Recombinant protein comprising human leukocyte antigen (HLA) and HLA binding peptide

Abstract

Provided herein, inter alia, are recombinant proteins including a human leukocyte antigen (HLA) heavy chain binding peptide, a beta-2 microglobulin (B2M) sequence, and an HLA heavy chain sequence. In embodiments, the HLA heavy chain binding peptide and the HLA heavy chain sequence are covalently attached by a disulfide bond.

Description

RECOMBINANT PROTEIN COMPRISING HUMAN LEUKOCYTE ANTIGEN (HLA) AND HLA BINDING PEPTIDECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United Kingdom Provisional Application No.2316281. 1, filed October 24, 2023, which is hereby incorporated by reference in its entirety and for all purposes.REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE

[0002] The contents of the electronic sequence listing (061404-509001 WO_Sequence_Listing_ST26.xml; Size 249,856 bytes; and Date of Creation: October 24, 2024) are hereby incorporated by reference in their entirety.BACKGROUND

[0003] Cellular therapies hold great promise for combating previously intractable diseases. While the use of autologous cells is generally optimal, this approach can be cost- prohibitive and burdensome. Although allogeneic pluripotent stem cells (PSCs) provide greater scalability and cost savings, their utility is limited by the need to match human leukocyte antigen (HLA) class I alleles, the most genetically polymorphic region in the human genome. Two categories of MHC molecules are involved in presenting antigenic peptides to the immune system, namely MHC I molecules recognized by CD8+ T cells and MHC II molecules recognized by CD4+ T cells.

[0004] Major mismatches in HLA class I haplotypes lead to the “self versus non-self ’ immune response that can result in the body’s rejection of transplanted therapeutic cells by either a T cell mediated response or an antibody-mediated rejection (where the T-helper cells are co-stimulated, with concurrent inflammatory response, leading to recognition of foreign HLA). Likewise, mismatch in one or more minor histocompatibility antigens may also lead to transplant rejection. One proposed mechanism for cellular therapeutics is to knock out HLAs in the cellular therapeutic to avoid the alloreactive response by T cells due to the mismatch between the HLA expressed by the cellular therapeutic and the patient’s HLA. This creates a “missing self’ problem where a patient’s NK cells will kill the HLA Class-I-negative cells administered in the cellular therapeutic. Consequently, there remains an unmet need for the development of engineered cellular therapeutics,pluripotent stem cells, and compositions for use with cellular therapeutics that circumvent both T cell- and NK cell-mediated immune responses, that reduce or avoid NK cell lysis (e.g., either the recipient’s or fratricide in the cellular therapeutic population), or that avoid a recipient’s immune surveillance, all towards the goal of enhancing the recipient’s immune system to more effectively fight disease, such as cancer.BRIEF SUMMARY

[0005] In an aspect is provided a recombinant protein including from the N-terminus to the C-terminus direction: a) a human leukocyte antigen-E (HLA-E) heavy chain binding peptide, wherein the HLA-E heavy chain binding peptide is 9 amino acids in length, and wherein the HLA-E heavy chain binding peptide includes a cysteine relative to position 5 or position 9 from the N-terminus of SEQ ID NO: 124- 126 or SEQ ID NO: 151-208; b) a first peptide linker; c) a beta-2 microglobulin (B2M) sequence; d) a second peptide linker; and e) an HLA-E heavy chain sequence, wherein the HLA-E heavy chain sequence includes a cysteine in a position equivalent to position 77 or position 155 of an HLA-E heavy chain.

[0006] In another aspect is provided a nucleic acid encoding the recombinant protein provided herein including embodiments thereof.

[0007] In another aspect is provided a cell including the recombinant protein provided herein including embodiments thereof or the nucleic acid provided herein including embodiments thereof.

[0008] In another aspect is provided a method of making a hypoimmunogenic cell, including delivering to a cell the nucleic acid provided herein including embodiments thereof, and culturing the cell under conditions to express the recombinant protein provided herein including embodiments thereof.

[0009] In some aspects herein is an amino acid construct comprising: one or more major histocompatibility complex (MHC) regions, at least one of the one or more MHC regions comprises a cluster of differentiation (CD) binding site, one or more linker regions, a beta microglobulin (BM) or portion thereof, and a peptide. In some embodiments, the peptide is between 3 and 15 amino acid residues in length. In some embodiments, the peptide is bound within an antigen presenting region of the one or more MHC regions, some embodiments, the peptide is a 9 amino acid sequence derived from an HLA leader sequence. In some embodiments, the peptide further comprises a mutation. In some embodiments, the mutation in the peptide comprises an amino acid substituted for a cysteine. In some embodiments, the cysteine is in the fifth position in the peptide. In someembodiments, the cysteine is in the ninth position in the peptide. In some embodiments, the cysteine forms a disulfide bridge within the construct. In some embodiments, the disulfide bridge is between the peptide and one of the one or more MHC regions of the construct. In some embodiments, the peptide is derived from an HLA-B, HLA-C, HLA-G, or H2A leader sequence. In some embodiments, the peptide is SEQ ID NO: 124. In some embodiments, the peptide is SEQ ID NO: 125. In some embodiments, the peptide is SEQ ID NO: 126. In some embodiments, the peptide is SEQ ID NO: 127. In some embodiments, the peptide is SEQ ID NO: 128. In some embodiments, the peptide is derived from an HLA-B *0702 leader sequence. In some embodiments, the peptide is any one of SEQ ID:151-208.

