In some embodiments, the linker does not block KIR binding when expressed on the surface of a cell. Thus, when a cell (e.g., an NK cell) containing KIR interrogates the construct, the KIR is not activated or is activated less than when it interacts with a reference (e.g., wild-type) construct. In some embodiments, the KIR comprises one or more of KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, KIR3DL3, KIR2DL5A, or KIR2DL5B. The ability of the linker to allow binding of KIR to the construct can be measured by assaying the binding affinity between the constructs (e.g., either in soluble or cell surface form) and / or by functional assays. In some embodiments, the functional assay is a NK cell killing assay. In some embodiments, the NK cell killing assay is a chromium release assay.

[0139] An example of a linker that is configured not to block KIR is shown in Figure 39. Figure 39 shows a 2.35 Å resolution X-ray crystal structure of an example synthetic human leukocyte antigen (synHLA) construct 3901 (SEQ ID NO: 125) described herein in complex with a killer cell immunoglobulin-like receptor (KIR2DL2, 3902), which results in successful KIR interaction and does not result in a "missing self" immune signal. Linker 13901b does not block KIR2DL2 interaction. Targeting moiety 3901a (exemplified here as a peptide) in complex with the HLA heavy chain of construct 3901 and KIR2DL2 3902 is shown as a black sphere.

[0140] In some embodiments, constructs that include a linker are characterized in that the affinity of the construct for a (e.g., inhibitory) KIR is higher than the reference (e.g., a construct that does not include a linker). In some embodiments, the presence of the linker increases the affinity of the KIR for the construct by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to a reference (e.g., a construct that does not include a linker, such as a wild-type construct). In some embodiments, the stability is increased by about 1% to about 100%. In some embodiments, the stability is about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5 ...80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, %, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60 %, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% increase by about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%.In some embodiments, the stability is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, the stability is increased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, the stability is increased by up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0141] In some embodiments, the presence of the linker increases the affinity of the KIR for the construct by about 2-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, about 100-fold, about 1000-fold, or more, compared to the refence (e.g., a construct that does not include a linker, such as a wild-type construct). In some embodiments, the stability is increased by about 1-fold to about 1,000-fold. In some embodiments, the stability is about 1-fold to about 2-fold, about 1-fold to about 3-fold, about 1-fold to about 4-fold, about 1-fold to about 5-fold, about 1-fold to about 10-fold, about 1-fold to about 100-fold, about 1-fold to about 1,000-fold, about 2-fold to about 3-fold, about 2-fold to about 4-fold, about 2-fold to about 5-fold, about 2-fold to about 10-fold, about 2-fold to about 100-fold, about 2-fold to about 1,000-fold, about 3-fold to about 4-fold, about 3-fold to about 5-fold, about 3-fold to about 10-fold, about 3-fold to about 100-fold, about 3-fold to about 1,000-fold, about 4-fold to about 5-fold, about 4-fold to about 10-fold, about 4-fold to about 100-fold, about 4-fold to about 1,000-fold, about 5-fold to about 10-fold, about 5-fold to about 100-fold, about 5-fold to about 1,000-fold, about 10-fold to about 100-fold, about 10-fold to about 1,000-fold, or about 100-fold to about 1,000-fold. In some embodiments, the stability is increased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold. In some embodiments, the stability is increased by at least about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 100-fold. In some embodiments, the stability is increased by up to about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold.

[0142] In some embodiments, the constructs comprising a linker are characterized by a lower amount of NK cell killing (e.g., as determined by a chromium release assay) than a reference (e.g., a construct without a linker). In some embodiments, the presence of the linker reduces NK cell killing by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to a reference (e.g., a construct without a linker, such as a wild-type construct). In some embodiments, NK cell killing is reduced by about 1% to about 100%. In some embodiments, killing by NK cells is about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5 ... % to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to About 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40 % to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%.In some embodiments, killing by NK cells is reduced by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, killing by NK cells is reduced by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, killing by NK cells is reduced by up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0143] In some embodiments, there is a reduction in NK cell killing of cells expressing the construct compared to the refence (e.g., a construct that does not contain a linker, such as a wild-type construct) that is about 2-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, about 100-fold, about 1000-fold, or lower. In some embodiments, NK cell killing is reduced by about 1-fold to about 1,000-fold. In some embodiments, killing by NK cells is about one-half to about one-half, about one-half to about one-third, about one-half to about one-quarter, about one-half to about one-fifth, about one-half to about one-tenth, about one-half to about one-hundredth, about one-half to about one-thousandth, about one-half to about one-third, about one-half to about one-quarter, about one-half to about one-fifth, about one-half to about one-tenth, about one-half to about one-hundredth, about one-half to about one-thousandth, about one-half to about one-quarter, about one-half to about one-quarter, about one-half to about one-tenth, about one-half to about one-hundredth, about one-half to about one-thousandth, about one-third to about one-quarter ... about one-third to about one-tenth, about one-third to about one-hundredth, about one-third to about one-thousandth, about one-quarter to about one-fifth, about one-quarter to about one-tenth, about one-quarter to about one-hundredth, about one-quarter to about one-thousandth, about one-fifth to about one-tenth, about one-fifth to about one-hundredth, about one-fifth to about one-thousandth, about one-tenth to about one-hundredth, about one-tenth to about one-thousandth, or about one-hundredth to about one-thousandth. In some embodiments, killing by NK cells is reduced by about one-fold, about one-half, about one-third, about one-quarter, about one-fifth, about one-tenth, about one-hundredth, or about one-thousandth. In some embodiments, NK cell killing is reduced by at least about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 100-fold. In some embodiments, NK cell killing is reduced by up to about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold.

[0144] In some embodiments, a linker of the one or more linkers comprises a sequence that is at least about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, or about 100% identical to a sequence listed in Table 3 below.

[0145] [Table 3]

[0146] In some embodiments, one or more human leukocyte antigens (HLA) comprise one or more mutations, which inhibit the one or more HLA from eliciting a T cell response when the construct is interrogated by one or more cluster of differentiation 8 (CD8) cells. In some embodiments, the one or more mutations may be located in the CD8 binding site. In some embodiments, the one or more mutations comprise one or more mutations at amino acid residues 84, 115, 122, 128, 194, 197, 198, 212, 214, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 243, 245, 248, 262, or any combination thereof. In some embodiments, the one or more mutations comprise a deletion of one or more of amino acid residues 84, 115, 122, 128, 194, 197, 198, 212, 214, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 243, 245, 248, 262, or any combination thereof. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 84. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 115. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 122. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 128. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 194. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 197. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 198. In some embodiments, the one or more mutations include a mutation at amino acid residue 212. In some embodiments, the one or more mutations include a mutation at amino acid residue 214. In some embodiments, the one or more mutations include a mutation at amino acid residue 222. In some embodiments, the one or more mutations include a mutation at amino acid residue 223. In some embodiments, the one or more mutations include a mutation at amino acid residue 224. In some embodiments, the one or more mutations include a mutation at amino acid residue 225. In some embodiments, the one or more mutations include a mutation at amino acid residue 226.In some embodiments, the one or more mutations include a mutation at amino acid residue 227. In some embodiments, the one or more mutations include a mutation at amino acid residue 228. In some embodiments, the one or more mutations include a mutation at amino acid residue 229. In some embodiments, the one or more mutations include a mutation at amino acid residue 230. In some embodiments, the one or more mutations include a mutation at amino acid residue 231. In some embodiments, the one or more mutations include a mutation at amino acid residue 232. In some embodiments, the one or more mutations include a mutation at amino acid residue 233. In some embodiments, the one or more mutations include a mutation at amino acid residue 243. In some embodiments, the one or more mutations include a mutation at amino acid residue 245. In some embodiments, the one or more mutations include a mutation at amino acid residue 248. In some embodiments, the one or more mutations include a mutation at amino acid residue 262.

[0147] In some embodiments, the construct further comprises one or more proteins or fragments thereof that inhibit an immune response via the complement system. In some embodiments, the one or more proteins or fragments thereof are selected from CD48, CD59, or a combination thereof. In some embodiments, the one or more proteins or fragments thereof are CD48. In some embodiments, the one or more proteins or fragments thereof are CD59. In some embodiments, the one or more proteins or fragments thereof are CD48 and CD59.

[0148] In some embodiments, the peptide comprises a second amino acid residue selected from L, M, S, I, F, T, V, and Y. In some embodiments, the second amino acid residue is selected from T, V, and Y. In some embodiments, the peptide comprises a final amino acid residue selected from V, I, F, W, Y, L, R, and K. In some embodiments, the final amino acid residue is selected from Y, L, R, and K.

[0149] In some embodiments, the peptide comprises a second amino acid residue selected from E, P, L, Q, A, R, H, S, T, V, M, D, and K. In some embodiments, the second amino acid residue is selected from E, P, L, Q, A, R, and H. In some embodiments, the peptide comprises a final amino acid residue selected from V, L, F, A, I, Y, M, W, P, and R. In some embodiments, the final amino acid residue is selected from V, L, and F.

[0150] In some embodiments, the peptide comprises a second amino acid residue selected from A, Y, S, T, V, I, L, F, Q, R, N, and W. In some embodiments, the second amino acid residue is selected from A and Y. In some embodiments, the peptide comprises a final amino acid residue selected from L, V, M, F, Y, and I. In some embodiments, the final amino acid residue is L.

[0151] Disulfide Staples vs. In some embodiments, the construct may include one or more disulfide staple pairs. The disulfide staple pair may include two cysteine ​​residues configured to form a disulfide bond under appropriate conditions (e.g., oxidizing conditions, such as in a cellular compartment). The two cysteine ​​residues may be located at any suitable location on the construct. In some embodiments, the disulfide staple pair is distributed across two portions of the construct, for example across the targeting portion (e.g., peptide) and the MHC region (e.g., HLA class I heavy chain), or across the peptide and the linker, or across the peptide and the HLA class I heavy chain. In some embodiments, the disulfide staple pair is contained entirely within one region of the construct, for example entirely within the linker, entirely within the HLA class I heavy chain, or entirely within the peptide. In some embodiments, one or more disulfide staple pairs are introduced into the construct by engineering (e.g., mutating by site-directed mutagenesis). Alternatively or additionally, one or more disulfide staple pairs are present in whole or in part in the wild-type or non-mutated sequence.

[0152] The disulfide staple pairs can be configured to confer a particular secondary, tertiary, or quaternary structure when the construct sequence is arranged in three-dimensional space (e.g., expressed in a host cell). The secondary, tertiary, or quaternary structure can be determined from experimental structural biology data (e.g., x-ray crystallography data, cryo-electron microscopy data, nuclear magnetic resonance data), biochemical data (e.g., mass spectrometry data, chromatography data, electrophoresis data), or computer simulation or modeling data (e.g., molecular dynamics simulation, de novo or ab initio prediction, homology modeling, fragment assembly, secondary structure prediction). Alternatively or additionally, one or more disulfide staple pairs can be configured to confer a particular functional outcome to the expressed construct. In some embodiments, one or more disulfide staple pairs enhance expression (e.g., cell surface expression) of the imaging agent, enhance stability of the construct, prevent peptide exchange, or some combination thereof. In some embodiments, one or more disulfide staple pairs enhance expression of the construct. In some embodiments, one or more disulfide staple pairs enhance the stability of the construct, hi some embodiments, one or more disulfide staple pairs prevent peptide exchange.

[0153] In some embodiments, the disulfide staple pairs can be configured to increase expression in a host cell (e.g., compared to a wild-type or other construct lacking the disulfide staple pairs). In some embodiments, the expression is cell surface expression. Expression can be measured, for example, by flow cytometry, fluorescence microscopy, mass spectrometry, or any other suitable quantitative method. In some embodiments, the presence of the disulfide staple pairs increases expression of the construct in a host cell compared to a reference (e.g., wild-type) construct. In some embodiments, expression is increased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more. In some embodiments, expression is increased by about 1% to about 100%.In some embodiments, expression is from about 1% to about 5%, from about 1% to about 10%, from about 1% to about 20%, from about 1% to about 30%, from about 1% to about 40%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, from about 1% to about 80%, from about 1% to about 90%, from about 1% to about 100%, from about 5% to about 10%, from about 5% to about 20%, from about 5% to about 30%, from about 5% to about 40%, from about 5% to about 50%, from about 5% to about 60%, %, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60 %, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%. In some embodiments, expression is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In some embodiments, expression is increased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, expression is increased by up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0154] In some embodiments, the disulfide staple pairs may be configured to increase the stability of the construct (e.g., compared to a construct that does not contain a disulfide staple pair, such as a wild-type construct). The presence of disulfide staple pairs may increase stability by allowing the formation of additional disulfide bonds that help the construct retain a particular secondary, tertiary, and / or quaternary structure, even in the presence of forces or conditions that tend to unfold the protein (e.g., chaotropic agents, elevated temperature). The stability of the construct may be measured by differential scanning calorimetry (DSC), pulse chase assays (e.g., bleach chase and cycloheximide chase assays), thermal shift assays, circular dichroism (CD) spectroscopy, UV-vis spectroscopy, nuclear magnetic resonance (NMR), gel filtration, isothermal calorimetry, light scattering, or any other suitable assay or instrument. In some embodiments, the stability of the construct is expressed in relative terms (e.g., percent or fold change in stability relative to a reference, such as a wild-type construct). In some embodiments, the stability of a construct is expressed in absolute terms (eg, with respect to a thermodynamic coordinate such as a melting or other phase transition temperature or free energy of folding).

[0155] In some embodiments, the presence of a disulfide staple pair increases the stability of a construct by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, or more, compared to a reference (e.g., a construct that does not contain a disulfide pair, such as a wild-type construct). In some embodiments, the stability is increased by between about 1% and about 100%. In some embodiments, the stability is about 1% to about 5%, about 1% to about 10%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5 ...80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 20%, %, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60 %, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%. In some embodiments, the stability is increased by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.In some embodiments, the stability is increased by at least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, the stability is increased by up to about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0156] In some embodiments, the presence of a disulfide staple pair increases the stability of the construct by about 2-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 50-fold, about 100-fold, about 1000-fold, or more, as compared to a refence (e.g., a construct that does not contain a disulfide staple pair, such as a wild-type construct). In some embodiments, the stability is increased by about 1-fold to about 1,000-fold. In some embodiments, the stability is about 1-fold to about 2-fold, about 1-fold to about 3-fold, about 1-fold to about 4-fold, about 1-fold to about 5-fold, about 1-fold to about 10-fold, about 1-fold to about 100-fold, about 1-fold to about 1,000-fold, about 2-fold to about 3-fold, about 2-fold to about 4-fold, about 2-fold to about 5-fold, about 2-fold to about 10-fold, about 2-fold to about 100-fold, about 2-fold to about 1,000-fold, about 3-fold to about 4-fold, about 3-fold to about 5-fold, about 3-fold to about 10-fold, about 3-fold to about 100-fold, about 3-fold to about 1,000-fold, about 4-fold to about 5-fold, about 4-fold to about 10-fold, about 4-fold to about 100-fold, about 4-fold to about 1,000-fold, about 5-fold to about 10-fold, about 5-fold to about 100-fold, about 5-fold to about 1,000-fold, about 10-fold to about 100-fold, about 10-fold to about 1,000-fold, or about 100-fold to about 1,000-fold. In some embodiments, the stability is increased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold. In some embodiments, the stability is increased by at least about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, or about 100-fold. In some embodiments, the stability is increased by up to about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold.

[0157] In some embodiments, the constructs described herein that include disulfide staple pairs have a higher melting temperature (T) compared to a reference (e.g., a construct that does not include a disulfide staple pair, such as a wild-type construct). m ) (e.g., as determined by a thermodynamic technique such as DSC). In some embodiments, T m is increased by about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 20° C., about 30° C., or more, as compared to the reference. m In some embodiments, T m is about 1°C to about 2°C, about 1°C to about 3°C, about 1°C to about 4°C, about 1°C to about 5°C, about 1°C to about 6°C, about 1°C to about 7°C, about 1°C to about 8°C, about 1°C to about 9°C, about 1°C to about 10°C, about 1°C to about 20°C, about 1°C to about 30°C, about 2°C to about 3°C, about 2°C to about 4°C, about 2°C to about 5°C, about 2°C to about 6°C, about 2°C to about 7°C, about 2°C to about 8°C, about 2°C to about 9°C, about 2°C to about 10°C, about 2°C to about 20°C, about 2°C to about 30°C, about 3°C ​​to about 4°C, about 3°C ​​to about 5°C, about 3°C ​​to about 6°C, about 3°C ​​to about 7°C, about 3°C ​​to about 8°C, about 3°C ​​to about 9°C, about 3°C ​​to about 10°C, about 3°C ​​to about 20°C, about 3°C ​​to about 30°C, about 4°C to about 5°C, about 4°C to about 6°C, about 4°C to about 7°C, about 4°C to about 8°C, about 4°C to about 9°C, about 4°C to about 10°C, about 4°C to about 20°C, about 4°C to about 30°C, about 5°C to about 6°C, about 5°C to about 7°C, about 5°C to about 8°C, about 5°C to about 9°C, about 5°C to about 10°C, about 5°C to about 20°C, about 5°C to about 30°C, about 6°C to about 7°C, about 6°C to about 8°C, about 6°C to about 9°C, about 6°C to about 10°C, about 6°C to about 20°C, about 6°C to about 30 ℃, about 7°C to about 8°C, about 7°C to about 9°C, about 7°C to about 10°C, about 7°C to about 20°C, about 7°C to about 30°C, about 8°C to about 9°C, about 8°C to about 10°C, about 8°C to about 20°C, about 8°C to about 30°C, about 9°C to about 10°C, about 9°C to about 20°C, about 9°C to about 30°C, about 10°C to about 20°C, about 10°C to about 30°C, or about 20°C to about 30°C. m increases by about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 20° C., or about 30° C. In some embodiments, T mincreases by at least about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., or about 20° C. In some embodiments, T m increases by up to about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 20°C, or about 30°C.

[0158] In some embodiments, the construct may be free of one or more disulfide staple pairs. In some embodiments, the construct may be engineered (e.g., mutated by site-directed mutagenesis) to remove one or more disulfide staple pairs. Such removal may be performed to reduce the likelihood that alternative or undesirable configurations of the construct components are formed under certain conditions (e.g., when expressed in a host cell).