[0010] In some aspects described herein is a construct that has an increased affinity for a NKG2A / CD94 receptor or a decreased affinity for a NKG2C / CD94. In some embodiments, the construct inhibits NK cell lysis when interacting with NK cells. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the one or more MHC regions comprise one or more human HLA class heavy chain sequences. In some embodiments, the one or more human HLA- class I heavy chain sequences are derived from HLA- A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or some combination thereof. In some embodiments, the one or more major histocompatibility construct (MHC) regions comprise an HLA-E region. In some embodiments, the HLA-E region is an HLA-E*01 :03 region. In some embodiments, the HLA-E region is an HLA-E*01 :01 region. In some embodiments, the one or more MHC regions comprise one or more mutations. In some embodiments, the one or more mutations are not present in the CD8 binding site. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the one or more mutations comprise a substitution of a cysteine residue at the 77 amino acid position of an HLA-E heavy chain In some embodiments, the one or more mutations comprise a substitution of a cysteine residue at the 155 amino acid position of an HLA-E heavy chain. In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the Q226 residue of HLA-E heavy chain, or In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the D227K residue of HLA-E heavy chain. In some embodiments, the Q226 residue of the CD8 binding site is deleted. In some embodiments, the construct further comprises one or more disulfide staple pairs. In some embodiments, the one or more disulfide staple pairs create a disulfide bond between the one or more MHC regions and the peptide. In some embodiments, the disulfide staple pairis formed between the residue of the one or more MHC regions corresponding to the N77 residue of an unmodified HLA-E heavy chain and a residue in the peptide. In some embodiments, the disulfide staple pair is formed between the residue of the one or more MHC regions corresponding to the H155 residue of an HLA-E heavy chain and a residue in the peptide.

[0011] In some aspects, described herein is one or more histocompatibility construct (MHC) regions comprising a truncated HLA heavy chain sequence. In some embodiments, the truncated HLA heavy chain sequence further comprises one or more C-terminus modifications. In some embodiments, the one or more C-terminus modifications comprise substituting one or more lysines. In some embodiments, the one or more major histocompatibility construct (MHC) regions comprise a truncated HLA-E heavy chain. In some embodiments, the truncated HLA-E heavy chain comprises removal of the amino acids in positions 315-337 of HLA-E heavy chain. In some embodiments, the C-terminus modification comprises mutations of one or more of the K310 and K311 residues. In some embodiments, the modification comprises a 310A substitution. In some embodiments, the modification comprises a 311A substitution. In some embodiments, the one or more major histocompatibility construct (MHC) regions comprise an HLA-G region. In some embodiments, the HLA-G region comprises an HLA-G*01 :01 sequence. In some embodiments, the HLA-G region comprises an HLA-G*01 :04 sequence.

[0012] In some aspects, described herein is a HLA-G region comprising one or more mutations. In some embodiments, the one or more mutations comprise a substitution at the Cys-42. In some embodiments, the one or more mutations comprise a substitution at the Cys-147. In some embodiments, a linker region of the one or more linker regions 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the construct comprises a first linker region and a second linker region. In some embodiments, the first linker region 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210, and wherein the second linker region 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210.

[0013] In some embodiments, the construct 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: 1-121. In some embodiments, the construct is soluble. In some embodiments, the construct is insoluble. In some embodiments, the construct is a single chain trimer (SCT). In some embodiments, the construct comprises a single chain dimer (SCD).

[0014] In some embodiments, the construct comprises in N-terminus to C-terminus order: a peptide, a first linker of the one or more linkers, a beta microglobulin (BM) or portion thereof, a second linker of the one or more linkers, and an MHC region of the one or more MHC regions. In some embodiments, the construct further comprises a disulfide staple pair configured to associate the peptide and the MHC region, or In some embodiments, the construct further comprises one or more signal sequences. 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 NO: 124-208. In some embodiments, the first linker 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the second linker 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the MHC region 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 NO:146-149. In some embodiments, the MHC region 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 NO:130-134.

[0015] In some aspects, described herein is a nucleic acid that encodes any one of the constructs described herein. In some embodiments, the nucleic acid further comprises a signal sequence. In some embodiments, the signal sequence is a B2M signal sequence. In some embodiments, the B2M signal sequence comprises SEQ ID NO: 135. In some aspects, described herein is an engineered vector encoding the nucleic acids described herein. In some embodiments, the vector is a plasmid, a minicircle, an adenoassociated virus (AAV) derived virion, a lentivirus, an adenovirus, or a herpes simplex virus (HSV).

[0016] In some aspects, described herein is a method of generating a hypoimmunogenic cell comprising administering to a cell the vectors described herein.

[0017] In some aspects described herein is a cell comprising a complex comprising: one or more major histocompatibility complex (MHC) regions, at least one of the one or more MHC regions comprise a cluster of differentiation (CD) binding site, a peptide, and one or more linker regions. In some embodiments, the peptide is between 3 and 15 amino acid sequence in length. In some embodiments, the peptide is bound within an antigen presenting region of the one or more MHC regions. In some embodiments, the peptide is a 9 amino acid sequence derived from an HLA leader sequence. In some embodiments, the peptide further comprises a mutation. In some embodiments, the mutation in the peptide comprises an amino acid substituted for a cysteine. In some embodiments, the cysteine is in the fifth position in the peptide. In some embodiments, the cysteine is in the ninth position in the peptide. In some embodiments, the cysteine forms a disulfide bridge within the complex. In some embodiments, the disulfide bridge is between the peptide and one of the one or more MHC- 155 regions of the complex. In some embodiments, the peptide is derived from an HLA-G, HLA-C, HLA-B, or HLA leader sequence. In some embodiments, the peptide is SEQ ID NO: 126. In some embodiments, the peptide is derived from an HLA-B*0702 leader sequence. In some embodiments, the peptide is SEQ ID NO: 124. In some embodiments, the peptide is derived from an HLA-Cw*03:02 leader sequence. In some embodiments, the peptide is SEQ ID NO: 125. In some embodiments, the peptide is SEQ ID NO: 150. In some embodiments, the peptide is SEQ ID NO: 127. In some embodiments, the peptide is SEQ ID NO: 128. In some embodiments, the complex has an increased affinity for a NKG2A / CD94 receptor or a decreased affinity for a NKG2C / CD94. In some embodiments, the complex inhibits NK cell lysis when interacting with NK cells. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the one or more MHC regions comprise one or more human HLA- class I heavy chain sequences. In some embodiments, the one or more human HLA class I heavy chain sequences are derived from HLA- A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or some combination thereof. In some embodiments, the one or more major histocompatibility complex (MHC) regions comprise an HLA-E region. In some embodiments, the HLA-E region is an HLA-E*01 :01 region. In some embodiments, the HLA-E region is an HLA-E*01 :03 region. In some embodiments, the one or more MHC regions comprise one or more mutations. In some embodiments, the one or more mutations are not present in the CD8 binding site. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the one or more mutations comprise a substitution of a cysteine residue at the amino acid position of an HLA-Eheavy chain In some embodiments, the one or more mutations comprise a substitution of a cysteine residue at the amino acid position of an HLA-E heavy chain.

[0018] In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the Q226 residue of HLA-E heavy chain. In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the D227K residue of HLA-E heavy chain. In some embodiments, the Q226 residue of the CD8 binding site is deleted. In some embodiments, the complex further comprising one or more disulfide staple pairs. In some embodiments, the one or more disulfide staple pairs create a disulfide bond between an MHC region of the one or more MHC regions and the peptide. In some embodiments, the disulfide staple pair is formed between the residue of the one or more MHC regions corresponding to the N77 residue of an HLA-E heavy chain and a residue in the peptide. In some embodiments, the disulfide staple pair is formed between the residue of the one or more MHC regions corresponding to the Hl 55 residue of an HLA-E heavy chain and a residue in the peptide. In some embodiments, the one or more major histocompatibility complex (MHC) regions comprise a truncated HLA-E heavy chain heavy chain sequence. In some embodiments, truncated HLA-E heavy chain heavy chain sequence further comprises one or more C-terminus modifications. In some embodiments, the one or more C-terminus modifications comprise substituting one or more lysines. In some embodiments, the one or more major histocompatibility complex (MHC) regions comprise a truncated HLA-E heavy chain. In some embodiments, the truncated HLA-E heavy chain comprises removal amino acids in positions 315-337 of HLA-E heavy chain. In some embodiments, the C-terminus modification comprises mutations of one or more of the lysine residues. In some embodiments, the modification comprises a 310A substitution. In some embodiments, the modification comprises a 311 A substitution.

[0019] In some embodiments, the one or more major histocompatibility complex (MHC) regions comprise an HLA-G region. In some embodiments, the HLA-G region comprises an HLA-G*01 :01 sequence. In some embodiments, the HLA-G region comprises an HLA- G*01 :04 sequence. In some embodiments, the HLA-G region comprises one or more mutations. In some embodiments, the one or more mutations comprise a substitution at the Cys-42. In some embodiments, the one or more mutations comprise a substitution at the Cys-147. In some embodiments, a linker region of the one or more linker regions 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: 192-198, comprising a first linker region and a second linker region. In some embodiments, the first linker regioncomprises 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: 192-198, and wherein the second linker region 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the complex 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 NO: l-121. In some embodiments, the complex is soluble. In some embodiments, the complex is insoluble. In some embodiments, the complex is a single chain trimer (SCT). In some embodiments, the complex comprises a single chain dimer (SCD). In some embodiments, the complex comprises in N-terminus to C-terminus order: a peptide, a first linker of the one or more linkers, a beta microglobulin (BM) or portion thereof, a second linker of the one or more linkers, and an MHC region of the one or more MHC regions. In some embodiments, the complex further comprises a disulfide staple pair configured to associate the peptide and the MHC region. In some embodiments, the complex comprises a signal sequence. 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 NO: 124-208. In some embodiments, the first linker 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 NO: 138- 144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the second linker 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the MHC region 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 NO: 146-149 or SEQ ID NO: 130-134.