[0159] In some embodiments, the disulfide staple pair is introduced by mutation of one or more residues to cysteine. In some embodiments, the mutation is in a targeting moiety (e.g., a peptide) described herein. In some embodiments, the mutation is in an HLA class I heavy chain described herein. In some embodiments, the disulfide staple pair comprises a residue corresponding to the Y84 residue of HLA-C (e.g., SEQ ID NO: 194). In some embodiments, the disulfide staple pair comprises a residue corresponding to the R69 residue of HLA-C. In some embodiments, the disulfide staple pair comprises a residue corresponding to the A150 residue of HLA-C. In some embodiments, the disulfide staple pair comprises a residue corresponding to the A73 residue of HLA-C. In some embodiments, the disulfide staple pair comprises a cysteine ​​at any one of positions 1-9 of a targeting moiety (e.g., a peptide) described herein. In some embodiments, the disulfide staple pair comprises the C5 residue of a peptide. In some embodiments, the disulfide staple pair comprises the C7 residue of a peptide. In some embodiments, the disulfide staple pair comprises a C8 residue of the peptide. In some embodiments, the disulfide staple pair comprises a cysteine ​​in a peptide listed in Table 2. In some embodiments, the disulfide staple pair comprises a cysteine ​​in a linker listed in Table 3. In some embodiments, the disulfide staple pair comprises a residue corresponding to the Y84 residue of HLA-C and a residue in the linker region. In some embodiments, the disulfide staple pair comprises a residue corresponding to the R69 residue of HLA-C and a residue in a targeting moiety (e.g., a peptide). In some embodiments, the disulfide staple pair comprises a residue corresponding to the R69 residue of HLA-C and a residue in the linker region. In some embodiments, the disulfide staple pair comprises a residue corresponding to the A150 residue of HLA-C and a targeting moiety (e.g., a peptide). In some embodiments, the disulfide staple pair comprises a residue corresponding to the A150 residue of HLA-C and a residue in the linker region.In some embodiments, the disulfide staple pair comprises a residue corresponding to the A73 residue of HLA-C and a targeting moiety (e.g., a peptide). In some embodiments, the disulfide staple pair comprises a residue corresponding to the A73 residue of HLA-C and a residue in the linker region.

[0160] N- and C-terminal additions In some embodiments, the constructs described herein include one or more N- or C-terminal additions. N- or C-terminal additions can be added, for example, for purposes of purification or targeting to specific cellular components (e.g., the nucleus). In some embodiments, the constructs include an N-terminal nuclear localization signal (NLS). In some embodiments, the constructs include a purification tag, such as a hexa-his tag. The purification tag can be located at the N- or C-terminus of the construct. In some embodiments, the N- or C-terminal addition includes a tobacco etch virus (TEV) protease cleavage site. In some embodiments, the tag includes a peptide sequence that includes a portion of a cognate binding pair. In some embodiments, the cognate binding pair includes an avidin-biotin binding pair or a streptavidin-biotin binding pair. In such embodiments, the tag can include a sequence configured to be biotinylated such that the biotinylated construct can bind to a streptavidin or avidin moiety. In some embodiments, the N- or C-terminal addition includes a furin cleavage site. In some embodiments, the N- or C-terminal addition comprises a 2A self-cleaving peptide (2A peptide). In some embodiments, the 2A peptide comprises a T2A peptide, a P2A peptide, an E2A peptide, or an F2A peptide. In some embodiments, a SGSG linker is placed between the 2A peptide and the remainder of the construct (e.g., an HLA class 1 heavy chain domain).

[0161] nucleic acid molecule Provided herein, in another aspect, is a nucleic acid molecule encoding a construct provided herein.

[0162] In some embodiments, the nucleic acid molecule comprises a deletion in an endogenous HLA locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A, HLA-B, or HLA-C locus, or any combination thereof. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-B locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-C locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A locus and an HLA-B locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A locus and an HLA-C locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-B locus and an HLA-C locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A locus, an HLA-B locus, and an HLA-C locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A locus, an HLA-B locus, and an HLA-C locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A locus, an HLA-B locus, and an HLA-C locus. In some embodiments, the deletion is a complete deletion of the endogenous HLA locus.

[0163] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human HLA class 1 heavy chain sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or any combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-B sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence and an HLA-B sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence and an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-B sequence and an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, an HLA-B sequence, and an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, an HLA-B sequence, and an HLA-C sequence.

[0164] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or any combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-A sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-B sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-A sequence and multiple alleles of an HLA-B sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-A sequence and multiple alleles of an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-B sequence and multiple alleles of an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-A sequence, multiple alleles of an HLA-B sequence, and multiple alleles of an HLA-C sequence.

[0165] In some embodiments, the alleles of the HLA-A sequence are * 02:01, HLA-A * 01:01, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 24:02, HLA-A * 29:02, HLA-A * 26:01, HLA-A * 32:01, HLA-A * 23:01, HLA-A * 68:02, HLA-A * 30:01, HLA-A * 30:02, HLA-A * 34:02, HLA-A * 31:01, HLA-A *33:03, HLA-A * 02:07, HLA-A * 02:06, and HLA-A * Selected from 02:03.

[0166] In some embodiments, the alleles of the HLA-B sequence are * 44:02, HLA-B * 07:02, HLA-B * 08:01, HLA-B * 40:01, HLA-B * 35:01, HLA-B * 51:01, HLA-B * 15:01, HLA-B * 53:01, HLA-B * 15:03, HLA-B * 58:01, HLA-B * 45:01, HLA-B * 42:01, HLA-B * 44:03, HLA-B * 18:01, HLA-B * 52:01, HLA-B * 14:02, HLA-B * 46:01, HLA-B * 38:02 and HLA-B * Selected from 15:02.

[0167] In some embodiments, the alleles of the HLA-C sequence are * 07:01, HLA-C * 07:02, HLA-C * 04:01, HLA-C * 05:01, HLA-C * 03:04, HLA-C * 06:02, HLA-C * 03:03, HLA-C * 12:03, HLA-C * 08:02, HLA-C * 02:02, HLA-C * 16:01, HLA-C * 17:01, HLA-C * 01:02, HLA-C* 02:01, HLA-C * 08:01, HLA-C * 03:02 and HLA-C * Selected from 14:02.

[0168] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, which is positioned between an HLA-B sequence and an HLA-C sequence.

[0169] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1700 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1600 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1500 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1400 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1300 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1200 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1100 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1000 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 900 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 800 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 700 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 600 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 500 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 450 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 400 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 350 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 300 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 250 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 200 bp.In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 150 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 100 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 50 bp.

[0170] In some embodiments, the HLA-A sequence, the HLA-B sequence, the HLA-C sequence, or a combination thereof, comprises one or more flanking sequences. In some embodiments, the HLA-A sequence comprises one or more flanking sequences. In some embodiments, the HLA-B sequence comprises one or more flanking sequences. In some embodiments, the HLA-C sequence comprises one or more flanking sequences. In some embodiments, the HLA-A sequence and the HLA-B sequence comprise one or more flanking sequences. In some embodiments, the HLA-A sequence and the HLA-C sequence comprise one or more flanking sequences. In some embodiments, the HLA-B sequence and the HLA-C sequence comprise one or more flanking sequences. In some embodiments, the HLA-A sequence, the HLA-B sequence, and the HLA-C sequence comprise one or more flanking sequences. In some embodiments, the HLA-A sequence, the HLA-B sequence, and the HLA-C sequence comprise one or more flanking sequences.

[0171] In some embodiments, the one or more flanking sequences comprise an endogenous HLA sequence. In some embodiments, the one or more flanking sequences are specific for one or more promoters. In some embodiments, the promoter comprises an HLA-A promoter, an HLA-B promoter, an HLA-C promoter, or a combination thereof. In some embodiments, the HLA-A sequence comprises an endogenous HLA-A promoter. In some embodiments, the HLA-B sequence comprises an endogenous HLA-B promoter. In some embodiments, the HLA-C sequence comprises an endogenous HLA-C promoter. In some embodiments, the HLA-A sequence comprises an endogenous HLA-A promoter and the HLA-B sequence comprises an endogenous HLA-B promoter. In some embodiments, the HLA-A sequence comprises an endogenous HLA-A promoter and the HLA-C sequence comprises an endogenous HLA-C promoter. In some embodiments, the HLA-B sequence comprises an endogenous HLA-B promoter and the HLA-C sequence comprises an endogenous HLA-C promoter. In some embodiments, the HLA-A sequence comprises an endogenous HLA-A promoter, the HLA-B sequence comprises an endogenous HLA-B promoter, and the HLA-C sequence comprises an endogenous HLA-C promoter.

[0172] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or a combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-A sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-B sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-A sequence or an HLA-B sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-A sequence or an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-B sequence or an HLA-C sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-A sequence, an HLA-B sequence, or an HLA-C sequence.

[0173] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human beta-2 microglobulin (B2M) peptide. In some embodiments, the nucleic acid molecule further comprises a sequence encoding an endogenous human beta-2 microglobulin peptide.

[0174] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a targeting moiety (e.g., a peptide). The peptide can comprise any sequence or feature disclosed herein.

[0175] In some embodiments, the nucleic acid molecule further comprises one or more sequences encoding one or more linkers between the sequence encoding the peptide and the sequence encoding the human HLA class 1 heavy chain sequence. In some embodiments, the sequence of the one or more sequences encoding the one or more linkers is disposed between the sequence encoding the peptide and the sequence encoding the human beta-2 microglobulin peptide, between the sequence encoding the human beta-2 microglobulin peptide and the sequence encoding the human HLA class 1 heavy chain sequence, or both. In some embodiments, the sequence of the one or more sequences encoding the one or more linkers is disposed between the sequence encoding the peptide and the sequence encoding the human beta-2 microglobulin peptide. In some embodiments, the sequence of the one or more sequences encoding the one or more linkers is disposed between the sequence encoding the human beta-2 microglobulin peptide and the sequence encoding the human HLA class 1 heavy chain sequence. In some embodiments, a first sequence of the one or more sequences encoding the one or more linkers is disposed between the sequence encoding the peptide and the sequence encoding the human beta-2 microglobulin peptide, and a second sequence of the one or more sequences encoding the one or more linkers is disposed between the sequence encoding the human beta-2 microglobulin peptide and the sequence encoding the human HLA class 1 heavy chain sequence.

[0176] In some embodiments, the nucleic acid molecule further comprises a sequence encoding one or more immune checkpoint modulators. In some embodiments, the nucleic acid further comprises a sequence encoding CD8. In some embodiments, the nucleic acid molecule further comprises a sequence encoding CD47. In some embodiments, the nucleic acid molecule further comprises a sequence encoding PD-L1. In some embodiments, the nucleic acid molecule further comprises a sequence encoding A2AR. In some embodiments, the nucleic acid molecule further comprises a sequence encoding B7-H3. In some embodiments, the nucleic acid molecule further comprises a sequence encoding B7-H4. In some embodiments, the nucleic acid molecule further comprises a sequence encoding BTLA. In some embodiments, the nucleic acid molecule further comprises a sequence encoding CTLA-4. In some embodiments, the nucleic acid molecule further comprises a sequence encoding IDO. In some embodiments, the nucleic acid molecule further comprises a sequence encoding KIR. In some embodiments, the nucleic acid molecule further comprises a sequence encoding LAG3. In some embodiments, the nucleic acid molecule further comprises a sequence encoding NOX2. In some embodiments, the nucleic acid molecule further comprises a sequence encoding PD-1. In some embodiments, the nucleic acid molecule further comprises a sequence encoding TIM-3. In some embodiments, the nucleic acid molecule further comprises a sequence encoding VISTA.In some embodiments, the nucleic acid molecule further comprises a sequence encoding SIGLEC7.

[0177] In some embodiments, the nucleic acid molecule further comprises a sequence encoding one or more knockout proteins corresponding to one or more immune checkpoint modulator receptors. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout CD47 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout PD-L1 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout A2AR receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout B7-H3 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout B7-H4 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout BTLA receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout CTLA-4 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout IDO receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout KIR receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout LAG3 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout NOX2 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout PD-1 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout TIM-3 receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout VISTA receptor. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knockout SIGLEC7 receptor.

[0178] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-E sequence or a fragment thereof, an HLA-F sequence or a fragment thereof, an HLA-G sequence or a fragment thereof, or any combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-E sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-F sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-G sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-E sequence or a fragment thereof and an HLA-F sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-E sequence or a fragment thereof and an HLA-G sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-F sequence or a fragment thereof and an HLA-G sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-E sequence or a fragment thereof, an HLA-F sequence or a fragment thereof, and an HLA-G sequence or a fragment thereof.

[0179] In some embodiments, at least one of the HLA-E sequence or a fragment thereof, the HLA-F sequence or a fragment thereof, the HLA-G sequence or a fragment thereof, or any combination thereof, is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the HLA-E sequence or a fragment thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the HLA-F sequence or a fragment thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the HLA-G sequence or a fragment thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the HLA-E sequence or a fragment thereof and the HLA-F sequence or a fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the HLA-E sequence or fragment thereof and the HLA-G sequence or fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the HLA-F sequence or fragment thereof and the HLA-G sequence or fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells. In some embodiments, the HLA-E sequence or fragment thereof, the HLA-F sequence or fragment thereof, and the HLA-G sequence or fragment thereof are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells.

[0180] In some embodiments, the nucleic acid molecule further comprises a sequence encoding one or more knockout proteins corresponding to class II major histocompatibility complex transactivator (CIITA). In some embodiments, the entire class II major histocompatibility complex transactivator (CIITA) locus is knocked out.

[0181] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a regulatory peptide. In some embodiments, the nucleic acid molecule further comprises a sequence encoding an apoptosis-inducing peptide. In some embodiments, the nucleic acid molecule further comprises a sequence encoding an apoptosis-inducing peptide that acts as a "kill switch."

[0182] In some embodiments, the nucleic acid molecule further comprises a sequence encoding an epitope configured to allow detection of the construct, hi some embodiments, the nucleic acid molecule further comprises a sequence encoding an epitope comprising 3,5-dinitrosalicylic acid.

[0183] In some embodiments, the nucleic acid molecule further comprises a sequence encoding one or more knocked out proteins. In some embodiments, the one or more knocked out proteins are selected from a blood group A antigen and a blood group B antigen. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knocked out blood group A antigen. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a knocked out blood group B antigen.

[0184] In some embodiments, the nucleic acid molecule is a. a peptide coding sequence; b. a first sequence encoding a first linker of the one or more sequences encoding one or more linkers; c. a sequence encoding a human beta-2 microglobulin peptide; d. a second sequence encoding a second linker of the one or more sequences encoding the one or more linkers; and e. a sequence encoding a human HLA class 1 heavy chain sequence Includes.

[0185] Provided herein, in another aspect, is a nucleic acid molecule comprising a sequence encoding a construct comprising one or more class 1 human leukocyte antigen (HLA) proteins, wherein the one or more class 1 HLA proteins are inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells, the nucleic acid molecule comprising: a. a sequence encoding a peptide, wherein the peptide is unable to activate one or more T cells; b. a first sequence encoding a first linker; and C. a sequence encoding one or more class 1 HLA proteins Includes; The first linker is a nucleic acid molecule comprising a three-dimensional structure configured so as not to block one or more killer cell immunoglobulin-like receptor (KIR) binding sites on a human HLA class 1 heavy chain sequence, the three-dimensional structure being further configured to resist proteolytic cleavage.

[0186] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human beta-2 microglobulin peptide between the sequence encoding the linker and the sequence encoding the human HLA class 1 heavy chain sequence. In some embodiments, the nucleic acid molecule further comprises a sequence encoding an endogenous human beta-2 microglobulin peptide between the sequence encoding the linker and the sequence encoding the human HLA class 1 heavy chain sequence.

[0187] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises one or more mutations that inhibit the human HLA class 1 heavy chain sequence from eliciting a T cell response when the human HLA class 1 heavy chain sequence is interrogated by one or more CD8 cells. In some embodiments, the one or more mutations comprise one or more of amino acid residues 84, 115, 122, 128, 194, 197, 198, 212, 214, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 243, 245, 248, 262, or any combination thereof. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 84. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 115. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 122. In some embodiments, the one or more mutations include a mutation at amino acid residue 128. In some embodiments, the one or more mutations include a mutation at amino acid residue 194. In some embodiments, the one or more mutations include a mutation at amino acid residue 197. In some embodiments, the one or more mutations include a mutation at amino acid residue 198. In some embodiments, the one or more mutations include a mutation at amino acid residue 212. In some embodiments, the one or more mutations include a mutation at amino acid residue 214. In some embodiments, the one or more mutations include a mutation at amino acid residue 222. In some embodiments, the one or more mutations include a mutation at amino acid residue 223. In some embodiments, the one or more mutations include a mutation at amino acid residue 224. In some embodiments, the one or more mutations include a mutation at amino acid residue 225. In some embodiments, the one or more mutations include a mutation at amino acid residue 226. In some embodiments, the one or more mutations include a mutation at amino acid residue 227. In some embodiments, the one or more mutations include a mutation at amino acid residue 228. In some embodiments, the one or more mutations include a mutation at amino acid residue 229. In some embodiments, the one or more mutations comprises a mutation at amino acid residue 230. In some embodiments, the one or more mutations comprises a mutation at amino acid residue 231.In some embodiments, the one or more mutations comprise a mutation at amino acid residue 232. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 233. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 243. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 245. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 248. In some embodiments, the one or more mutations comprise a mutation at amino acid residue 262.

[0188] In some embodiments, the nucleic acid molecule further comprises a sequence encoding one or more proteins or fragments thereof that inhibit an immune response via the complement system. In some embodiments, the one or more proteins or fragments thereof are selected from CD48, CD59, or a combination thereof. In some embodiments, the one or more proteins or fragments thereof are CD48. In some embodiments, the one or more proteins or fragments thereof are CD59. In some embodiments, the one or more proteins or fragments thereof are CD48 and CD59.