[0020] For the cell provided herein, in some embodiments, the cell is a stem cell. In some embodiments, the cell is a pluripotent cell. T In some embodiments, the cell has reduced immunogenicity when compared to a similar cell comprising an unmodified HLA-E heavy chain or HLA-G heavy chain.

[0021] In some aspects described herein is a method of generating a cell comprising: generating a complex comprising one or more targeting moieties, one or more linker regions, and one or more major histocompatibility complex (MHC) regions, at least one ofthe one or more MHC regions comprise a cluster of differentiation 8 (CD8) binding site, transfecting the cell with a polynucleotide cassette, the cassette configured to express at least one of the complexes described herein, and expressing the complex in the cell. In some embodiments, the peptide is between 3 and 15 amino acid residues in length. In some embodiments, the peptide is held within the antigen presenting region of the one or more MHC regions. In some embodiments, the peptide is a 9 amino acid sequence derived from an HLA-G leader sequence. In some embodiments, the peptide further comprises a mutation. In some embodiments, the mutation in the peptide comprises an amino acid substituted for a cysteine. In some embodiments, the cysteine is in the fifth position in the peptide. In some embodiments, the cysteine is in the ninth position in the peptide. In some embodiments, the cysteine forms a disulfide bridge within the complex. In some embodiments, the disulfide bridge is between the peptide and one of the one or more MHC regions of the complex. In some embodiments, the peptide is derived from an HLA-B, HLA-C, or HLA-G leader sequence. In some embodiments, the peptide is SEQ ID NO: 126. In some embodiments, the peptide is derived from an HLA-B*0702 leader sequence. In some embodiments, the peptide is SEQ ID NO: 124. In some embodiments, the peptide is derived from an HLA-Cw*03:02 leader sequence. In some embodiments, the peptide is SEQ ID NO: 125. In some embodiments, the peptide is SEQ ID NO: 127. In some embodiments, the peptide is SEQ ID NO: 132. In some embodiments, the peptide is SEQ ID NO: 128. In some embodiments, the complex has an increased affinity for a NKG2A / CD94 receptor or a decreased affinity for a NKG2C / CD94.

[0022] In some aspects, described herein is a complex that inhibits NK cell lysis when interacting with NK cells. In some embodiments, the CD binding site comprises one or more mutations. In some embodiments, the one or more MHC regions comprise one or more human HLA- class I heavy chain sequences. In some embodiments, the one or more human HLA class I heavy chain sequences are derived from HLA- A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or some combination thereof. In some embodiments, the one or more major histocompatibility complex (MHC) regions comprise an HLA-E region. In some embodiments, the HLA-E region is an HLA-E*01 :01 region. In some embodiments, the HLA-E region is an HLA-E*01 :03 region. In some embodiments, the one or more MHC regions comprise one or more mutations. In some embodiments, the one or more mutations are not present in the CD8 binding site. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the one or more mutations comprise a substitution of a cysteine residue at the 77 amino acid position of an HLA-Eheavy chain In some embodiments, the one or more mutations comprise a substitution of a cysteine residue at the 155 amino acid position of an HLA-E heavy chain In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the Q226 residue of HLA-E heavy chain. In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the D227K residue of HLA-E heavy chain. In some embodiments, the Q residue of the CD binding site is deleted. In some embodiments, the complex further comprising one or more disulfide staple pairs. In some embodiments, the one or more disulfide staple pairs couple an MHC region of the one or more MHC regions and the peptide. In some embodiments, the disulfide staple pair is formed between the residue of the one or more MHC regions corresponding to the N77 residue of an HLA-E heavy chain and a residue in the peptide. In some embodiments, the disulfide staple pair is formed between the residue of the one or more MHC regions corresponding to the Hl 55 residue of an HLA-E heavy chain and a residue in the peptide.

[0023] In some aspects, described herein is one or more major histocompatibility complex (MHC) regions comprising a truncated HLA- heavy chain sequence. In some embodiments, the truncated HLA heavy chain sequence further comprises one or more C- terminus modifications. In some embodiments, the one or more C-terminus modifications comprise substituting one or more lysines. In some embodiments, the one or more major histocompatibility complex (MHC) regions comprise a truncated HLA-E heavy chain. In some embodiments, the truncated HLA-E heavy chain comprises removal amino acids in positions 315-337 of HLA-E heavy chain. In some embodiments, the C-terminus modification comprises mutations of one or more of the K310 and K311 residues. In some embodiments, the modification comprises a 310A substitution. In some embodiments, the modification comprises a 311A substitution.

[0024] In some embodiments, the one or more major histocompatibility complex (MHC) regions comprise an HLA-G region. In some embodiments, the HLA-G region comprises an HLA-G*01 :01 sequence. In some embodiments, the HLA-G region comprises an HLA- G*01 :04 sequence. In some embodiments, the HLA-G region comprises one or more mutations. In some embodiments, the one or more mutations comprise a substitution at the Cys-42. In some embodiments, the one or more mutations comprise a substitution at the Cys-147. In some embodiments, a linker region of the one or more linker regions 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:209, or SEQ ID NO:210, comprising a first linker regionand a second linker region. In some embodiments, the first linker region 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 NO: 138- 144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210, and wherein the second linker region 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the complex 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 NO:1-121. In some embodiments, the complex is a single chain trimer (SCT). In some embodiments, the complex comprises in N-terminus to C-terminus order: a peptide, a first linker of the one or more linkers, a beta microglobulin (BM) or portion thereof, a second linker of the one or more linkers, and an MHC region of the one or more MHC regions. In some embodiments, the complex further comprises a disulfide staple pair configured to associate the peptide and the MHC region. In some embodiments, the complex comprises one or more signal sequences. 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: 128-191. In some embodiments, the first linker 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO:123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the second linker 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 NO: 138-144, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:209, or SEQ ID NO:210. In some embodiments, the MHC region 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 NO: 146-149.

[0025] In some embodiments, the nucleic acid provided herein is contained within an engineered vector In some embodiments, the vector is a plasmid, a minicircle, an adenoassociated virus (AAV) derived virion, a lentivirus, an adenovirus, or a herpes simplex virus (HSV). In some embodiments, the cell is a stem cell. In some embodiments, the cell is a pluripotent cell. In some embodiments, the cell has reduced immunogenicity when compared to a similar cell comprising an unmodified HLA-E heavy chain or HLA-G heavy chain.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 displays a schematic showing the single chain trimer domain structure of exemplary recombinant proteins including synHLA-E or synHLA-G. Recombinant proteins including synHLA-G may or may not include a portion or all of the cytoplasmic tail.

[0027] FIG. 2 displays a model of a folded synHLA-E or synHLA-G based constructs showing examples of construct architecture and linkers. The model was created using an AlphaFold model of synHLA-E based on the peptide, beta-2 microglobulin and heavy chain coordinates from the crystal structure of HLA-E in complex with NKG2A / CD94 (PDB ID 3CDG; PubMed: 18332182). In comparison to HLA-G, wild-type HLA-E comprises an extended cytoplasmic tail which is shown and labelled. Both HLA-E and HLA-G comprise a transmembrane and cytosolic region, which is shown and labelled; the construct of the invention may or may not include a portion of or all of the tail on sequences derived from HLA-E or HLA-G. The cytosolic tail may be further modified to alter migration along the secretory trafficking pathway, surface expression, and subcellular localization.

[0028] FIG. 3 displays a model of an example of a folded synHLA-E or synHLA-G based constructs in complex with a receptor NKG2A / CD94. The model was created using the crystal structure of HLA-E in complex with NKG2A / CD94 (PDB ID 3CDG; PubMed ID: 18332182) and an AlphaFold model of synHLA-E based on the peptide, beta-2 microglobulin and heavy chain coordinates of the same crystal structure.

[0029] FIG. 4 displays a model of an example of a folded synHLA-E or synHLA-G based construct in complex with a CD8 receptor. This structural model highlights the location of the CD8 knock-out (KO) mutations Q226A / D227K. The model was built using the HLA coordinates from the crystal structure of HLA-E in complex with NKG2A / CD94 (PDB ID 3CDG; PubMed ID: 18332182) and the CD8 coordinates from the crystal structure of HLA-A2 in complex with CD8 (PDB ID 1AKJ; PubMed ID: 9177355).

[0030] FIG. 5. Phenotypic analysis and analysis of cell surface expression of synHLA-E and synHLA-G constructs 6 days post-thaw. Mean fluorescence Intensity (MFI) is shown for each construct using mCherry, and antibodies specific for B2M and heavy chain portions of the molecule. Loaded peptides include VMAPRTVLL (SEQ ID NO: 124), VMAPRTLIL (SEQ ID NO: 125), VMAPRTLFL(SEQ ID NO: 126), VMAPCTLFL(SEQ ID NO: 127), VMAPRTLFC(SEQ ID NO: 128), and RIIPRHLQL(SEQ ID NO: 150).

[0031] FIG.s 6A-6D. Cell surface expression data for the synHLA-E construct that showed highest expression levels. Expression levels of the synHLA-E construct, whichincludes a disulfide staple (SEQ NO 13; HLA-E_pHLA-G_lllong_pC5_hcC155) was compared to a control construct (SEQ NO 9; HLA-E_pHLA- G il long). FIG. 6A) A schematic showing synHLA-E expressed on the surface of K562 cells. FIG. 6B) Molecular representation of synHLA-E showing B2M and heavy chain binding regions of the antibodies used in staining. FIG. 6C) FACS data showing expression of synHLA-E constructs. FIG. 6D) Bar graph illustrating level of cell surface expression of synHLA-E construct (SEQ NO: 13) relative to HLA-E control construct (SEQ ID NO:9). Depending on which antibody was used for staining, the synHLA-E construct of SEQ NO 13 shows between 6-fold and 12-fold increase in surface expression relative to the HLA-E control construct (SEQ ID NO:9).

[0032] FIG.s 7A-7C. Binding analysis of the NKG2A / CD94 to the synHLA-E construct including a disulfide staple (SEQ NO 13; HLA-E_pHLA-G_lllong_pC5_hcC155) compared to a control HLA-E construct (SEQ NO 9; HLA-E_pHLA-G_lllong). FIG. 7A) Schematic illustrating the binding assay. FIG. 7B) Data illustrating NKG2A / CD94 binding normalized to HLA-E / synHLA-E surface expression. FIG. 7C) Binding affinities of the SynHLA-E contract or control HLA-E construct (EC50) to NKG2A / CD94.