[0189] Provided herein, in another aspect, is a method for generating a nucleic acid molecule provided herein, comprising placing a sequence encoding a region configured to receive a sequence comprising a deletion at an HLA locus, a sequence encoding a human HLA class 1 heavy chain sequence, or any combination thereof. In some embodiments, the method comprises placing a sequence encoding a region configured to receive a sequence comprising a deletion at an HLA locus. In some embodiments, the method comprises placing a sequence encoding a region configured to receive a sequence encoding a human HLA class 1 heavy chain sequence. In some embodiments, the method comprises placing a sequence encoding a region configured to receive a sequence comprising a deletion at an HLA locus and a sequence encoding a human HLA class 1 heavy chain sequence.

[0190] Immunocompetent cells Provided herein, in another aspect, is a method of generating an immunocompromised cell, the method comprising administering to a cell a construct provided herein or a nucleic acid molecule provided herein.

[0191] In some embodiments, the nucleic acid molecule is delivered to the genome of the cell, hi some embodiments, the cell is incubated with the construct.

[0192] In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the stem cell is an embryonic stem cell (ESC). In some embodiments, the stem cell is a mesenchymal stem cell (MSC). In some embodiments, the stem cell is a hematopoietic stem cell (HSC). In some embodiments, the cell is a chimeric antigen receptor (CAR) T cell. In some embodiments, the cell is a chimeric antigen receptor macrophage (CAR-M) cell. In some embodiments, the cell is a chimeric antigen receptor natural killer (CAR-NK) cell.

[0193] In some embodiments, the immunologically incompetent cells are suitable for use in cell therapy, hi some embodiments, the immunologically incompetent cells are suitable for administration to a subject without eliciting an immune response.

[0194] Other methods of gene therapy / writing Vectors and Nucleic Acids Various nucleic acids can be introduced into cells to obtain expression of genes for knockout purposes or other purposes. Nucleic acid constructs that can be used to generate transgenic cells containing target nucleic acid sequences. As used herein, the term nucleic acid or "nucleic acid molecule" includes DNA, RNA, and nucleic acid analogs, as well as nucleic acids that are double-stranded or single-stranded (i.e., sense or antisense single stranded). Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve the stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. Modifications at the sugar moiety include modification of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids (where each base moiety is linked to a six-membered morpholino ring) or peptide nucleic acids (where the deoxyphosphate backbone is replaced by a pseudopeptide backbone and four bases are retained). See Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7(3):187; and Hyrup et al. (1996) Bioorgan. Med. Chem. 4:5. Additionally, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoramidite, or an alkyl phosphotriester backbone.

[0195] The target nucleic acid sequence may be operably linked to a regulatory region, such as a promoter. The regulatory region may be from any species. As used herein, operably linked refers to the positioning of the regulatory region relative to the nucleic acid sequence so as to permit or facilitate transcription of the target nucleic acid.

[0196] Any type of promoter may be operably linked to the target nucleic acid sequence. Examples of promoters include, but are not limited to, tissue-specific promoters, constitutive promoters, and promoters that are responsive or unresponsive to a particular stimulus. Suitable tissue-specific promoters may result in preferential expression of nucleic acid transcripts in beta cells, including, for example, the human insulin promoter. Other tissue-specific promoters may result in preferential expression in, for example, hepatocytes or cardiac tissue, including the albumin or alpha-myosin heavy chain promoters, respectively. In other embodiments, promoters that promote the expression of nucleic acid molecules without significant tissue or time specificity may be used (i.e., constitutive promoters). For example, beta-actin promoters such as chicken beta-actin gene promoter, ubiquitin promoter, mini-CAG promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, or 3-phosphoglycerate kinase (PGK) promoter, as well as viral promoters such as herpes simplex virus thymidine kinase (HSV-TK) promoter, SV40 promoter, or cytomegalovirus (CMV) promoter may be used. In some embodiments, a fusion of the chicken beta actin gene promoter and CMV enhancer is used as the promoter. See, e.g., Xu et al. (2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther. 7:821.

[0197] An example of an inducible promoter is the tetracycline (tet)-on promoter system, which can be used to regulate transcription of a nucleic acid. In this system, a mutant Tet repressor (TetR) is fused to the activation domain of the herpes simplex virus VP16 transactivator protein to create a tetracycline-controlled transcription activator (tTA), which is regulated by tet or doxycycline (dox). In the absence of antibiotics, transcription is minimal, but in the presence of tet or dox, transcription is induced. Alternative inducible systems include the ecdysone system or the rapamycin system. Ecdysone is an insect molting hormone whose production is controlled by a heterodimer of the ecdysone receptor and the product of the ultraspiracle gene (USP). Expression is induced by treatment with ecdysone or an analog of ecdysone (e.g., muristerone A). An agent administered to a subject to induce an inducible system is called an inducer.

[0198] Additional regulatory regions that may be useful in nucleic acid constructs include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., internal ribosome entry segments, IRES), enhancers, inducible elements, or introns. Such regulatory regions may increase expression by affecting transcription, mRNA stability, translation efficiency, etc., but may not be necessary. Such regulatory regions may be included in the desired nucleic acid construct to obtain optimal expression of the nucleic acid in cells. However, sufficient expression may be obtained without such additional elements.

[0199] Nucleic acid constructs encoding signal peptides or selectable markers may be used. Signal peptides can be used to direct the encoded polypeptide to a specific cellular location (e.g., the cell surface). Non-limiting examples of selectable markers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase, thymidine kinase (TK), and xanthine-guanine phosphoribosyltransferase (XGPRT). Such markers are useful for selecting stable transformants in culture. Other selectable markers include fluorescent polypeptides, such as green fluorescent protein or yellow fluorescent protein.

[0200] In some embodiments, the sequence encoding the selectable marker may be flanked by recognition sequences for a recombinase, such as Cre or Flp. For example, the selectable marker may be flanked by loxP recognition sites (a 34 bp recognition site recognized by Cre recombinase) or FRT recognition sites so that the selectable marker can be excised from the construct. See Orban, et al., Proc. Natl. Acad. Sci. (1992) 89:6861, for a review of Cre / lox technology, and Brand and Dymecki, Dev. Cell (2004) 6:7. Transposons containing a Cre or Flp activatable transgene interrupted by a selectable marker gene may also be used to obtain transgenic cells with conditional expression of the transgene.

[0201] In some embodiments, the target nucleic acid encodes a polypeptide. The nucleic acid sequence encoding the polypeptide may include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection). The tag sequence can be inserted into the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include glutathione S-transferase (GST) and FLAG™ tags (Kodak, New Haven, Conn.).

[0202] In other embodiments, the target nucleic acid sequence induces RNA interference to the target nucleic acid, so that the expression of the target nucleic acid is reduced.For example, the target nucleic acid sequence can induce RNA interference to the nucleic acid encoding Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) polypeptide.For example, the expression of the DNA can be reduced by using double-stranded small interfering RNA (siRNA) or short hairpin RNA (shRNA) homologues to CFTR DNA. Constructs for siRNA can be produced as described, for example, in Fire et al. (1998) Nature 391:806; Romano and Masino (1992) Mol. Microbiol. 6:3343; Cogoni et al. (1996) EMBO J. 15:3153; Cogoni and Masino (1999) Nature 399:166; Misquitta and Paterson (1999) Proc. Natl. Acad. Sci. USA 96:1451; and Kennerdell and Carthew (1998) Cell 95:1017. Constructs for shRNA can be produced as described in McIntyre and Fanning (2006) BMC Biotechnology 6:1. In general, shRNA is transcribed as a single-stranded RNA molecule that contains a complementary region that can anneal to form a short hairpin.

[0203] Nucleic acid constructs can be introduced into any type of embryonic, fetal, or adult cell, including, for example, germ cells such as oocytes or eggs, progenitor cells, adult or embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, primordial germ cells, kidney cells such as PK-15 cells, pancreatic islet cells, beta cells, liver cells, or fibroblasts such as skin fibroblasts, using a variety of techniques. Non-limiting examples of techniques include the use of transposon systems, recombinant viruses capable of infecting cells, or liposomes, or other non-viral methods capable of delivering nucleic acids to cells (e.g., electroporation, microinjection, or calcium phosphate precipitation).

[0204] In the transposon system, the transcription unit of the nucleic acid construct, i.e., the regulatory region operably linked to the target nucleic acid sequence, is flanked by the inverted repeats of the transposon. For example, several transposon systems have been developed to introduce nucleic acid into cells, including Sleeping Beauty (see U.S. Pat. No. 6,613,752 and U.S. Patent Application Publication No. 2005 / 0003542); FrogPrince (Miskey et al. (2003) Nucleic Acids Res. 31:6873); Tol2 (Kawakami (2007) Genome Biology 8 (Suppl. 1):S7, Minos (Pavlopoulos et al. (2007) Genome Biology 8 (Suppl. 1):S2); Hsmar1 (Miskey et al. (2007)) Mol Cell Biol. 27:4589); and Passport. Sleeping Beauty and Passport transposons are particularly useful. The transposase can be delivered as a protein encoded on the same nucleic acid construct as the target nucleic acid, can be introduced on a separate nucleic acid construct, or can be provided as an mRNA (e.g., an in vitro transcribed, capped mRNA).

[0205] Insulator elements can also be included in the nucleic acid construct to maintain expression of target nucleic acid and inhibit undesired transcription of host genes. For example, see US Patent Application Publication No. 2004 / 0203158. Typically, insulator elements flank both sides of the transcription unit and are internal to the inverted repeat of the transposon. Non-limiting examples of insulator elements include matrix attachment region (MAR) type insulator elements and border type insulator elements. For example, see US Patent Application Publication No. 6,395,549, US Patent Application Publication No. 5,731,178, US Patent Application Publication No. 6,100,448 and US Patent Application Publication No. 5,610,053, and US Patent Application Publication No. 2004 / 0203158.

[0206] Nucleic acids can be incorporated into vectors. Vector is a broad term that includes any specific DNA segment designed to transfer from a carrier to a target DNA. A vector may also be called an expression vector or vector system, which is a set of components required to effect DNA insertion into a genome or other target DNA sequence, such as an episome, a plasmid, or even a virus / phage DNA segment. Vector systems, such as viral vectors (e.g., retroviruses, adeno-associated viruses, and integrative phage viruses) and non-viral vectors (e.g., transposons) used for gene delivery in subjects, have two basic components: 1) a vector composed of DNA (or RNA that is reverse transcribed into cDNA), and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and the DNA target sequence and inserts the vector into the target DNA sequence. A vector most often contains one or more expression cassettes that include one or more expression control sequences, where the expression control sequences are DNA sequences that control and regulate the transcription and / or translation of another DNA sequence or mRNA, respectively.

[0207] Many different types of vectors are known. For example, plasmids and viral vectors, such as retroviral vectors, are known. Mammalian expression plasmids typically have an origin of replication, a suitable promoter and optional enhancer, as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non-transcriptional sequences. Examples of vectors include plasmids (which may be carriers of other types of vectors), adenoviruses, adeno-associated viruses (AAV), lentiviruses (e.g., HIV-1, SIV or FIV), retroviruses (e.g., ASV, ALV or MoMLV), herpes simplex viruses (HSV) and transposons (e.g., Sleeping Beauty, P elements, Tol-2, Frog Prince, piggyBac).

[0208] In another aspect, provided herein is an additional method of delivering one or more human leukocyte antigen (HLA)-encoding nucleic acid molecules to cells. In some embodiments, the method comprises delivering one or more HLA-encoding nucleic acid molecules via a viral vector. In some embodiments, the method comprises delivering one or more HLA-encoding nucleic acid molecules via a non-viral vector. In some embodiments, the viral vector is derived from a lentivirus.

[0209] In some embodiments, the nucleic acid molecule comprises a deletion in an endogenous HLA locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A, HLA-B, or HLA-C locus, or any combination thereof. In some embodiments, the deletion is a complete deletion of the endogenous HLA locus.

[0210] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human HLA class 1 heavy chain sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or any combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or any combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, and the HLA-A sequence is disposed between the HLA-B sequence and the HLA-C sequence.

[0211] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1700 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 500 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 250 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 150 bp.

[0212] In some embodiments, the HLA-A sequence, HLA-B sequence, HLA-C sequence, or combinations thereof, comprises one or more flanking sequences. In some embodiments, the one or more flanking sequences comprise endogenous HLA sequences. In some embodiments, the one or more flanking sequences are specific to one or more promoters. In some embodiments, the promoter comprises an HLA-A promoter, an HLA-B promoter, an HLA-C promoter, or combinations thereof.

[0213] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or a combination thereof.

[0214] In some embodiments, the nucleic acid molecule encoding the human HLA class 1 heavy chain sequence comprises an HLA-E sequence or a fragment thereof, an HLA-F sequence or a fragment thereof, an HLA-G sequence or a fragment thereof, or any combination thereof. In some embodiments, at least one of the HLA-E sequence or a fragment thereof, the HLA-F sequence or a fragment thereof, the HLA-G sequence or a fragment thereof, or any combination thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells.

[0215] In some embodiments, the nucleic acid molecule encoding the human HLA class 1 heavy chain sequence comprises one or more mutations, and a cell comprising the mutant human HLA class 1 heavy chain sequence comprising one or more mutations does not elicit an immune response when the cell is probed by one or more CD8 cells. In some embodiments, the mutant human HLA class 1 heavy chain sequence encodes an HLA comprising one or more mutations at one or more of amino acid residues 115, 122, 128, 194, 197, 198, 212, 214, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 243, 245, 248, 262, or any combination thereof.

[0216] Templated and Non-templated Repair Zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats / CRISPR-associated endonuclease cas9 (CRISPR / Cas9) can be used to introduce insertions and / or deletions (indels) into the genome of a species, for example by non-homologous end joining (NHEJ), to disrupt gene function. However, the indels introduced by NHEJ are variable in size and sequence, which makes it difficult to screen for functionally disrupted clones and does not allow for precise modification. TALENs or CRISPR / Cas9-mediated homology-directed repair (HDR) supports the introduction of defined nucleotide changes in eukaryotic cells.

[0217] The subject may be modified using other genetic engineering tools, including TALEN, zinc finger nucleases, or various vectors known in the art. The genetic modification made by such tools may include gene inactivation. The term gene inactivation refers to preventing the formation of a functional gene product. A gene product is functional only if it fulfills its normal (wild type) function. Materials and methods for genetically modifying a subject are further described in U.S. Patent Application No. 13 / 404,662, filed February 24, 2012, U.S. Patent Application No. 13 / 467,588, filed May 9, 2012, and U.S. Patent Application No. 12 / 622,886, filed November 10, 2009, which are incorporated herein by reference for all purposes. In case of conflict, the present specification takes precedence. The term trans-acting refers to the process of acting on a target gene from different molecules (i.e., between molecules). A trans-acting element is usually a DNA sequence that contains a gene. This gene codes for a protein (or a microRNA or other diffusible molecule) that is used in the regulation of the target gene. A trans-acting gene may be on the same chromosome as the target gene, but its activity is mediated by an intermediate protein or RNA that it codes for. Inactivation of a gene using dominant negative generally involves a trans-acting element. The term cis-regulatory or cis-acting refers to an action that does not code for a protein or RNA; in the context of gene inactivation, this generally refers to the inactivation of the coding portion of a gene, or the promoter and / or operator required for the expression of a functional gene.

[0218] A variety of techniques known in the art can be used to introduce the nucleic acid construct into non-humans and humans to generate founder lines in which the nucleic acid construct has been integrated into the genome. Such techniques include, but are not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191), retroviral-mediated gene transfer into the germ line (Van der Putten et al. (1985) Proc. Natl. Acad. Sci. USA 82, 6148-1652), gene targeting into embryonic stem cells (Thompson et al. (1989) Cell 56, 313-321), electroporation of embryos (Lo (1983) Mol. Cell. Biol. 3, 1803-1814), sperm-mediated gene transfer (Lavitrano et al. (2002) Proc. Natl. Acad. Sci. USA 99, 14230-14235; Lavitrano et al. al. (2006) Reprod. Fert. Develop. 18, 19-23), and in vitro transformation of somatic cells, such as cumulus or mammary cells, or adult, fetal, or embryonic stem cells, followed by nuclear transfer (Wilmut et al. (1997) Nature 385, 810-813; and Wakayama et al. (1998) Nature 394, 369-374). Pronuclear microinjection, sperm-mediated gene transfer, and somatic cell nuclear transfer are particularly useful techniques, as are cytoplasmic injection, primordial germ cell transfer (Brinster), and blastocyst chimera production, in which germ cells are expanded in the embryo.

[0219] TALENs, zinc finger nucleases, CRISPR nucleases (e.g., CRISPR / Cas9) and recombinase fusion proteins can be used with or without a template. A template is exogenous DNA that is added to a cell to be used as a guide (template) for the cellular repair machinery to repair double-strand breaks (DSBs) in DNA. This process is generally called homology-directed repair (HDR). Template-free processes involve creating DSBs and providing the cellular machinery to create repairs, often less than complete, such that insertions or deletions (indels) are created. A cellular pathway called non-homologous end joining (NHEJ) typically mediates non-template repair of DSBs. The term NHEJ is generally used to refer to all such non-template repairs, regardless of whether NHEJ or alternative cellular pathways are involved.

[0220] Targeted Nuclease Systems Genome editing tools such as transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have impacted the fields of biotechnology, gene therapy and functional genomics research in many organisms. More recently, RNA-guided endonucleases (RGENs) are directed to their target sites by complementary RNA molecules. The Cas9 / CRISPR system is an RGEN. tracrRNA is another such tool. These are examples of targeted nuclease systems: they have a DNA-binding member that localizes the nuclease to the target site. This site is then cleaved by the nuclease. TALENs and ZFNs have a nuclease fused to a DNA-binding member. Cas9 / CRISPRs are cognate to each other found on the target DNA. The DNA-binding members have cognate sequences in chromosomal DNA. The DNA-binding members are typically designed with the intended cognate sequence in mind to obtain nucleolytic action at or near the intended site. Certain embodiments are applicable to all such systems, including, but not limited to, embodiments that minimize nuclease re-cleavage, embodiments for creating SNPs precisely at intended residues, embodiments for creating indels precisely at intended residues, and positioning of the introgressed allele at the DNA binding site.

[0221] Zinc finger nucleases (ZFNs) Zinc finger nuclease (ZFN) is an artificial restriction enzyme that is generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target a desired DNA sequence, which allows the zinc finger nuclease to target a unique sequence within the construct genome. By utilizing endogenous DNA repair mechanisms, these reagents can be used to modify the genome of higher organisms. ZFN can be used in methods to inactivate genes.

[0222] Zinc finger DNA binding domains have about 30 amino acids and fold into a stable structure. Each finger binds primarily to a triplet in the DNA substrate. Amino acid residues at key positions contribute most of the sequence-specific interactions with the DNA site. These amino acids can be changed while maintaining the remaining amino acids to conserve the required structure. Binding to longer DNA sequences is achieved by linking several domains in tandem. Other functionalities such as a non-specific FokI cleavage domain (N), a transcriptional activator domain (A), a transcriptional repressor domain (R) and a methylase (M) can be fused to the ZFP to form ZFNs, zinc finger transcriptional activators (ZFA), zinc finger transcriptional repressors (ZFR) and zinc finger methylases (ZFM), respectively.

[0223] Transcription activator-like effector nucleases (TALENs) The term TALEN as used herein is broad and includes monomeric TALENs that can cleave double-stranded DNA without assistance from another TALEN, for example, as in Beurdeley, M. et al. Compact designer TALENs for efficient genome engineering. Nat. Commun. 4:1762 doi:10.1038 / ncomms2782 (2013). The term TALEN is also used to refer to one or both members of a pair of TALENs that are engineered to act together to cleave DNA at the same site. TALENs that act together may be referred to as left TALENs and right TALENs, which refers to the handedness of the DNA or TALEN pair.

[0224] In some embodiments, monomeric TALENs can be used. TALENs typically function as dimers across a bipartite recognition site with a spacer, where two TAL effector domains are each fused to the catalytic domain of FokI restriction enzyme, and the resulting DNA recognition sites of each TALEN are separated by a spacer sequence, such that binding of each TALEN monomer to its recognition site causes FokI to dimerize and create a double-stranded break within the spacer. However, monomeric TALENs can also be constructed such that a single TAL effector is fused to a nuclease that does not require dimerization to function. One such nuclease is, for example, a single-chain variant of FokI, where the two monomers are expressed as a single polypeptide. Other naturally occurring or engineered monomeric nucleases can also fulfill this role. The DNA recognition domain used for monomeric TALENs can be derived from a naturally occurring TAL effector. Alternatively, the DNA recognition domain can be engineered to recognize a specific DNA target. Engineered single-stranded TALENs may be easier to construct and deploy, since they only require one engineered DNA recognition domain. Dimeric DNA sequence-specific nucleases can be generated using two different DNA binding domains (e.g., one TAL effector binding domain and one binding domain from another type of molecule). TALENs can function as dimers across a bipartite recognition site with a spacer. This nuclease structure can also be used for target-specific nucleases generated from, for example, one TALEN monomer and one zinc finger nuclease monomer. In such cases, the DNA recognition sites of the TALEN and zinc finger nuclease monomer can be separated by a spacer of appropriate length. Binding of the two monomers can allow FokI to dimerize, generating a double-stranded break within the spacer sequence. DNA binding domains other than zinc fingers, such as homeodomains, myb repeats or leucine zippers, can also be fused to FokI and function as partners with TALEN monomers to generate functional nucleases.

[0225] In some embodiments, TAL effectors can be used to target other protein domains (e.g., non-nuclease protein domains) to specific nucleotide sequences. For example, TAL effectors can be linked to protein domains derived from proteins that interact with or modify other proteins, such as, but not limited to, DNA20-interacting enzymes (e.g., methylases, topoisomerases, integrases, transposases, or ligases), transcriptional activators or repressors, or histones. Applications of such TAL effector fusions include, for example, creating or modifying epigenetic regulatory elements, making site-specific insertions, deletions, or repairs in DNA, controlling gene expression, and altering chromatin structure.

[0226] The spacer of the target sequence can be selected or changed to modulate TALEN specificity and activity.The flexibility of spacer length indicates that the spacer length can be selected to target specific sequences with high specificity.In addition, activity variations have been observed for different spacer lengths, indicating that the spacer length can be selected to achieve the desired level of TALEN activity.

[0227] Alternative embodiments use alternative mRNA polymerases and cognate binding sites, such as T7 or SP6. Other embodiments relate to the use of any of several modifications of the UTR sequence; these may be beneficial for translation of the mRNA. Some examples are the addition of a cytoplasmic polyadenylation element binding site in the 3'UTR, or replacing the Xenopus β-globin UTR with a UTR sequence from a gene from human, pig, cow, sheep, goat, zebrafish, i.e., including B-globin. UTRs from genes may be selected for regulation of embryo development or expression in cells. Some examples of UTRs that may be useful include β-actin, DEAH (SEQ ID NO: 527), TPT1, ZF42, SKP1, TKT, TP3, DDX5, EIF3A, DDX39, GAPDH, CDK1, Hsp90ab1, Ybx1 fEif4b Rps27a Stra13, Myc, Paf1 and Foxo1, or CHUK. Such vector or mRNA improvements can be used to direct specific or transient expression of ectopic TALENs for gene depletion studies at desired stages of development.

[0228] In some embodiments, monomeric TALENs can be used. TALENs typically function as dimers spanning a bipartite recognition site with a spacer, such that two TAL effector domains are each fused to the catalytic domain of FokI restriction enzyme, and the resulting DNA recognition sites of each TALEN are separated by a spacer sequence, allowing binding of each TALEN monomer to its recognition site to dimerize FokI and generate a double-stranded break within the spacer. However, monomeric TALENs can also be constructed such that a single TAL effector is fused to a nuclease that does not require dimerization to function. One such nuclease is, for example, a single-chain variant of FokI, where the two monomers are expressed as a single polypeptide. Other naturally occurring or engineered monomeric nucleases can also fulfill this role. The DNA recognition domain used for monomeric TALENs can be derived from a naturally occurring TAL effector. Alternatively, the DNA recognition domain can be engineered to recognize a specific DNA target. Engineered single-stranded TALENs may be easier to construct and deploy, since they only require one engineered DNA recognition domain. Dimeric DNA sequence-specific nucleases can be generated using two different DNA binding domains (e.g., one TAL effector binding domain and one binding domain from another type of molecule). TALENs can function as dimers across a bipartite recognition site with a spacer. This nuclease structure can also be used for target-specific nucleases generated from, for example, one TALEN monomer and one zinc finger nuclease monomer. In such cases, the DNA recognition sites of the TALEN and zinc finger nuclease monomer can be separated by a spacer of appropriate length. Binding of the two monomers can allow FokI to dimerize, generating a double-stranded break within the spacer sequence. DNA binding domains other than zinc fingers, such as homeodomains, myb repeats or leucine zippers, can also be fused to FokI and function as partners with TALEN monomers to generate functional nucleases.

[0229] The term nuclease includes exonucleases and endonucleases. The term endonuclease refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids in DNA or RNA molecules, preferably DNA molecules. Non-limiting examples of endonucleases include type II restriction endonucleases such as FokI, HhaI, HindIII, NotI, BbvCl, EcoRI, BglII, and AhwI. Endonucleases also include rare-cutting endonucleases, which typically have a polynucleotide recognition site of about 12-45 base pairs (bp) in length, more preferably 14-45 bp. Rare-cutting endonucleases induce DNA double-strand breaks (DSBs) at defined loci. The rare-cutting endonuclease may be, for example, a homing endonuclease, a chimeric zinc finger nuclease (ZFN) resulting from the fusion of an engineered zinc finger domain with the catalytic domain of a restriction enzyme such as FokI, or a chemical endonuclease. In chemical endonucleases, a chemical or peptide cleaving agent is conjugated to either a polymer of nucleic acid or another DNA that recognizes a specific target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical endonucleases also encompass synthetic nuclease-like conjugates of orthophenanthroline, DNA cleavage molecules, and triplex-forming oligonucleotides (TFOs), which are known to bind to specific DNA sequences. Such chemical endonucleases are included in the term "endonuclease" according to the present invention.Examples of such endonucleases are I-See I, I-Chu L I-Cre I, I-Csm I, PI-See L PI-Tti L PI-Mtu I, I-Ceu I, I-See IL 1-See III, HO, PI-Civ I, PI-Ctr L PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra L PI-May L PI-Meh I, PI-Mfu L PI-Mfl I, PI-Mga L PI-Mgo I, PI-Min L PI-Mka L PI-Mle I, PI-Mma I, PI-30 Msh L PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu L PI-Rma I, PI-Spb I, PI-Ssp L PI-Fae L PI-Mja I, PI-Pho L Examples of PI-Tag L include PI-Thy I, PI-Tko I, PI-Tsp I, and I-Msol.

[0230] The genetic modification performed by TALEN or other tools can be selected from the list consisting of, for example, insertion, deletion, insertion of exogenous nucleic acid fragment, and substitution. The term "insertion" is used broadly to mean either literal insertion into a chromosome or the use of an exogenous sequence as a template for repair. In general, a target DNA site is identified and a TALEN pair is created that specifically binds to that site. TALEN is delivered to a cell or embryo, for example, as a protein, mRNA, or by a vector that codes for the TALEN. TALEN cleaves DNA to create a double-strand break, which is then repaired, often resulting in the creation of an indel or incorporating a sequence or polymorphism contained in the accompanying exogenous nucleic acid that is inserted into the chromosome or acts as a template for repair of the break by the modified sequence. This template-driven repair is a useful process for changing chromosomes, resulting in effective changes to cell chromosomes.

[0231] The term exogenous nucleic acid refers to a nucleic acid that is added to a cell or embryo, regardless of whether the nucleic acid is the same as or different from a naturally occurring nucleic acid sequence in the cell. In some cases, the exogenous nucleic acid differs in sequence from any nucleic acid sequence that naturally occurs in the cell. The term nucleic acid fragment is broad and includes chromosomes, expression cassettes, genes, DNA, RNA, mRNA, or portions thereof.

[0232] The genetic modification of the cell may also include the insertion of a reporter. The reporter may be, for example, a fluorescent marker, such as green fluorescent protein and yellow fluorescent protein. The reporter may be a selection marker, such as puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase, thymidine kinase (TK), or xanthine-guanine phosphoribosyltransferase (XGPRT). The vector for the reporter, selection marker, and / or one or more TALENs may be a plasmid, TALENs can target multiple DNA sites. These sites can be separated by thousands or even thousands of base pairs. The DNA can be respliced ​​by cellular machinery, thereby resulting in deletion of the entire region between the sites. Embodiments include sites separated by distances of, for example, between 1 and 5 megabases, or between 50% and 80% of the chromosome, or between about 100 and about 1,000,000 base pairs. One of skill in the art will readily appreciate that all ranges and values ​​within the explicitly stated ranges are contemplated, for example, between about 1,000 and about 10,000 base pairs or between about 500 and about 500,000 base pairs. Alternatively, exogenous DNA can be added to the cell or embryo to insert the exogenous DNA or perform template-driven repair of the DNA between the sites. Modification at multiple sites can be used to generate genetically modified cells, embryos, ungulates, and livestock. One or more genes can be selected, including sexual maturation genes or their cis-acting factors, for complete or at least partial deletion.

[0233] RecombinaseAn embodiment of the present invention includes administering one or more TALENs together with a recombinase or other DNA binding protein involved in DNA recombination. The recombinase forms a filament with a nucleic acid fragment, and in effect searches the cellular DNA for DNA sequences that are substantially homologous to its sequence. Some embodiments of TALEN recombinase embodiments include combining the recombinase with a nucleic acid sequence that serves as a template for HDR. The HDR template sequence has substantial homology to the site targeted for cleavage by the TALEN / TALEN pair. As described herein, the HDR template provides changes to the native DNA by positioning alleles, creating indels, inserting exogenous DNA, or with other changes. The TALEN is placed in the cell or embryo by the methods described herein as a protein, mRNA, or by using a vector. The recombinase is combined with the HDR template to form a filament and is placed in the cell. The recombinase and / or the HDR template combined with the recombinase can be placed in the cell or embryo as a protein, mRNA, or with a vector encoding the recombinase. The term recombinase refers to a genetic recombinase that enzymatically catalyzes the joining of relatively short pieces of DNA between two relatively long DNA strands in a cell. Recombinases include Cre recombinase, Hin recombinase, RecA, RAD51, Cre, and FLP. Cre recombinase is a type I topoisomerase from P1 bacteriophage that catalyzes site-specific recombination of DNA between loxP sites. Hin recombinase is a 21 kD protein composed of 198 amino acids found in the bacterium Salmonella. Hin belongs to the serine recombinase family of DNA invertases that depend on an active site serine to initiate DNA cleavage and recombination. RAD51 is a human gene. The protein encoded by this gene is a member of the RAD51 protein family that assists in the repair of DNA double-strand breaks. RAD51 family members are homologous to bacterial RecA and yeast Rad51 genes.Cre recombinase is an enzyme used in experiments to delete specific sequences adjacent to loxP sites. FLP refers to flippase recombinase (FLP or Flp) derived from the 2μ plasmid of the baker's yeast Saccharomyces cerevisiae.

[0234] As used herein, "RecA" or "RecA protein" refers to a family of RecA-like recombination proteins that have essentially all or most of the same functions, in particular: (i) the ability to properly position oligonucleotides or polynucleotides on their homologous targets for subsequent extension by DNA polymerase; (ii) the ability to topologically prepare double-stranded nucleic acids for DNA synthesis; and (iii) the ability of RecA / oligonucleotide or RecA / polynucleotide constructs to efficiently find and bind complementary sequences. The best-characterized RecA protein is in E. coli, and in addition to the original allelic form of the protein, several mutant RecA-like proteins, e.g., RecA803, have been identified. Furthermore, many organisms, including, for example, yeast, Drosophila, mammals including humans, and plants, possess RecA-like strand transfer proteins. These proteins include, for example, Rec1, Rec2, Rad51, Rad51B, Rad51C, Rad51D, Rad51E, XRCC2, and DMC1. An embodiment of the recombination protein is the RecA protein of E. coli. Alternatively, the RecA protein can be the mutant RecA-803 protein of E. coli, a RecA protein from another bacterial source, or a homologous recombination protein from another organism.

[0235] RecA is known for its recombinase activity, which catalyzes strand exchange during the repair of double-strand breaks by homologous recombination (McGrew and Knight, 2003) Radding, et al., 1981; Seitz et al., 1998). RecA has also been shown to catalyze the proteolysis of, for example, LexA and X repressor proteins and to have DNA-dependent ATPase activity. After a double-strand break occurs from ionizing radiation or some other insult, the exonuclease chews back the DNA end 5' to 3', thereby exposing one strand of DNA (Cox, 1999; McGrew and Knight, 2003). Single-stranded DNA is stabilized by single-stranded binding proteins (SSBs). After binding of SSBs, RecA binds to single-stranded (ss) DNA and forms helical nucleoprotein filaments (called filaments or presynaptic filaments). During DNA repair, the homology-searching function of RecA directs the filament to homologous DNA and catalyzes homologous base pairing and strand exchange, resulting in the formation of a DNA heteroduplex. After strand invasion, DNA polymerase extends the ssDNA based on the homologous DNA template to repair the DNA break, forming a crossover structure or Holliday junction. RecA also exhibits a motor function involved in the movement of the crossover structure (Campbell and Davis, 1999).

[0236] Recombinase activity includes several different functions. For example, a polypeptide sequence having recombinase activity can bind to single-stranded DNA in a non-sequence-specific manner to form a nucleoprotein filament. Such a recombinase-bound nucleoprotein filament can interact with a double-stranded DNA molecule in a non-sequence-specific manner, search for a sequence in the double-stranded molecule that is homologous to a sequence in the filament, and if such a sequence is found, position one of the strands of the double-stranded molecule to allow base pairing between the sequence in the filament and the complementary sequence of one of the strands of the double-stranded molecule. Such steps are collectively called "synapsis".

[0237] Thus, recombinase activities include, but are not limited to, single-stranded DNA binding, synapsis, homology search, double-stranded invasion by single-stranded DNA, heteroduplex formation, ATP hydrolysis, and proteolysis. The prototypic recombinase is the RecA protein from Escherichia coli (E. coli). See, for example, U.S. Pat. No. 4,888,274. Prokaryotic RecA-like proteins have also been described in Salmonella, Bacillus, and Proteus species. A thermostable RecA protein from Thermus aquaticus is described in U.S. Pat. No. 5,510,473. The UvsX protein, a bacteriophage T4 homolog of RecA, has been described. RecA mutants with altered recombinase activity have been described, for example, in U.S. Pat. Nos. 6,774,213; 7,176,007, and 7,294,494. Plant RecA homologues are described, for example, in U.S. Patent Nos. 5,674,992; 6,388,169 and 6,809,183. RecA fragments containing recombinase activity are described, for example, in U.S. Patent No. 5,731,411. Mutant RecA proteins with recombinase activity, such as RecA803, have been described. See, for example, Madiraju et al. (1988) Proc. Natl. Acad. Sci. USA 85:6592-6596.

[0238] A eukaryotic homolog of RecA, which also has recombinase activity, is the Rad51 protein, first identified in the yeast Saccharomyces cerevisiae. See Bishop et al., (1992) Cell 69:439-56; Shinohara et al., (1992) Cell:457-70; Aboussekhra, et al., (1992) Mol. Cell. Biol. 72, 3224-3234 and Basile et al., (1992) Mol. Cell. Biol. 12, 3235-3246. Plant Rad51 sequences are described in U.S. Patent Nos. 6,541,684; 6,720,478; 6,905,857 and 7,034,117. Another yeast protein that is homologous to RecA is the Dmcl protein. RecA / Rad51 homologs in organisms other than E. coli and S. cerevisiae have been described. Morita et al. (1993) Proc. Natl. Acad. Sci. USA 90:6577-6580; Shinohara et al. (1993) Nature Genet. 4:239-243; Heyer (1994) Experientia 50:223-233; Maeshima et al. (1995) Gene 160:195-200; U.S. Patent Nos. 6,541,684 and 6,905,857.