[0033] FIG.s 8A-8B. NK cytotoxicity assay for the most highly expressed synHLA-E construct (SEQ NO 13; HLA-E_pHLA-G_lllong_pC5_hcC155 containing a disulfide staple) compared to the control (SEQ NO 9; HLA-E_pHLA- G il long). FIG. 8A) Schematic illustrating assay including co-culture of effector NK cells and target cells. FIG. 8B) Data illustrating percent NK cell killing. Effector and target cells were cocultured at different E:T ratios. Effector cells were provided by 2 different NK donors. Cytotoxicity was measured using 7-AAD staining and flow-cytometry. The data illustrates that the synHLA-E construct decreases NK killing similarly to HLA-E control.

[0034] FIG.s 9A-9B. ELISPOT assay for the synHLA-E construct showing highest expression levels (SEQ NO:13; HLA-E_pHLA-G_lllong_pC5_hcC155) compared to the control (SEQ NO 9; HLA-E_pHLA-G_lllong). FIG. 9A) Schematic illustrating co-culture assay format. FIG. 9B) ELISPOT data showing that synHLA-E does not re-stimulate antigen-experienced T cells, from 4 different HLA-A2 healthy donors. 24 hour cocultures. CERI: MHC-I-restricted peptide pool consisting of 124 epitopes from Cytomegalovirus (30 peptides), Epstein Barr Virus (59 peptides), Human Respiratory Syncytial Virus (11 peptides), and Influenza A Virus (24 peptides).

[0035] FIG.s 10A-10E. synHLA-E design. FIG. 10A) Engagement of cell-surface synHLA-E with NKG2A / CD94 on NK cells. FIG. 10B) Model of synHLA- E:NKG2A / CD94 complex. FIG. 10C) Molecular representation illustrating disulfide staples between the synHLA-E heavy chain and HLA-E heavy chain binding peptide. FIG. 10D) Selected synHLA-E heavy chain binding peptides (top to bottom: SEQ ID NO: 126, 125, and 124) FIG. 10E) Schematic illustrating design of synHLA-E recombinant proteins.

[0036] FIG.s 11A-11B. NK cytotoxicity assay for synHLA-E constructs. FIG. 11A). Illustrates expression levels of the constructs on K526 cells. FIG. 11B) Illustrates percent NK cell killing. Effector cells were provided by 2 different NK cell donors. Loaded peptides include VMAPRTVLL (SEQ ID NO: 124), VMAPRTLIL (SEQ ID NO: 125), VMAPRTLFL (SEQ ID NO: 126), and VMAPRTLFC (SEQ ID NO: 128).

[0037] FIG.s 12A-12B. NK cytotoxicity assay for various synHLA-E constructs as shown. Donor NK cells were provided by a FIG. 12A) first donor and a FIG. 12B) second donor.DETAILED DESCRIPTION

[0038] While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0039] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

[0040] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0041] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g.,Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0042] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. 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.

[0043] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. 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.

[0044] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof; or nucleosides (e.g., deoxyribonucleosides or ribonucleosides). In embodiments, “nucleic acid” does not include nucleosides. The terms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term “nucleoside” refers, in the usual and customary sense, to a glycosylamine including a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non limiting examples, of nucleosides include, cytidine, uridine, adenosine, guanosine, thymidine and inosine. The term “nucleotide” refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acid, e.g. polynucleotides contemplated herein include any types of RNA, e.g. mRNA, siRNA, miRNA, and guide RNA and any types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragmentsthereof. The term “duplex” in the context of polynucleotides refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like.

[0045] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three- dimensional structures, and may perform various functions, known or unknown. Nonlimiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. For example, the nucleic acid provided herein may be part of a vector. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0046] A polynucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Polynucleotides may optionally include one or more non-standard nucleotide(s), nucleotide analog(s) and / or modified nucleotides.

[0047] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g.,hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.

[0048] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0049] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0050] An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). Due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N-terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0051] The terms "numbered with reference to" or "corresponding to," when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue when it occupies the same essential structural position within the protein as the given residue. One skilled in the art will immediately recognize the identity and location of residues corresponding to a specific position in a protein in other proteins with different numbering systems. For example, by performing a simple sequence alignment with a protein the identity and location of residues corresponding to specific positions of the protein are identified in other protein sequences aligning to the protein. For example, a selected residue in a selected protein corresponds to glutamic acid at position 138 when the selected residue occupies the same essential spatial or other structural relationship as a glutamic acid at position 138. In some embodiments, where a selected protein is aligned for maximum homology with a protein, the position in the aligned selected protein aligning with glutamic acid 138 is the to correspond to glutamic acid 138. Instead of a primary sequence alignment, a three- dimensional structural alignment can also be used, e.g., where the structure of the selected protein is aligned for maximum correspondence with the glutamic acid at position 138, and the overall structures compared. In this case, an amino acid that occupies the same essential position as glutamic acid 138 in the structural model is the to correspond to the glutamic acid 138 residue. Reference to an amino acid position of an HLA-E heavy chain is relative to the N-terminus of SEQ ID NO: 146 or SEQ ID NO: 147. Reference to an amino acid position of an HLA-G heavy chain is relative to the N-terminus of SEQ ID NO: 148 or SEQ ID NO:149.