[0239] Further description of proteins having recombinase activity can be found in, for example, Fugisawa et al. (1985) Nucl. Acids Res. 13:7473; Hsieh et al. (1986) Cell 44:885; Hsieh et al. (1989) J. Biol. Chem. 264:5089; Fishel et al. (1988) Proc. Natl. Acad. Sci. USA 85:3683; Cassuto et al. (1987) Mol. Gen. Genet. 208:10; Ganea et al. (1987) Mol. Cell Biol. 7:3124; Moore et al. (1990) J. Biol. Chem.:11108; Keene et al. (1984) Nucl. Acids Res. Res.12:3057;Kimiec(1984)Cold Spring Harbor Symp.48:675;Kimeic(1986)Cell 44:545;Kolodner et al.(1987)Proc.Natl.Acad..USA 84:5560;Sugino et al.(1985)Proc.Natl.Acad,Sci.USA 85:3683;Halbrook et al.(1989)J.Biol.Chem.264:21403;Eisen et al.(1988)Proc.Natl.Acad.Sci.USA 85:7481;McCarthy et al.(1988)Proc.Natl.Acad.Sci.USA 85:5854;and Lowenhaupt et al. al. (1989) J. Biol. Chem. 264:20568, which are incorporated herein by reference. See also Brendel et al. (1997) J. Mol. Evol. 44:528.

[0240] Examples of proteins with recombinase activity include recA, recA803, uvsX, and other recA mutants and recA-like recombinases (Roca (1990) Crit. Rev. Biochem. Molec. Biol. 25:415), (Kolodner et al. (1987) Proc. Natl. Acad. Sci. USA 84:5560; Tishkoff et al. (1991) Molec. Cell. Biol. 11:2593), RuvC (Dunderdale et al. (1991) Nature 354:506), DST2, KEM1, and XRN1 (Dykstra et al. (1991) Molec. Cell. Biol. 11:2583), STPa / DST1 (Clark et al. al. (1991) Molec. Cell. Biol. 11:2576), HPP-1 (Moore et al. (1991) Proc. Natl. Acad. Sci. USA 88:9067), other eukaryotic recombinases (Bishop et al. (1992) Cell 69:439; and Shinohara et al. (1992) Cell 69:457), which are incorporated herein by reference.

[0241] In vitro evolved proteins with recombinase activity are described in U.S. Patent No. 6,686,515. Further publications on recombinases include, for example, U.S. Patent Nos. 7,732,585, 7,361,641, and 7,144,734. For a review of recombinases, see Cox (2001) Proc. Natl. Acad. Sci. USA 98:8173-8180.

[0242] Nucleoprotein filaments, or "filaments," may be formed. In the context of forming a structure with a recombinase, the term filament is a term known to those of skill in the art. The nucleoprotein filaments so formed can then be contacted, for example, with another nucleic acid or introduced into a cell. Methods for forming nucleoprotein filaments, where the filaments comprise a polypeptide sequence and a nucleic acid having recombinase activity, are well known in the art. See, for example, Cui et al. (2003) Marine Biotechnol. 5:174-184 and U.S. Pat. Nos. 4,888,274; 5,763,240; 5,948,653 and 7,199,281, the disclosures of which are incorporated by reference for the purposes of disclosing exemplary techniques for binding a recombinase to a nucleic acid to form a nucleoprotein filament.

[0243] In general, a molecule having recombinase activity is contacted with a linear single-stranded nucleic acid. The linear single-stranded nucleic acid may be a probe. Methods for preparing such single-stranded nucleic acids are known. The reaction mixture typically contains magnesium ions. Optionally, the reaction mixture is buffered and optionally also contains ATP, dATP or a non-hydrolyzable ATP analog, such as γ-thio-ATP (ATP-γ-S) or γ-thio-GTP (GTP-γ-S). The reaction mixture may also optionally contain an ATP generating system. The double-stranded DNA molecules may be denatured (e.g., by heat or alkali) either before or during filament formation. Optimization of the molar ratio of recombinase to nucleic acid is within the skill of the art. For example, a series of different concentrations of recombinase may be added to a fixed amount of nucleic acid, and filament formation may be assayed by mobility in agarose or acrylamide gels. Since the bound protein retards the electrophoretic mobility of the polynucleotide, filament formation is evidenced by a retardation of the mobility of the nucleic acid. Either the greatest degree of retardation, or the greatest amount of nucleic acid migrating with retarded mobility, can be used to indicate the optimal recombinase:nucleic acid ratio. Protein-DNA association can also be quantified by measuring the ability of the polynucleotide to bind to nitrocellulose.

[0244] Provided herein, in another aspect, is an additional method of delivering one or more human leukocyte antigen (HLA)-encoding nucleic acid molecules to cells. In some embodiments, the method comprises delivery of one or more HLA-encoding nucleic acid molecules via transcription activator-like effector nuclease (TALEN). In some embodiments, the method comprises delivery of one or more HLA-encoding nucleic acid molecules via zinc finger nuclease (ZFN). In some embodiments, the method comprises delivery of one or more HLA-encoding nucleic acid molecules via clustered regularly interspaced short palindromic repeats / CRISPR-associated endonuclease cas9 (CRISPR / Cas9).

[0245] In some embodiments, the nucleic acid molecule comprises a deletion in an endogenous HLA locus. In some embodiments, the deletion comprises a deletion in an endogenous HLA-A, HLA-B, or HLA-C locus, or any combination thereof. In some embodiments, the deletion is a complete deletion of the endogenous HLA locus.

[0246] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human HLA class 1 heavy chain sequence. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or any combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises multiple alleles of an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or any combination thereof. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises an HLA-A sequence, and the HLA-A sequence is disposed between the HLA-B sequence and the HLA-C sequence.

[0247] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 1700 base pairs (bp). In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 500 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 250 bp. In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence comprises less than 150 bp.

[0248] In some embodiments, the HLA-A sequence, HLA-B sequence, HLA-C sequence, or combinations thereof, comprises one or more flanking sequences. In some embodiments, the one or more flanking sequences comprise endogenous HLA sequences. In some embodiments, the one or more flanking sequences are specific to one or more promoters. In some embodiments, the promoter comprises an HLA-A promoter, an HLA-B promoter, an HLA-C promoter, or combinations thereof.

[0249] In some embodiments, the sequence encoding the human HLA class 1 heavy chain sequence does not include at least a portion of an HLA-A sequence, an HLA-B sequence, an HLA-C sequence, or a combination thereof.

[0250] In some embodiments, the nucleic acid molecule encoding the human HLA class 1 heavy chain sequence comprises an HLA-E sequence or a fragment thereof, an HLA-F sequence or a fragment thereof, an HLA-G sequence or a fragment thereof, or any combination thereof. In some embodiments, at least one of the HLA-E sequence or a fragment thereof, the HLA-F sequence or a fragment thereof, the HLA-G sequence or a fragment thereof, or any combination thereof is inhibited from eliciting a T cell response when the construct is interrogated by one or more T cells.

[0251] In some embodiments, the nucleic acid molecule encoding the human HLA class 1 heavy chain sequence comprises one or more mutations, and a cell comprising the mutant human HLA class 1 heavy chain sequence comprising one or more mutations does not elicit an immune response when the cell is probed by one or more CD8 cells. In some embodiments, the mutant human HLA class 1 heavy chain sequence encodes an HLA comprising one or more mutations at one or more of amino acid residues 115, 122, 128, 194, 197, 198, 212, 214, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 243, 245, 248, 262, or any combination thereof.

[0252] Treatment method Provided herein, in another aspect, is a method of treating a disease or disorder in a subject in need thereof, comprising administering a therapeutically effective amount of a nucleic acid molecule provided herein or an immunocompromised cell provided herein.

[0253] In some embodiments, the disease is an autoimmune disease. In some embodiments, the disease is type 1 diabetes. In some embodiments, the disease is rheumatoid arthritis. In some embodiments, the disease is psoriasis. In some embodiments, the disease is psoriatic arthritis. In some embodiments, the disease is multiple sclerosis. In some embodiments, the disease is systemic lupus erythematosus. In some embodiments, the disease is inflammatory bowel disease. In some embodiments, the disease is Addison's disease. In some embodiments, the disease is Graves' disease. In some embodiments, the disease is Sjogren's syndrome. In some embodiments, the disease is Hashimoto's thyroiditis. In some embodiments, the disease is myasthenia gravis. In some embodiments, the disease is autoimmune vasculitis. In some embodiments, the disease is pernicious anemia. In some embodiments, the disease is celiac disease. In some embodiments, the disease is vasculitis.

[0254] In some embodiments, the disease is cancer. In some embodiments, the disease is lung cancer. In some embodiments, the disease is breast cancer. In some embodiments, the disease is colorectal cancer. In some embodiments, the disease is prostate cancer. In some embodiments, the disease is skin cancer. In some embodiments, the disease is gastric cancer. In some embodiments, the disease is leukemia. In some embodiments, the disease is lymphoma. In some embodiments, the disease is bladder cancer. In some embodiments, the disease is renal cancer. In some embodiments, the disease is endometrial cancer. In some embodiments, the disease is pancreatic cancer. In some embodiments, the disease is thyroid cancer. In some embodiments, the disease is liver cancer. In some embodiments, the disease is ovarian cancer. In some embodiments, the disease is cervical cancer.

[0255] In some embodiments, the disease is a degenerative disease. In some embodiments, the disease is Alzheimer's disease. In some embodiments, the disease is amyotrophic lateral sclerosis. In some embodiments, the disease is Friedreich's ataxia. In some embodiments, the disease is Huntington's disease. In some embodiments, the disease is Lewy body disease. In some embodiments, the disease is Parkinson's disease. In some embodiments, the disease is spinal muscular atrophy. In some embodiments, the disease is multiple sclerosis. In some embodiments, the disease is muscular dystrophy. In some embodiments, the disease is cystic fibrosis. In some embodiments, the disease is Creutzfeldt-Jakob disease. In some embodiments, the disease is Tay-Sachs disease.

[0256] In some embodiments, administration of the immunologically incompetent cells provided herein treats a disease or disorder without eliciting an immune response.

[0257] When one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein are administered to a human subject, the dosage will typically be determined by a physician, and the dosage will generally vary according to the age, weight, and response of the individual subject, as well as the severity of the subject's symptoms.

[0258] The actual dosage used may vary depending on the requirements of the subject and the severity of the condition being treated. The determination of the appropriate dosage for a particular situation is within the capabilities of one of ordinary skill in the art. In general, treatment is initiated with a smaller dosage that is less than the optimal dose of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein. Thereafter, dosage is increased by small increments until the optimal effect is reached under the circumstances.

[0259] The amount and frequency of administration of one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein, and, if applicable, other chemotherapeutic agents and / or radiation therapy, will be regulated according to the judgment of the attending clinician (physician), taking into account factors such as the age, condition and size of the subject, and the severity of the disease being treated.

[0260] The chemotherapy and / or radiation therapy can be administered according to a treatment protocol well known in the art. It is clear to those skilled in the art that the administration of chemotherapy and / or radiation therapy can vary depending on the disease to be treated and the known effect of chemotherapy and / or radiation therapy on the disease. Also, according to the knowledge of a skilled clinician, the treatment protocol (e.g., dosage and number of administrations) can be changed taking into account the observed effect of the administered therapeutic agent (i.e., anti-neoplastic agent or radiation) on the subject and the observed response of the disease to the administered therapeutic agent.

[0261] Also, generally, one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein do not need to be administered in the same pharmaceutical composition as the chemotherapeutic agent, and may be administered by different routes due to different physical and chemical properties. The determination of the mode of administration and the appropriateness of administration in the same pharmaceutical composition is well within the knowledge of a skilled clinician, if possible. Initial administration can be performed according to established protocols known in the art, and then the dosage, mode of administration, and time of administration can be modified by the skilled artisan based on the observed effects.

[0262] The particular selection of one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein (as well as chemotherapeutic agents and / or radiation, if appropriate) will depend on the diagnosis of the attending physician and their judgment of the subject's condition and the appropriate treatment protocol.

[0263] One or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein (and, if appropriate, chemotherapeutic agents and / or radiation) may be administered concurrently (e.g., simultaneously, essentially simultaneously, or within the same treatment protocol) or sequentially, depending on the nature of the proliferative disease, the condition of the subject, and the actual choice of chemotherapeutic agents and / or radiation to be administered in combination (i.e., within a single treatment protocol) with one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein.

[0264] In combination applications and uses, one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein, and chemotherapeutic agents and / or radiation do not need to be administered simultaneously or essentially simultaneously, and the initial order of administration of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein, and chemotherapeutic agents and / or radiation may not be important. Thus, one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein may be administered first, followed by chemotherapeutic agents and / or radiation; or chemotherapeutic agents and / or radiation may be administered first, followed by one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein. This alternating administration may be repeated during a single treatment protocol. The determination of the order of administration of each therapeutic agent during a treatment protocol and the number of repeated administrations is well within the knowledge of a skilled physician after evaluation of the disease being treated and the condition of the subject. For example, chemotherapeutic agents and / or radiation may be administered first, then treatment may be continued with administration of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein, followed by administration of chemotherapeutic agents and / or radiation, etc., if determined to be advantageous, until the treatment protocol is completed.

[0265] Thus, according to experience and knowledge, the practitioner can modify each protocol for administration of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein for treatment according to the needs of the individual subject as the treatment progresses.

[0266] Usage Provided herein, in another aspect, is a method of inhibiting a human leukocyte antigen (HLA), comprising contacting the HLA with a peptide that does not contain a T cell receptor binding residue or fragment.

[0267] In some embodiments, the peptide binds to one or more HLA binding groove domain residues of HLA. In some embodiments, the peptide modulates the conformation of HLA. In some embodiments, the conformation prevents T cells from binding to HLA.

[0268] In some embodiments, the HLA is synthetic.

[0269] Treatment Efficacy In determining whether the treatment is effective at the administered dosage, the attending physician will consider the subject's general health and more specific signs such as relief of disease-related symptoms, inhibition of tumor growth, actual shrinkage of the tumor, or inhibition of metastasis. The size of the tumor can be measured by standard methods, for example, radiological studies, such as CAT or MRI scans, and serial measurements can be used to determine whether tumor growth has been slowed or reversed. Relief of disease-related symptoms, such as pain, and improvement of overall condition can also be used to help determine the effectiveness of treatment.

[0270] In some embodiments, therapeutic efficacy is measured based on the effectiveness of treating a proliferative disorder such as cancer. In general, therapeutic efficacy of the methods and compositions of the present invention can be measured by the extent to which the methods and compositions promote inhibition of tumor cell proliferation, inhibition of tumor angiogenesis, eradication of tumor cells, slowing the growth rate of a tumor, and / or reducing the size of at least one tumor, with respect to the treatment of a proliferative disorder (e.g., benign or malignant cancer). Several parameters considered in determining therapeutic efficacy are discussed herein. The appropriate combination of parameters for a particular situation can be established by the clinician. The progress of the methods of the present invention in treating cancer (e.g., reduction in tumor size or eradication of cancer cells) can be confirmed using any suitable method, such as methods currently used in clinics to track tumor size and cancer progression. The primary efficacy parameter used to evaluate the treatment of cancer with the methods and compositions of the present invention is preferably the reduction in tumor size. Tumor size can be determined using any suitable technique, such as measuring dimensions or estimating tumor volume using available computer software, such as FreeFlight software developed at Wake Forest University, which allows for accurate estimation of tumor volume. Tumor size may be determined by tumor visualization using, for example, CT, ultrasound, SPECT, spiral CT, MRI, photography, etc. In embodiments in which the tumor is surgically removed after completion of the treatment period, the presence of tumor tissue and tumor size may be determined by gross analysis of the resected tissue and / or by pathological analysis of the resected tissue.

[0271] In some desirable embodiments, tumor growth is stabilized as a result of the methods and compositions of the invention (i.e., one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20% and / or do not metastasize). In some embodiments, tumors are stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or longer. In some embodiments, tumors are stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer. In some embodiments, tumors are stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or longer. Preferably, the methods of the invention reduce the size of a tumor by at least about 5% (e.g., at least about 10%, 15%, 20%, or 25%). More preferably, the tumor size is reduced by at least about 30% (e.g., at least about 35%, 40%, 45%, 50%, 55%, 60%, or 65%). Even more preferably, the tumor size is reduced by at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, or 95%). Most preferably, the tumor is completely eliminated or reduced to below detection levels. In some embodiments, the subject remains tumor-free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or longer, following treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer, following treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years, or longer, following treatment.

[0272] In some embodiments, the effectiveness of the methods of the present invention in reducing tumor size can be determined by measuring the percentage of necrotic (i.e., dead) tissue of the surgically resected tumor after completion of the treatment period. In some further embodiments, the treatment is therapeutically effective when the necrotic percentage of the resected tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), more preferably about 90% or greater (e.g., about 90%, 95%, or 100%). Most preferably, the necrotic percentage of the resected tissue is 100%, i.e., no or no detectable tumor tissue is present.

[0273] The efficacy of the method of the present invention can be determined by several secondary parameters. Examples of secondary parameters include, but are not limited to, detection of new tumors, detection of tumor antigens or markers (e.g., CEA, PSA, or CA-125), biopsy, surgical downstaging (i.e., converting the surgical stage of a tumor from unresectable to resectable), PET scans, survival, disease progression-free survival, time to disease progression, quality of life assessments such as Clinical Benefit Response Assessment, etc., all of which can indicate the overall progression (or regression) of cancer in humans. Biopsy is particularly useful for detecting the eradication of cancer cells in tissue. Radioimmunodetection (RAID) is used to localize and stage tumors using serum levels of markers (antigens) produced by and / or associated with tumors ("tumor markers" or "tumor-associated antigens"), which can be useful as a pretreatment diagnostic predictive value, a posttreatment diagnostic indicator of recurrence, and a posttreatment indicator of therapeutic efficacy. Examples of tumor markers or tumor-associated antigens that can be evaluated as indicators of therapeutic efficacy include, but are not limited to, carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), CA-125, CA19-9, ganglioside molecules (e.g., GM2, GD2, and GD3), MART-1, heat shock proteins (e.g., gp96), sialyl Tn (STn), tyrosinase, MUC-1, HER-2 / neu, c-erb-B2, KSA, PSMA, p53, RAS, EGF-R, VEGF, MAGE, and gp100. Other tumor-associated antigens are known in the art. RAID technology in combination with an endoscopic detection system can also effectively distinguish small tumors from surrounding tissues (see, for example, U.S. Pat. No. 4,932,412).

[0274] In further desirable embodiments, treatment of cancer in a human patient by the methods of the invention is evidenced by one or more of the following results: (a) complete disappearance of the tumor (i.e., complete response); (b) about a 25% to about a 50% decrease in tumor size for at least 4 weeks after completion of the treatment period, compared to the size of the tumor before treatment; (c) at least about a 50% decrease in tumor size for at least 4 weeks after completion of the treatment period, compared to the size of the tumor before the treatment period; and (d) at least a 2% decrease (e.g., about a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decrease) in the level of a particular tumor-associated antigen for about 4 to 12 weeks after completion of the treatment period, compared to the tumor-associated antigen level before the treatment period. Although at least a 2% decrease in tumor-associated antigen level is preferred, any decrease in tumor-associated antigen level is evidence of treatment of cancer in a patient by the methods of the invention. For example, for unresectable locally advanced pancreatic cancer, treatment can be evidenced by at least a 10% decrease in CA19-9 tumor associated antigen levels 4-12 weeks after completion of the treatment period compared to the CA19-9 levels prior to the treatment period. Similarly, for locally advanced rectal cancer, treatment can be evidenced by at least a 10% decrease in CEA tumor associated antigen levels 4-12 weeks after completion of the treatment period compared to the CEA levels prior to the treatment period.

[0275] For quality of life assessments, e.g., Clinical Benefit Response Criteria, the therapeutic benefit of treatment according to the invention may be demonstrated in terms of pain intensity, analgesic consumption, and / or Karnofsky Performance Scale score. Alternatively, or in addition, treatment of cancer in a human patient is evidenced by: (a) at least a 50% reduction (e.g., at least a 60%, 70%, 80%, 90%, or 100% reduction) in pain intensity reported by the patient, such as for any 4 consecutive weeks in a 12 week period after completion of treatment, compared to the pain intensity reported by the patient prior to treatment; (b) at least a 50% reduction (e.g., at least a 60%, 70%, 80%, 90%, or 100% reduction) in pain medication consumption reported by the patient, such as for any 4 consecutive weeks in a 12 week period after completion of treatment, compared to the pain medication consumption reported by the patient prior to treatment; and / or (c) at least a 20 point increase (e.g., at least a 30 point, 50 point, 70 point, or 90 point increase) in the Karnofsky Performance Scale score reported by the patient, such as for any 4 consecutive weeks in a 12 week period after completion of the treatment, compared to the Karnofsky Performance Scale score reported by the patient prior to the treatment period.

[0276] Treatment of a proliferative disorder (e.g., cancer, whether benign or malignant) in a human patient will desirably be evidenced by one or more (any combination) of the aforementioned results, although alternative or additional results from the referenced tests and / or other tests may evidence therapeutic efficacy.

[0277] In some embodiments, tumor size is reduced as a result of the method of the present invention, preferably without significant adverse events in the subject. Adverse events are classified or "graded" by the Cancer Therapy Evaluation Program (CTEP) of the National Cancer Institute (NCI), with grade 0 representing minimal adverse side effects and grade 4 representing the most severe adverse events. Desirably, the method of the present invention is associated with minimal adverse events, for example, grade 0, grade 1, or grade 2 adverse events as graded by CTEP / NCI. However, as discussed herein, reduction in tumor size, although preferred, is not required in that the actual size of the tumor may not decrease despite eradication of tumor cells. Eradication of cancerous cells is sufficient to achieve a therapeutic effect. Similarly, any reduction in tumor size is sufficient to achieve a therapeutic effect.

[0278] Detection, monitoring and evaluation of various cancers in humans are further described in Cancer Facts and Figures 2001, American Cancer Society, New York, NY and International Patent Application No. 01 / 24684. Thus, clinicians may use standard tests to determine the effectiveness of various embodiments of the methods of the present invention in treating cancer. However, in addition to tumor size and spread, clinicians may also consider the patient's quality of life and survival in assessing the effectiveness of treatment.

[0279] In some embodiments, administration of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein provides improved therapeutic efficacy. Improved efficacy can be measured using any method known in the art, including but not limited to the methods described herein. In some embodiments, improved therapeutic efficacy is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 1000% or more improvement using an appropriate measure (e.g., reduction in tumor size, period of tumor size stability, period free of metastatic events, disease-free survival). Improved efficacy may also be expressed as at least about a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 1000-fold, 10000-fold or greater improvement using an appropriate scale (e.g., reduction in tumor size, duration of tumor size stability, duration free of metastatic events, disease-free survival).

[0280] Pharmaceutical Compositions and Formulations The present disclosure provides compositions, including pharmaceutical compositions, that comprise one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein.

[0281] In some embodiments, one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein are formulated into pharmaceutical compositions.In a specific embodiment, the pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers that include excipients and auxiliaries that facilitate the processing of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein into medicament-usable preparations.The appropriate formulation depends on the route of administration selected. Any pharma- ceutically acceptable techniques, carriers, and excipients may be used as appropriate to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0282] Provided herein is a pharmaceutical composition comprising one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein and a pharma- ceutically acceptable diluent(s), excipient(s), or carrier(s). In certain embodiments, one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein are administered as a pharmaceutical composition in which one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein are mixed with other active ingredients, such as in combination therapy. In a specific embodiment, the pharmaceutical composition comprises one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein.

[0283] Pharmaceutical composition, as used herein, refers to a mixture of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents, and / or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein to an organism. In some embodiments, in practicing the methods of treatment or use provided herein, a therapeutically effective amount of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein is administered in a pharmaceutical composition to a mammal having a disease or condition to be treated. In specific embodiments, the mammal is a human. In certain embodiments, the therapeutically effective amount varies depending on the severity of the disease, the age and relative health of the subject, and other factors. One or more constructs, nucleic acid molecules, or immunocompromised cells provided herein are used alone or in combination with one or more therapeutic agents as components of a mixture.

[0284] In one embodiment, one or more constructs, nucleic acid molecules, or immunologically incompetent cells provided herein are formulated in an aqueous solution. In a specific embodiment, the aqueous solution is selected from physiologically compatible buffers, such as, by way of example only, Hank's solution, Ringer's solution, or saline buffer. In other embodiments, one or more constructs, nucleic acid molecules, or immunologically incompetent cells provided herein are formulated for transmucosal administration. In a specific embodiment, the transmucosal formulation comprises a penetrant appropriate for the barrier to be permeated. In yet other embodiments, where one or more constructs, nucleic acid molecules, or immunologically incompetent cells provided herein are formulated for other parenteral injections, suitable formulations comprise aqueous or non-aqueous solutions. In a specific embodiment, such solutions comprise physiologically compatible buffers and / or excipients.

[0285] In yet other embodiments, one or more constructs, nucleic acid molecules, or immunologically incompetent cells provided herein are formulated for parenteral injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, the formulation for injection is provided in unit dosage form (e.g., ampoules) or in multi-dose containers. A preservative is optionally added to the injection formulation. In still other embodiments, the pharmaceutical composition of one or more constructs, nucleic acid molecules, or immunologically incompetent cells provided herein is formulated in a form suitable for parenteral injection as a sterile suspension, solution, or emulsion in an oily or aqueous vehicle. The parenteral injection formulation optionally contains formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. In specific embodiments, the pharmaceutical formulation for parenteral administration comprises an aqueous solution of one or more constructs, nucleic acid molecules, or immunologically incompetent cells provided herein in water-soluble form. In further embodiments, the suspension of one or more constructs, nucleic acid molecules, or immunologically incompetent cells provided herein is prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.In certain specific embodiments, aqueous injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran.Optionally, the suspension contains suitable stabilizers or agents that increase the solubility of one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein, to allow the preparation of highly concentrated solutions.Alternatively, in other embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0286] In certain embodiments, the pharmaceutical composition is formulated in any conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate the processing of one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein into pharma- ceutically usable preparations. The appropriate formulation depends on the route of administration selected. Any pharma- ceutically acceptable techniques, carriers, and excipients are optionally used appropriately. The pharmaceutical composition comprising one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein is prepared in a conventional manner, for example, by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping, or compressing processes, for example.

[0287] In some embodiments, pharmaceutical compositions comprising one or more constructs, nucleic acid molecules, or immunocompromised cells provided herein are illustratively in the form of a liquid, in which the agent is in solution, in suspension, or both. Typically, when the composition is administered as a solution or suspension, a first portion of the agent is in solution, and a second portion of the agent is in particulate form in suspension in a liquid matrix. In some embodiments, the liquid composition comprises a gel formulation. In other embodiments, the liquid composition is aqueous.

[0288] In certain embodiments, useful aqueous suspensions contain one or more polymers as suspending agents. Useful polymers include water-soluble polymers such as cellulose polymers (e.g., hydroxypropylmethylcellulose) and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein include mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran.

[0289] Useful pharmaceutical compositions also optionally include a solubilizing agent to aid in the solubility of one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein. The term "solubilizing agent" generally includes agents that result in the formation of a micellar or true solution of the agent. Certain acceptable non-ionic surfactants, such as polysorbate 80, are useful as solubilizing agents, as are ophthalmically acceptable glycols, polyglycols (e.g., polyethylene glycol 400), and glycol ethers.

[0290] In addition, useful pharmaceutical compositions optionally contain one or more pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases, such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane; and buffers, such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within an acceptable range.

[0291] In addition, useful compositions also optionally contain one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0292] Other useful pharmaceutical compositions optionally contain one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merphen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0293] Still other useful compositions contain one or more surfactants to enhance physical stability or for other purposes. Suitable non-ionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10, octoxynol 40.

[0294] Yet other useful compositions can include, where required, one or more antioxidants to enhance chemical stability. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0295] In certain embodiments, the aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers are used, in which case it is typical to include a preservative in the composition.

[0296] In alternative embodiments, other delivery systems are used. Liposomes and emulsions are examples of delivery vehicles or carriers useful herein. In further embodiments, one or more of the constructs, nucleic acid molecules, or immunocompromised cells provided herein are delivered using sustained release systems, such as semipermeable matrices of solid hydrophobic polymers containing therapeutic agents. A variety of sustained release materials are useful herein. Depending on the chemical nature and biological stability of the therapeutic reagent, further strategies for protein stabilization are used.

[0297] In certain embodiments, the formulations described herein include one or more antioxidants, metal chelators, thiol-containing compounds, and / or other general stabilizing agents. Examples of such stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0298] Route of administration Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration.Further, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injection.

[0299] In certain embodiments, the compositions comprising immunologically incompetent cells provided herein are administered in a local rather than systemic manner, for example, by injecting the composition directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long-acting formulations are administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Furthermore, in other embodiments, the compositions are delivered in targeted drug delivery systems, for example, in liposomes coated with organ-specific antibodies. In such embodiments, the liposomes are targeted to and selectively taken up by the organ. In yet other embodiments, the compositions comprising immunologically incompetent cells provided herein are provided in the form of a rapid release formulation, a sustained release formulation, or an intermediate release formulation.

[0300] Kits and manufactured products Kits and articles of manufacture are also provided for use in the therapeutic applications described herein. In some embodiments, such kits include a carrier, package, or container that is compartmentalized to accommodate one or more containers, such as vials, tubes, etc., each of the container(s) includes one of the separate elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. Containers are formed from a variety of materials, such as glass or plastic.

[0301] The articles of manufacture provided herein contain packaging materials. Packaging materials used for packaging pharmaceutical products include, for example, those found in U.S. Patent Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment. For example, the container(s) optionally contain one or more nucleic acid molecules or immunocompromised cells described herein in a composition. The container(s) optionally have a sterile access port (e.g., the container is an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). Such kits optionally contain the composition with an identifying description or label or instructions for its use in the methods described herein.

[0302] For example, a kit typically includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and / or devices) that are desirable from a commercial and user perspective for the use of the compositions described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels that list the contents and / or instructions for use, and package inserts that include instructions for use. Typically, a set of instructions is also included. The label is optionally on or associated with the container. For example, a label is on the container when letters, numbers or other symbols that form the label are attached, molded or etched into the container itself, and a label is associated with the container when the label is present in a receptacle or carrier that also holds the container, for example as a package insert. In addition, the label is used to indicate that the contents are to be used for a particular therapeutic application. In addition, the label indicates instructions for the use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical composition is provided in a pack or dispenser device containing one or more unit dosage forms comprising one or more constructs, nucleic acid molecules, immunologically incompetent cells, or pharmaceutical compositions thereof provided herein. The pack contains, for example, metal or plastic foil, such as a blister pack. Or the pack or dispenser device is accompanied by instructions for administration. Or the pack or dispenser is affixed with a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the approval by the government agency of the form of the drug for human or veterinary administration. Such notice is, for example, a label approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. In some embodiments, a composition comprising immunologically incompetent cells formulated in a compatible pharmaceutical carrier is prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. EXAMPLES

[0303] Example 1: Identification of Anchor Amino Acids of Peptides Most HLA class 1 alleles bind preferentially to 9- to 12-mer peptides, and the majority of alleles accommodate peptides with anchor residues in the second and final positions because these residues are buried in the peptide-binding groove.

[0304] Analysis of 9-mer peptides was performed to determine peptide binding preferences for all alleles of HLA-A, HLA-B, and HLA-C. Amino acid diversity was determined for anchor residues at the second (e.g., P2) and last (e.g., P9 for the 9-mer) positions. Longer peptides also bind at these anchor positions, with the extra length being accommodated by either a bulge in the middle of the peptide binding groove or an overhang on the outside.

[0305] The HLA-A alleles investigated included HLA-A * 01:01, HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, HLA-A * 02:05, HLA-A * 02:06, HLA-A * 02:07, HLA-A * 02:11, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 11:02, HLA-A * 23:01, HLA-A * 24:02, HLA-A * 24:07, HLA-A * 25:01, HLA-A * 26:01, HLA-A * 29:02, HLA-A * 30:01, HLA-A * 30:02, HLA-A * 31:01, HLA-A * 32:01, HLA-A * 33:01, HLA-A *33:03, HLA-A * 34:01, HLA-A * 34:02, HLA-A * 36:01, HLA-A * 66:01, HLA-A * 68:01, HLA-A * 68:02 and HLA-A * 74:01 was included.

[0306] The conserved anchor residues for each HLA-A allele examined are summarized in Table 4.

[0307] [Table 4]

[0308] The frequencies of the second amino acid anchor residues among the HLA-A alleles examined are summarized in Table 5:

[0309] [Table 5]

[0310] The frequencies of the final amino acid anchor residues in the HLA-A alleles examined are summarized in Table 6.

[0311] [Table 6]

[0312] The HLA-B alleles investigated were HLA-B * 07:02, HLA-B * 07:04, HLA-B * 08:01, HLA-B * 13:01, HLA-B * 13:02, HLA-B * 14:02, HLA-B * 15:01, HLA-B * 15:02, HLA-B * 15:03, HLA-B * 15:10, HLA-B* 15:17, HLA-B * 18:01, HLA-B * 27:05, HLA-B * 35:01, HLA-B * 35:03, HLA-B * 35:07, HLA-B * 37:01, HLA-B * 38:01, HLA-B * 38:02, HLA-B * 40:01, HLA-B * 40:02, HLA-B * 40:06, HLA-B * 42:01, HLA-B * 44:02, HLA-B * 44:03, HLA-B * 45:01, HLA-B * 46:01, HLA-B * 49:01, HLA-B * 50:01, HLA-B * 51:01, HLA-B * 52:01, HLA-B * 53:01, HLA-B * 54:01, HLA-B * 55:01, HLA-B * 55:02, HLA-B * 56:01, HLA-B * 57:01, HLA-B * 57:03, HLA-B * 58:01, and HLA-B * Including 58:02.

[0313] The conserved anchor residues for each HLA-B allele examined are summarized in Table 7.

[0314] [Table 7]

[0315] The frequencies of the second amino acid anchor residues among the HLA-B alleles examined are summarized in Table 8.

[0316] [Table 8]

[0317] The frequencies of the final amino acid anchor residues among the HLA-B alleles examined are summarized in Table 9.

[0318] [Table 9]

[0319] The HLA-C alleles investigated were HLA-C * 01:02, HLA-C * 02:02, HLA-C * 03:02, HLA-C * 03:03, HLA-C * 03:04, HLA-C * 04:01, HLA-C * 04:03, HLA-C * 05:01, HLA-C * 06:02, HLA-C * 07:01, HLA-C * 07:02, HLA-C * 07:04, HLA-C * 08:01, HLA-C * 08:02, HLA-C * 12:02, HLA-C * 12:03, HLA-C * 14:02, HLA-C * 14:03, HLA-C * 15:02, HLA-C * 16:01, and HLA-C * Including 17:01.

[0320] The conserved anchor residues for each HLA-C allele examined are summarized in Table 10:

[0321] [Table 10]

[0322] The frequencies of the second amino acid anchor residues among the HLA-C alleles examined are summarized in Table 11.

[0323] [Table 11]

[0324] The frequencies of the final amino acid anchor residues among the HLA-C alleles examined are summarized in Table 12.

[0325] [Table 12]

[0326] Example 2: Preparation of Nucleic Acid Molecules The nucleic acid molecule comprising the synthetic HLA sequence is prepared according to a method known in the art. For example, the nucleic acid molecule is prepared by solid-phase oligonucleotide synthesis using nucleoside phosphoramidites. Alternatively, portions of the nucleic acid molecule are prepared by solid-phase oligonucleotide synthesis using nucleoside phosphoramidites, and the portions are assembled into a complete nucleic acid molecule according to a method known in the art. For example, the portions are assembled using endonuclease-mediated assembly, site-specific recombination, or long overlap-based assembly.