[0052] The terms "corresponding to" and “at a position equivalent to” when used in the context of the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. An amino acid residue in a protein "corresponds" to a given residue or is “at a position equivalent to” another position when it occupies the same essential structural position within the protein as the given residue. For example, precise amino acid numbering assignments my change between homologous proteins or between versions of the same proteins that differ in length (e.g. due to elimination of a protein domain). Thus, an amino acid residue “at a position equivalent to” another position may be the precise same amino acid positionwithin the context of a given protein domain, but its number assignment may differ due to length between two version of the same protein. An amino acid or nucleotide base "position" is denoted by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-end). As descried above, due to deletions, insertions, truncations, fusions, and the like that must be taken into account when determining an optimal alignment, in general the amino acid residue number in a test sequence determined by simply counting from the N- terminus will not necessarily be the same as the number of its corresponding position in the reference sequence. For example, in a case where a variant has a deletion relative to an aligned reference sequence, there will be no amino acid in the variant that corresponds to a position in the reference sequence at the site of deletion. Where there is an insertion in an aligned reference sequence, that insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of truncations or fusions there can be stretches of amino acids in either the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence. By aligning sequences using methods known in the art, a given amino acid position that “corresponds to” or is “equivalent to” a given numbers position is easily identified.

[0053] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, "conservatively modified variants" refers to those nucleic acids that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a number of nucleic acid sequences will encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.

[0054] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or proteinsequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.

[0055] The following eight groups each contain amino acids that are conservative substitutions for one another:1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);6) Phenylalanine (E), Tyrosine (Y), Tryptophan (W);7) Serine (S), Threonine (T); and8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0056] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0057] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visualinspection (see, e.g., NCBI web site http: / / www.ncbi.nlm.nih.gov / BLAST / or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. The preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0058] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of, e.g., a full length sequence or from 20 to 600, about 50 to about 200, or about 100 to about 150 amino acids or nucleotides in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement)).

[0059] An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0060] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0061] For specific proteins described herein, the named protein includes any of the protein’s naturally occurring forms, variants or homologs that maintain activity of the protein (e.g., within at least 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the native protein). In some embodiments, variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring form. In other embodiments, the protein is the protein as identified by its NCBI sequence reference. In other embodiments, the protein is the protein as identified by its NCBI sequence reference, homolog or functional fragment thereof.

[0062] The term “HLA-E heavy chain protein” or “HLA-E heavy chain” as used herein includes any of the recombinant or naturally-occurring forms of HLA class Ihistocompatibility antigen, alpha chain E (HLA-E), also referred to as MHC class I antigen E or variants or homologs thereof that maintain HLA-E heavy chain activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HLA-E heavy chain). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HLA-E heavy chain protein. In embodiments, HLA-E heavy chain is substantially identical to the protein identified by the UniProt reference number Pl 3747 or a variant or homolog having substantial identity thereto. In embodiments, HLA-E heavy chain has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) of SEQ ID NO: 146. In embodiments, HLA-E heavy chain has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) of SEQ ID NO: 147.

[0063] The term “HLA-G heavy chain protein” or “HLA-G heavy chain” as used herein includes any of the recombinant or naturally-occurring forms of HLA class I histocompatibility antigen, alpha chain G (HLA-G), also referred to as HLA G antigen, MHC class I antigen G, or variants or homologs thereof that maintain HLA-G heavy chain activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to HLA-G heavy chain). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring HLA-G heavy chain protein. In embodiments, HLA-G heavy chain is substantially identical to the protein identified by the UniProt reference number P13747 or a variant or homolog having substantial identity thereto. In embodiments, HLA-G heavy chain has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) of SEQ ID NO: 148. In embodiments, HLA-G heavy chain has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) of SEQ ID NO: 149.

[0064] The term “CD8 protein” or “CD8” as used herein includes any of the recombinant or naturally-occurring forms of CD8, also referred to as T-cell surfaceglycoprotein CD8 alpha chain, T-cell surface glycoprotein CD8 beta chain, or variants or homologs thereof that maintain CD8 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD8). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD8 protein. In embodiments, CD8 is substantially identical to the protein identified by the UniProt reference number P01732 or a variant or homolog having substantial identity thereto. In embodiments, CD8 is substantially identical to the protein identified by the UniProt reference number Pl 0966 or a variant or homolog having substantial identity thereto.

[0065] The term “NKG2A protein” or “NKG2A” as used herein includes any of the recombinant or naturally-occurring forms of NKG2A, also referred to as NKG2A / NKG2B type II integral membrane protein, CD 159 antigen- like family member A, NK cell receptor A, or variants or homologs thereof that maintain NKG2A activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to NKG2A). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring NKG2A protein. In embodiments, NKG2A is substantially identical to the protein identified by the UniProt reference number P26715 or a variant or homolog having substantial identity thereto.

[0066] The term “NKG2C protein” or “NKG2C” as used herein includes any of the recombinant or naturally-occurring forms of NKG2C, also referred to as NKG2-C type II integral membrane protein, CD 159 antigen- like family member C, NK cell receptor C, or variants or homologs thereof that maintain NKG2C activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to NKG2C). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring NKG2C protein. In embodiments, NKG2C is substantially identical to the protein identified by the UniProt reference number P26717 or a variant or homolog having substantial identity thereto.