[0327] Example 3: Preparation of constructs A recombinant plasmid containing a nucleic acid molecule containing a synthetic HLA sequence is prepared according to known procedures for the preparation of recombinant plasmids, for example, by cleaving the plasmid with a restriction enzyme and introducing the nucleic acid molecule containing the synthetic HLA sequence into the plasmid using DNA ligase.

[0328] The recombinant plasmid is then transformed into a cell population. Successfully transformed cells are selected according to methods known in the art, such as using a selection antibiotic. The selected cells are cultured and induced to produce the construct. The cells are then lysed and the construct is purified according to protein purification techniques known in the art, such as size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, free-flow electrophoresis, immunoaffinity chromatography, immunoprecipitation, or high performance liquid chromatography.

[0329] Alternatively, the construct is prepared by solid phase peptide synthesis according to methods known in the art.

[0330] Example 4: Preparation of immunocompetent cells Immunocompetent cells, such as immunocompetent stem cells, are prepared by introducing the nucleic acid molecule of Example 2 into the genome of cells according to methods known in the art.For example, the nucleic acid molecule is delivered to cells via a viral vector.Alternatively, the nucleic acid molecule is delivered to cells via non-viral methods, such as using naked DNA injection, electroporation, gene gun, sonoporation, magnetofection, lipoplex, dendrimer, inorganic nanoparticle, CRISPR, mRNA, or siRNA.

[0331] Alternatively, immunologically incompetent cells, such as immunologically incompetent stem cells, are prepared by incubating cells with the construct of Example 3.

[0332] Example 5: Immune cell proliferation assay Immune cells, e.g., T cells, are cultured and treated with the immunocompromised cells of Example 4 and radiolabeled nucleotides, e.g. 3 H-thymidine. After incubation, the immune cells are centrifuged, washed, and the radioactivity is measured and compared to a control group of cells that were not treated with the immune-incompetent cells of Example 4.

[0333] Example 6: Treatment of Diseases or Disorders The immunocompromised cells of Example 4 are administered to a patient suffering from a disease or disorder. The patient's condition is monitored according to a treatment regimen appropriate for the disease or disorder.

[0334] Example 7: Transgenic cloning of synHLA constructs into B2Mnull EBV and K562 cells.

[0335] B2M - / - Generation of EBV-transformed B cell lines CRISPR / Cas9 is used to mutate β2M in two EBV strains (9031 and JK). EBV cells are transfected with the Cas9 construct and HLA class I low / negative EBV cells are selected. Cells are expanded and the absence of surface HLA class I is confirmed.

[0336] Generation of NK cell lines and K562 and / or B2M for susceptibility to NK cell-mediated killing - / - Testing of EBV-transformed B-cell lines NK cells (CD3 - , CD56 + ) are selected and expanded in the presence of IL-2, IL-15 and IL-21. NK cells from PBMCs are selected and expanded in cytokines (IL-2 and IL-15). Purity of NK cell lines can be confirmed by staining with CD3, CD4, CD8, CD56. K562 and / or WT and B2M / HLA class I - / - EBV cell killing is compared.

[0337] Expressing synHLA proteins in K562 and / or EBV-transformed B cell lines The synHLA gene encoding the synHLA construct described herein is cloned into either a lentiviral vector (pRRLSIN) or an EBV episomal vector (pCE). A gene block encoding synHLA that is codon-optimized for expression in mammalian cells is constructed. The gene can be cloned into pRRLSIN or pCE, for example, using Infusion cloning. The construct can be sequenced to ensure the sequence is correct, and a plasmid is prepared. pRRLSIN can be used to generate lentivirus that is used to transduce K562 cells. pCE is electroporated into EBV cells, and transfected cells are selected by antibiotic selection. Transduced K562 are stained for the synHLA construct and selected by FACS sorting. Transfected EBV cells (WT and B2MB2M / HLA class I) are ... - / - ) are selected in the presence of antibodies and expression of the synHLA constructs is assessed by flow cytometry using monoclonal antibodies. K562 or EBV cells expressing the synHLA constructs are tested for recognition and killing by the NK cell lines generated as described above.

[0338] EBV cells or K562 cells expressing synHLA express antigen-specific CD8 + Determine whether the T cell clone can be activated. Use of either EBV transformed B cell lines: with or without HLA class I; with or without synHLA, or K562 transduced with the appropriate HLA class I construct, to test: influenza MP as above pulsed with cognate peptide 58-66 Epitope-specific CD8 + Responses of T cell clones. Primary allogeneic CFSE-labeled CD8 T cells purified by magnetic beads or flow cytometry were analyzed in response to the K562 and / or EBV-transformed B cell lines described above using the method of Mannering, SI et al. +T cell proliferation. A sensitive method for detecting proliferation of rare autoantigen-specific human T cells. J Immunol Methods, 2003, 283, 173-183. T cell killing of transduced cells is also assessed by measuring interferon-gamma secretion.

[0339] Example 8: Recombinant bacterial expression of HLA constructs HLA constructs, such as synthetic HLA constructs (synHLA) and / or single chain dimers or trimers (e.g., those listed in Table 1) described herein, are recombinantly expressed in bacteria. Briefly, bacterial cells are transformed with a nucleic acid encoding the sequence of a synHLA construct described herein. In some cases, the proteins may contain tags and additional sequence motifs, for example, to aid in purification, to facilitate cleavage of the purification tag, or for other purposes, including (but not limited to) 6xHis tags, AviTag™, and / or TEV cleavage sites. Optionally, the purification tag(s) are removed by cleavage with TEV protease according to standard protocols. Optionally, the proteins are biotinylated on their AviTag™ according to standard protocols.

[0340] Proteins may be isolated and purified from bacteria. If the protein is present as an inclusion body, it may be refolded (e.g., by denaturing with a chaotropic agent and transferring to a dilute aqueous environment). After refolding, the refolded protein may be purified (e.g., by immobilized metal affinity chromatography (IMAC), anion exchange chromatography, size exclusion chromatography (SEC)). The isolated protein may be subjected to multiple quality control procedures, such as visualization by Coomassie blue stained SDS-PAGE gels in the presence or absence of a reducing agent (e.g., dithiothreitol; DTT), or verified by techniques such as time-of-flight mass spectrometry (TOF-MS) and dynamic light scattering (DLS).

[0341] Example 9: Expression of synthetic HLA constructs in bacteria The synthetic HLA proteins described herein (SYNC4-1, SEQ ID NO:39; SYNA1-1, SEQ ID NO:31; SYNC5-1, SEQ ID NO:42; SYNC6-1, SEQ ID NO:44; SCTC1-1, SEQ ID NO:10) were expressed, isolated, refolded, purified, and analyzed as described in Example 8. Briefly, DNA encoding the synthetic HLA proteins was inserted into an expression vector and then introduced into bacteria under antibiotic selection. The protein construct format was as described in Figure 1, but without the N-terminal signal peptide and with an additional C-terminal purification tag detailed in Table 13. Cells carrying the appropriate sequences were grown to mid-log phase and expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation, lysed using a high pressure homogenizer, and centrifuged to isolate soluble and insoluble fractions. The insoluble inclusion bodies containing the synthetic HLA proteins were washed and resuspended in denaturing buffer. Synthetic HLA proteins were refolded from the inclusion body preparations by dilution in refolding buffer followed by incubation for up to 72 hours at 4° C. The resulting proteins were purified by a combination of IMAC, size exclusion and / or ion exchange chromatography.

[0342] [Table 13] * Length of linker 1 (L1), L=long (GGGGSGGGGSGGGGS) (SEQ ID NO: 179), M=medium (GGGGSGGGGSGS) (SEQ ID NO: 177), S=short (GGGGSGGGGS) (SEQ ID NO: 175), +C=glycine at position 2 of L1 linker mutated to cysteine; and length of linker 2 (L2), L=long (GGGGSGGGGSGGGGS; SEQ ID NO: 183); $WT=wild type sequence of Q226, D227, Y84 or C1. ^Protein tags: His, Hexahis tag; AVI, AviTag™. A TEV protease cleavage site is included at the N-terminus of the Hexahis tag. #Position 1 in the HLA domain.

[0343] The results of SDS-PAGE analysis of each construct are shown in Figures 10 and 11. Separation by SDS-PAGE followed by Coomassie blue staining showed that SYNA1-1 migrated as a single band under non-reducing conditions, suggesting homogeneity of bisulfite bond formation (Figure 10). SYNA1-1 binds to HLA-A * Contains 02 scaffold. HLA-C * The synthetic HLA proteins SCTC1-1, SYNC4-1, SYNC5-1 and SYNC6-1, which contain the 07 scaffold, migrated as multiple species under non-reducing conditions (Figures 10 and 11). Both SYNC4-1 and SYNC5-1 migrated as two separate species, and SYNC6-1 migrated as three separate species (Figure 10). The collapse of these bands into a single band under reducing conditions is likely due to the lack of HLA-C scaffold activity. * As a result of an extra unpaired cysteine ​​present at residue number 1 (C1) in the 07 domain, HLA domain, it is suggested that multiple disulfide-bonded proteins exist.

[0344] Proteins were analyzed by TOF-MS and their masses were confirmed using standard equipment and protocols. Briefly, 10 μg of protein was heated to 50° C. for 5 min in the presence or absence of 10 mM reducing agent (e.g., TCEP, DTT) before analysis by TOF-MS. TOF-MS experiments revealed that SYNC4-1, SYNC5-1 and SYNC6-1 constructs were approximately 438 Da heavier than the predicted mass (SYNC4-1, predicted 47771.4 Da, observed 48209.9 Da; SYNC5-1 predicted 47727.5 Da, observed 48165.9 Da; SYNC6-1 predicted 47803.6 Da, observed 48242.2 Da).

[0345] Example 10: Modulation of the probability of disulfide bond formation As described in Example 9, HLA-C *The cysteine ​​present in the 07 domain was mutated to glycine (C1G) to reduce the possibility of mismatched disulfide formation and thus improve protein expression. This C1G mutation was introduced into two proteins, SYNC22-1 (SEQ ID NO:80) and SCTC3-1 (SEQ ID NO:14) (Table E).

[0346] Furthermore, to overcome the potential problem of the first residue of the peptide preventing cleavage of the initiating methionine in bacterial expression systems, an R to S mutation was introduced at the first position of the peptide domain (R1S) in SYNC22-1 and SCTC3-1, predicted to restore the ability of the bacterial machinery to remove the initiating methionine of recombinantly synthesized HLA proteins.

[0347] SYNC22-1 and SCTC3-1 were expressed, isolated, refolded, purified and analyzed as described in Examples 8 and 9. The protein construct format was as described in Figure 1, but without the N-terminal signal peptide and with or without an additional C-terminal purification tag (e.g., hexa-His tag or AviTag™) as detailed in Table E. The resulting recombinant proteins migrated as a single species under non-reducing conditions, suggesting homogeneity of disulfide bond formation (Figure 15). Furthermore, when these proteins were analyzed by TOF-MS, the predicted mass matched the observed mass (SYNC22-1, predicted 47656.2 Da, observed 47657.5 Da; SCTC3-1, predicted 47039.6 Da, observed 47040.6 Da), suggesting that the initiator methionine was successfully cleaved. The results are summarized in Table 13. The observed masses are within the allowed error range (±2 Da) for TOF-MS on a QTOF 5600 instrument.

[0348] Depending on the downstream application of the recombinant proteins, they were subjected to TEV protease cleavage or biotinylation according to standard protocols. Briefly, TEV protease was incubated with synthetic HLA proteins at a ratio of 1:100 w / w overnight at 4° C. and separated by size-exclusion chromatography. Biotinylation of AviTag™ was achieved by incubating synthetic HLA proteins with bacterially expressed recombinant GST-tagged BirA overnight at 4° C. in the presence of 10 mM ATP, 10 mM MgCl2 and 50 μM D-biotin. BirA was separated from the reaction mixture using Glutathione Sepharose 4b resin. Biotinylated synthetic HLA proteins were further purified by size-exclusion chromatography.

[0349] To assess the homogeneity of the protein samples, dynamic light scattering (DLS) was used to analyze SCTC3-1 according to standard protocols. Briefly, samples were added to a sample cuvette and analyzed on a Zetasizer. Figure 18 shows that a double peak was observed for SCTC3-1, indicating that more than one protein species may have been present in the sample and may represent a proportion of unfolded material. SCTC3-1 appeared to be homogeneous by SDS-PAGE. The results are summarized in Table 16.

[0350] Therefore, all subsequent synHLA constructs for recombinant protein expression were generated in the context of R1S in the peptide domain and C1G in the HLA domain.

[0351] [Table 14] * Linker 1 (L1) length, L = long (GGGGSGGGGSGGGGS; SEQ ID NO: 179), M = medium (GGGGSGGGGSGS; SEQ ID NO: 177), S = short (GGGGSGGGGS; SEQ ID NO: 175). +C = glycine at position 2 of the L1 linker mutated to cysteine. 及びLinker 2 (L2) length, L = long (GGGGSGGGGSGGGGS; SEQ ID NO: 183). $WT = wild type sequence of Q226, D227 or Y84. ^ Protein tags: His, Hexa-his tag; AVI, AviTag™. A TEV protease cleavage site is contained at the N-terminus of the Hexa-his tag. # Position 1 in the HLA domain.

[0352] Example 11: Expression of further synthetic HLA proteins in bacteria carrying R1S or C1G mutations in peptide and HLA domains Synthetic HLA proteins carrying the R1S and / or C1G mutations along with other variations (Table 15) were expressed, isolated, refolded, purified and analyzed as described in Examples 8 and 9. The protein construct format was as described in Figure 1 but without the N-terminal signal peptide and with or without an additional C-terminal purification tag (e.g., hexa-His tag or AviTag™) as detailed in Table 15.

[0353] Separation by SDS-PAGE followed by Coomassie Blue staining showed that the synthetic HLA proteins tested migrated as a single band under non-reducing conditions, with the exception of SYNC27-1, suggesting uniformity of disulfide bond formation for all proteins listed in Table 15. The results of SDS-PAGE analysis for representative constructs are shown in FIG. 17. SYNC27-1 contains two extra cysteines that are predicted to create a disulfide staple between residue 84 (Y84C) of the HLA domain and the second residue of linker 1. The presence of these two extra cysteine ​​residues is predicted to have resulted in a proportion of recombinant protein containing mismatched disulfide bonds, as evidenced in a doublet by SDS-PAGE under non-reducing conditions.

[0354] Depending on the downstream application of the recombinant proteins, they were subjected to TEV protease cleavage or biotinylation according to standard protocols. Briefly, TEV protease was incubated with synthetic HLA proteins at a ratio of 1:100 w / w overnight at 4° C. and separated by size-exclusion chromatography. Biotinylation of AviTag™ was achieved by incubating synthetic HLA proteins with bacterially expressed recombinant GST-tagged BirA overnight at 4° C. in the presence of 10 mM ATP, 10 mM MgCl2 and 50 μM D-biotin. BirA was separated from the reaction mixture using Glutathione Sepharose 4B resin. Biotinylated synthetic HLA proteins were further purified by size-exclusion chromatography.

[0355] TOF-MS experiments revealed that the observed masses were consistent with the predicted masses, confirming the absence of cleavage of the initiator methionine and S-glutathionylation at +305 Da after removal of the extra unpaired cysteine. The predicted and observed masses are listed in Table 16 and a representative spectrum is shown in Figure 18 (SYNC20-1, after cleavage of the His-tag by TEV protease). The purification strategy, downstream processing, yields and analysis are summarized in Table 16.

[0356] To assess the homogeneity of the protein samples, DLS was used to analyze SYNC25-1, SYNC26-1 and SYNC27-1 according to standard protocols. Briefly, samples were added to a sample cuvette and analyzed on a Zetasizer. Figures 19A-F show a single peak indicative of a homogenous protein sample for SYNC25-1 and SYNC26-1, but a double peak was observed for SYNC27-1. This indicates that there may be more than one protein species in the sample, which may be part of the unfolded material, consistent with the doublet observed on the gel for SYNC27-1. The results are summarized in Table 16.

[0357] [Table 15] * Linker 1 (L1) long, L = long (GGGGSGGGGSGGGGS; SEQ ID NO: 179), M = medium (GGGGSGGGGSGS; SEQ ID NO: 177), S = short (GGGGSGGGGS; SEQ ID NO: 175), +C = glycine at position 2 of the L1 linker mutated to cysteine. * Linker 2 (L2) long, L = long (GGGGSGGGGSGGGGS; SEQ ID NO: 183), M = medium (GGGGSGGGGSGS; SEQ ID NO: 177) $ WT = wild type sequence of Q226, D227 or Y84. ^ Protein tags: His, Hexahis tag; AVI, AviTag™. A TEV protease cleavage site is contained at the N-terminus of the Hexahis tag. # 1 in the HLA domain.

[0358] [Table 16] * Ni (NiNTA IMAC), Ni(r) (NiNTA resin clarification), D (DEAE AEX), MQ (monoQ AEX), DS (desalting), S (SEC), B (biotinylation) and C (protease tag-cleavage). $ DLS, dynamic light scattering; ND, not performed. @ RL9 = RL9 peptide, RYRPGTVAL (SEQ ID NO: 159). # +305Da S-glutathionylation

[0359] Example 12: Recombinant bacterial expression of immune receptors The synthetic immunoreceptor proteins described herein (listed in Table 17) were expressed, isolated, refolded, purified, and analyzed as described in Examples 8 and 9. Briefly, DNA encoding the immunoreceptors was inserted into an expression vector and then introduced into bacteria under antibiotic selection. The proteins contain the tags listed in Table 17. Cells carrying the appropriate sequences were grown to mid-log phase and expression was induced with isopropyl BD-1-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation, lysed using a high-pressure homogenizer, and centrifuged to isolate soluble and insoluble fractions. Insoluble inclusion bodies containing the immunoreceptor proteins were washed and resuspended in denaturing buffer. Recombinant immunoreceptor proteins were refolded from the inclusion body preparations by dilution in refolding buffer followed by incubation at 4° C. for up to 72 hours. The resulting proteins were purified by a combination of IMAC, SEC, or anion exchange chromatography. Depending on the downstream application of the recombinant immunoreceptors, they were subjected to PreScission protease cleavage or biotinylation according to standard protocols. Briefly, bacterially expressed recombinant GST-tagged PreScission protease was incubated with the immunoreceptors at a 1:100 w / w ratio and incubated overnight at 4°C. Glutathione Sepharose 4B resin was used to separate PreScission protease from the reaction mixture. The cleaved immunoreceptors were further purified by size-exclusion chromatography. Briefly, biotinylation of AVI-tagged immunoreceptors was achieved by overnight incubation at 4°C with bacterially expressed recombinant GST-tagged BirA in the presence of 10 mM ATP, 10 mM MgCl2, and 50 μM D-biotin. Glutathione Sepharose 4B resin was used to separate BirA from the reaction mixture. The biotinylated immunoreceptors were further purified by size-exclusion chromatography.

[0360] The purification strategy, downstream processing, yields and analyses are summarized in Table 18.

[0361] [Table 17] ^ Protein tags: His, Hexahis tag; Avi, AviTag™. A PreScission protease site is included at the N-terminus of the Hexahis tag.

[0362] [Table 18]

[0363] Ni (NiNTA IMAC), Ni(r) (NiNTA resin clarification), D (DEAE AEX), MQ (monoQ AEX), DS (desalting), S (SEC), B (biotinylation) and C (protease tag-cleavage).

[0364] Example 13: Thermostability assay Thermostability assays were performed to monitor protein unfolding by applying a temperature gradient from 10 to 100 °C at 1.0 °C / min in a Bio-Rad CFX96™ Real-Time System RT PCR. Protein unfolding was measured by monitoring the increase in signal from the fluorescent dye SYPRO™ Orange, which binds to hydrophobic regions of proteins as they unfold. Proteins were assayed at 5 μM and measured individually and in combination as described below. Protein melting points, T m (the temperature at the midpoint of the melting transition). The first derivative (negative mode) of the protein melting curve is T m A maximum corresponding to one step protein unfolding event is identified, where one step protein unfolding events have a single maximum, whereas two step unfolding events have two maxima, etc.

[0365] The melting curve of SYNC4-1 has T mWhile the combination of SYNC4-1 and KIR2DL2 showed a pronounced two-step unfolding event corresponding to a T of 54.8°C and 57.4°C (Figure 12A), m This resulted in a large shift of the first maximum to the right, corresponding to the SYNC4-1-associated chromatin (T). The prominent double maximum of SYNC4-1 alone became a condensed double peak (FIG. 12B). m The right shift of KIR2DL2 suggests that KIR2DL2 stabilized SYNC4-1 through a protein-protein interaction.

[0366] Compared to the KIR2DL2 melting curve and first derivative (FIG. 13), the two maxima are further condensed into a single maximum with a slight shoulder on the left, which corresponds to T m corresponding to 58.8° C. and 57.6° C. Again, this strongly suggests that there is a protein-protein interaction between SYNC4-1 and KIR2DL2.

[0367] In contrast to SYNC1-1, SYNA1-1, which has the influenza peptide GILGFVFTL and a relatively long linker sequence that may interfere with KIR binding, did not result in a right shift when combined with KIR2DL2 (Figure 14), indicating that KIR2DL2 did not increase the thermal stability of SYNA1-1 (SYNA1-1T m =48℃, SYNA1-1+KIR2DL2T m = 48.2°C). Instead, when combined with SYNA1-1, the thermal stability of KIR2DL2 was decreased (Figure 14), with Tms of 58°C and 48°C, respectively. These data suggest that there was no increase in thermal stability when SYNA1-1 was combined with KIR2DL2, suggesting little or no interaction between the two proteins.

[0368] Example 14: Interaction of soluble synthetic HLA proteins with immune receptors To investigate the ability of soluble synthetic HLA proteins to evade the immune system, their interactions with immune receptors are examined. Recombinant immune receptors such as the killer cell immunoglobulin-like receptor (KIR) on natural killer cells, and the T cell receptor and CD8 co-receptor on T cells are examined. Standard protein-protein interaction techniques such as surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA), thermal melting assay, and circular dichroism are used to investigate this interaction. When compared to controls, soluble synthetic HLA proteins carrying specific mutations show impaired binding to recombinant activating immune receptors and maintain or improve binding to inhibitory receptors.

[0369] Competitive cell assays are used to examine the interaction of soluble synthetic HLA proteins with immune receptors on cell surfaces. In these assays, soluble synthetic HLA proteins are incubated with mammalian cells expressing wild-type class I HLA molecules. The ability of wild-type cells to survive in the presence of immune cells (e.g., T cells and / or NK cells) is then measured. When compared to controls, soluble HLA proteins carrying specific mutations show reduced reactivity with activating receptors on immune cells and maintained or improved reactivity with inhibitory receptors, resulting in increased killing of wild-type mammalian cells.

[0370] Example 15: Production of recombinant HLA proteins from separate transcription units To potentially overcome the heterogeneity observed when disulfide staples were introduced into SYNC27-1 (Figure 17), synthetic HLA proteins were produced from separate transcription units to create the same protein lacking linker 2 (SCD2-1 / HHCC1-1; SEQ ID NO:21 / 131). DNA encoding either SCD2-1 or HHCC1-1 was inserted into a vector and then introduced into bacteria under antibiotic selection. The construct format was as described in Figure 15. Briefly, the peptide and beta-2 microglobulin domains were expressed as a single protein joined by a linker (Linker L1) (SCD2-1, SEQ ID NO:21), and the HLA heavy chain domain (HHCC1-2, SEQ ID NO:132) was produced as a separate protein. Cells carrying the appropriate constructs (SCD2-1 and HHCC1-2 listed in Table 19) were grown to mid-log phase and expression was induced with isopropyl BD-1-thiogalactopyranoside (IPTG). Cells were harvested by centrifugation, lysed using a cell disrupter, and centrifuged to isolate soluble and insoluble fractions. Insoluble inclusion bodies containing the appropriate domains of the synthetic HLA proteins were washed and resuspended in denaturing buffer. The peptide and B-2 microglobulin protein were first refolded for 30 min before the HLA heavy chain domain was added to the refolding solution. The resulting refolded protein was purified by a combination of IMAC, SEC and / or anion exchange chromatography. The purification strategy, yield, and analysis (TOF-MS, DLS) are summarized in Table 16. Coomassie blue staining showed that the assembled final product separated into two separate polypeptide chains only under reducing conditions and migrated as a single species under non-reducing conditions, suggesting that it was correctly folded (Figure 20).

[0371] Additionally, a wild type class I HLA protein (WTC1) was produced using a similar approach as above, except that the peptide B-2 microglobulin domain was produced without linker 1 and peptide domain. The construct format is as depicted in FIG. 21 and contains the peptide domain (RL9 peptide, SEQ ID NO: 159), the B-2 microglobulin domain (B2M, SEQ ID NO: 195) and the HLA domain (HLA-C * 07:02, SEQ ID NO: 194). Protein was produced as above, except that 10 μM RYRPGTVAL (SEQ ID NO: 195) peptide (synthesized by Mimotopes, Melbourne, Australia) was added to the rifold solution. The results of SDS-PAGE analysis are shown in FIG. 22. Separation by SDS-PAGE followed by Coomassie blue staining showed that the assembled final product migrated as two species slightly smaller than the expected molecular weight under non-reducing conditions and at the expected molecular weight under reducing conditions, suggesting that it was correctly folded FIG. 22. The purification strategy, yield, and analysis (TOF-MS, DLS) are summarized in Table 16. The masses of the HLA domains were consistent with S-glutathionylation of the cysteine ​​residues.

[0372] To assess the homogeneity of the protein samples, DLS was used to analyze SCD2-1 / HHCC1-1 and WTC1 according to standard protocols. Briefly, samples were added to a sample cuvette and analyzed on a Zetasizer. Figures 19A and 19F show that a single peak was observed for WTC1 and SCD2-1 / HHCC1-1, indicating a homogeneous protein sample. The results are summarized in Table 16.

[0373] [Table 19] * Linker 1 (L1) long, L = long (GGGGSGGGGSGGGGS; SEQ ID NO: 179), M = medium (GGGGSGGGGSGS; SEQ ID NO: 177), S = short (GGGGSGGGGS; SEQ ID NO: 175), +C = glycine at position 2 of the L1 linker mutated to cysteine.* Linker 2 (L2) length, L=long (GGGGSGGGGSGGGGS; SEQ ID NO: 183), M=medium (GGGGSGGGGSGS; SEQ ID NO: 177). $ WT = wild type sequence of Q226, D227 or Y84. ^ Protein tags: His, Hexahis tag; AVI, AviTag™. A TEV protease cleavage site is contained at the N-terminus of the Hexahis tag. # 1 in the HLA domain. @ RL9 = RL9 peptide, RYRPGTVAL (SEQ ID NO: 159).

[0374] Example 16: Analysis of the interaction of synthetic HLA constructs with KIR proteins by surface plasmon resonance. The interaction of synthetic HLA constructs with KIR immunity proteins was investigated by SPR according to standard protocols. Briefly, histidine-tagged KIR proteins (ligands) were immobilized via nickel-NTA coupling with nitrilotriacetic acid (NTA)-derivatized carboxymethyl dextran sensor chips (NiD50L, XanTec Bioanalytics). Recombinant HLA proteins (analytes) (SYNC20-1, SYNC23-1, SYNC25-1, SYNC26-1, SCD2-1 / HHCC1-2, and wild-type control protein WTC1) were then flowed over the surface to generate sensorgrams, which are graphs of SPR responses tracked over time during biomolecular interaction analysis (Figure 23 shows a representative example). All experiments were performed at 25 °C and both analyte and ligand were diluted in SPR buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 50 μM EDTA, 0.05% (v / v) Tween-20). The flow cell was primed with 500 μM NiCl (10 μL × min). -1 , 1 minute).

[0375] Prior to use in SPR assays, synHLA proteins (SYNC20-1, SYNC23-1, SYNC25-1, SYNC26-1, SCD2-1 / HHCC1-2) were modified by TEV cleavage as described in Example 4. Briefly, TEV protease was incubated with synthetic HLA proteins at a 1:100 w / w ratio overnight at 4° C. and separated by size exclusion chromatography.

[0376] Analyte injection responses at the end of the equilibration phase were double-referenced against the reference cell and blank injection values ​​before normalization to the respective ligand immobilization responses. The relative responses of each interaction are shown in Figure 24. The SPR data show that all synHLA proteins tested bound to KIR2DL2 and KIR2DL3, but with reduced relative responses compared to WTC1 (Figure 24).

[0377] The synHLA proteins with the highest relative response to KIR2DL2 were SYNC26-1 and SYNC23-1. Both of these proteins contained four point mutations in the HLA heavy chain (C1G, Y84A, Q226A, D227K), a 15-residue linker 2, and either a short (SYNC26-1) or medium (SYNC23-1) linker 1. When a longer linker 1 was introduced into the same protein scaffold to create SYNC20-1, reduced binding to KIR2DL2 was observed. This suggests that the presence of a longer linker 1 may impair binding to KIR2DL2.

[0378] When SYNC26-1 was produced without the CD8 mutations (SYNC25-1, wt Q226, D227), reduced binding to KIR2DL2 was observed. When a disulfide staple was introduced into the same construct lacking linker 2, binding to KIR2DL2 was further reduced (compare SYNC25-1 and SCD2-1 / HHCC1-2), suggesting that the presence of this disulfide staple and / or the absence of linker 2 may impair KIR2DL2 binding.

[0379] The same overall trends as above were observed for the binding of synHLA proteins to KIR2DL3, however smaller differences between proteins were generally observed and error bars overlapped.

[0380] Example 17: B2M - / - Generation of EBV-transformed B cell lines Using CRISPR / Cas9, B2M was mutated in two EBV strains (9031 and JK) such that B2M expression was ablated. EBV cells were transfected with the Cas9 construct and HLA class I low / negative EBV cells were selected. Cells were grown and the absence of surface HLA class I was confirmed by antibody staining using anti-HLA antibody W6 / 32. Binding of W6 / 32 to the cell surface was detected with phycoerythrin-labeled goat anti-mouse antibody (GAM-PE) as shown in FIG. 25. After three rounds of selection, homogenous class I-deficient strains were obtained. The left column of FIG. 25 shows class I and negative control staining of unmodified EBV cells (referred to as wild type). The right column of FIG. 25 shows the equivalent staining of B2M-deficient cells. The numbers on the plots are the mean fluorescence intensity (MFI) of the staining. The negative control is staining in the absence of primary antibody (W6 / 32) but in the presence of secondary antibody alone (GAM-PE). The shaded plot shows the staining profile of the negative control staining (no primary antibody). The open histogram with a solid box represents the staining profile with W6 / 32 detected by GAM-PE.

[0381] Example 18: Relative expression of synthetic HLA proteins on the surface of B2Mnull EBV cells DNA encoding the synthetic HLA protein controls described herein (SCTA2-M1, SEQ ID NO: 5) SCDA1-M1, SEQ ID NO: 17) was cloned into the EBV episomal vector pCE. The vector carrying the construct was introduced into B2M null EBV cells by electroporation according to standard protocols, followed by antibiotic selection. Expression of the synthetic HLA protein controls on the cell surface was then examined by antibody staining and fluorescence-activated cell sorting (FACS) (Figure 26). Expression of SCTA2-M1 and SCTDA1-M1 was detected in approximately 20 and 40% of the cell population, respectively, demonstrating that the synthetic HLA protein controls can be expressed on the cell surface.

[0382] To facilitate more rapid reporting of successful transfection, the EBV episomal vector pCE (Addgene#41858) was modified to encode a fluorescent reporting protein (green fluorescent protein, GFP) in a multigene expression system (Lewis, Jo E., et al. "The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo." Journal of neuroscience methods 256(2015):22-29, which is incorporated by reference in its entirety). The construct format and predominant products are shown in FIG. 27, with GFP fluorescence providing a readout of successful transfection.

[0383] DNA encoding the synHLA proteins (control and test) described herein (Figure 28) was cloned into the EBV episomal vector pCE modified for multigene expression (Figure 27). The vectors carrying the constructs were electroporated into B2M null EBV cells by electroporation, and the cells were cultured according to standard protocols. After 1-13 days of culture, the cells were stained for HLA-C expression using antibody DT-9 (mouse, IgG2b, AlexaFluor 647 labeled, Biolegend catalog number 400330). Transfected cells were detected by their GFP expression, and HLA-C expression was detected within the GFP+ve cell population, as expected (see Figures 29 and 30). The percentage of HLA-C expressing cells was determined and expressed as a percentage of transfected GFP+ cells minus staining with an isotype-matched control antibody (clone MCP-11, mouse, IgG2b, AlexaFluor 647 labeled, Biolegend catalog number 373307) (Figure 28). The highest expressed proteins (detected in >50% of GFP positive cells) were SYNC37-M1 (SEQ ID NO: 118), SYNC31-M1 (SEQ ID NO: 106) and SYNC34-M1 (SEQ ID NO: 112) (Figure 28). Maximal cell surface expression of HLA-C was detected 5 days after transfection (Figure 31). Western blot analysis using an antibody specific for GFP (mAb B34, supplied by Biolegend) revealed that GFP was mainly in a free form, rather than linked to the synHLA protein (Figure 32).

[0384] Example 19: Preparation of NK cells. NK cells were prepared in two ways: (1) NK cells were purified from peripheral blood mononuclear cells (PBMCs) using the Miltenyi NK Cell Isolation Kit (Cat. No. 130-092-657). Purified NK cells were expanded using bead-coupled antibodies from the Miltenyi NK Activation / Expansion Kit (Cat. No. 130-094-483) in Miltenyi's NK medium supplemented with 5% pooled human serum and 500 U / mL IL-2. Alternatively, PBMCs were cultured as above until NK cells were greater than 90% of cells present, which takes at least 4 weeks.

[0385] Following NK cell expansion by either method, the purity of the NK cell population was determined by flow cytometry. This was done by staining the cells with fluorescently conjugated antibodies specific for CD3 and CD56. The percentage of CD3- and CD56+ cells among the total viable cells was determined (see FIG. 33). The purity of the NK cells used in the experiments was generally greater than 95%, allowing the obtaining of highly pure NK cells.

[0386] Example 20: Relative susceptibility to NK-mediated lysis of EBV cells expressing synHLA proteins. To examine the susceptibility of cells expressing synthetic HLA molecules to NK cell-mediated killing, purified NK cells are incubated with cells expressing a subset of synthetic HLA proteins as described elsewhere herein. NK cells are prepared and analyzed as described elsewhere herein. To prepare target cells, B2M-deficient EBV cells are transfected with pCE plasmids encoding the expression of different HLA-C constructs. Five days after transfection, GFP+, HLA-C+ (DT-9 staining) cells are sorted by flow cytometry. Target cells 51 After loading with Cr for 1 hour and then washing three times, 5,000 51Cr-labeled target cells are cultured with 100,000 NK cells at an effector to target cell ratio of greater than 1:1. The chronic myeloid leukemia cell line K562 is included as a positive control for NK-mediated killing. After 5 hours of culture, the supernatant is collected and assayed for gamma radiation in the supernatant. 51 The release of Cr is measured. Results are expressed as the percentage of net killing compared to target cells cultured alone (background) and maximum lysis, target cells cultured with 2% Triton X-100. Lysis of cells expressing the various synHLA constructs is compared to controls.

[0387] Example 21: Relative ability of EBV cells expressing synHLA proteins to stimulate CD8+ T cell responses. CD8 + T cells are purified from peripheral blood of healthy volunteer donors using magnetic bead positive selection by staining with mAb specific for CD8α (clone SK1, phycoerythrin (PE) conjugated) followed by purification using Miltenyi anti-PE microbeads (Miltenyi Cat. No. 130-048-801) or Milteny REALease CD8 Microbead Kit (Cat. No. 130-117-036). After purification, CD8 + The purity of T cells is determined by staining the cells with an antibod...

Claims

[Claim 1] It is a structure, a. One or more target regions; b. One or more major histocompatibility complex (MHC) regions, wherein at least one of the one or more MHC regions includes a differentiation antigen group 8 (CD8) binding site; and c. One or more linker regions A structure that includes this.

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