[0067] The term “CD94 protein” or “CD94” as used herein includes any of the recombinant or naturally-occurring forms of CD94, also referred to as Natural killer cellsantigen CD94, KP43, Killer cell lectin-like receptor subfamily D member 1, or variants or homologs thereof that maintain CD94 activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to CD94). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD94 protein. In embodiments, CD94 is substantially identical to the protein identified by the UniProt reference number QI 3241 or a variant or homolog having substantial identity thereto.

[0068] The term “B2M protein” or “B2M” as used herein includes any of the recombinant or naturally-occurring forms of Beta-2-microglobulin (B2M), or variants or homologs thereof that maintain B2M activity (e.g. within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to B2M). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring B2M protein. In embodiments, B2M is substantially identical to the protein identified by the UniProt reference number P61769 or a variant or homolog having substantial identity thereto. In embodiments, B2M has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid portion) of SEQ ID NO: 145.

[0069] The term "gene" means the segment of DNA involved in producing a protein. In embodiments, a gene includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0070] The word “expression” or “expressed” as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of a recombinant protein (e.g. construct) provided herein may be detected by standard protein detection methods well known in the art, including UV absorbance assays, Bradford assay, fluorescence microscopy, fluorescent dye methods, ELISA, flow cytometry, mass spectrometry, etc.Expression may include any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g. , ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc ).

[0071] The terms "plasmid", "vector" or "expression vector" refer to a nucleic acid molecule that encodes for genes and / or regulatory elements necessary for the expression of genes. Expression of a gene from a plasmid can occur in cis or in trans. If a gene is expressed in cis, the gene and the regulatory elements are encoded by the same plasmid. Expression in trans refers to the instance where the gene and the regulatory elements are encoded by separate plasmids.

[0072] As used herein, the term “construct” is intended to mean any recombinant nucleic acid or polypeptide molecule. In embodiments, a recombinant nucleic acid construct includes an expression cassette, plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular, single-stranded or double-stranded, DNA or RNA polynucleotide molecule. A construct may be derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, e.g., operably linked. In embodiments, the construct is a recombinant protein. In embodiments, the recombinant protein construct includes an HLA-E heavy chain binding peptide, a first peptide linker, a second peptide linker, and an HLA-E heavy chain. In embodiments, the recombinant protein construct is a fusion protein. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences (e.g. an HLA heavy chain, a B2M sequence, an HLA-E heavy chain binding peptide) that are recombinantly expressed as a single moiety.

[0073] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule (i.e. DNA not naturally found in the cell) or a protein into a cell. Nucleic acids are introduced to a cell using non- viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non- viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non- viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heatshock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. In embodiments, a nucleic acid is introduced by transfecting or transducing a virus or viral vector (e.g. a CMV vector, a lentivirus vector, etc.) into the cell (e.g. a CAR T cell). For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8: 1-4 and Prochiantz (2007) Nat. Methods 4: 119-20.

[0074] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species to become sufficiently proximal to react, interact or physically touch. In embodiments, contacting includes allowing a nucleic acid encoding a recombinant protein provided herein including embodiments thereof to interact with a cell (e.g. in close enough proximity for the nucleic acid to be delivered into the cell). In embodiments, contacting includes allowing a nucleic acid to interact with a cell, thereby resulting in transduced cell.

[0075] A “cell” as used herein, refers to a cell carrying out metabolic or other function sufficient to preserve or replicate its genomic DNA. A cell can be identified by well- known methods in the art including, for example, presence of an intact membrane, staining by a particular dye, ability to produce progeny or, in the case of a gamete, ability to combine with a second gamete to produce a viable offspring. Cells may include prokaryotic and eukaroytic cells. Prokaryotic cells include but are not limited to bacteria. Eukaryotic cells include but are not limited to yeast cells and cells derived from plants and animals, for example mammalian, insect (e.g., spodoptera) and human cells. Cells may be useful when they are naturally nonadherent or have been treated not to adhere to surfaces, for example by trypsinization. In embodiments, the cell is a stem cell. In 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 embodiments, the stem cell is a hematopoietic stem cell (HSC). In embodiments, the cell is a chimeric antigen receptor (CAR) T cell. In embodiments, the cell is a chimeric antigen receptor macrophage (CAR-M) cell. In embodiments, the cell isa chimeric antigen receptor natural killer (CAR-NK) cell. In embodiments, the cell is an engineered T-cell Receptor (TCR) T cell. In embodiments, the cell is a TCR macrophage (TCR-M) cell. In embodiments, the cell is a TCR natural killer (TCR-NK) cell.

[0076] The term “culture” or “cell culture” means the maintenance of cells, for example neuronal cells, in an artificial, in vitro environment. A “cell culture system” is used herein to refer to culture conditions in which a population of cells may be grown as monolayers or in suspension. “Culture medium” is used herein to refer to a nutrient solution for the culturing, growth, proliferation, or differentiation of cells (e.g. a cell including a recombinant protein provided herein, a cell including a nucleic acid provided herein). In embodiments, the culture includes a nutrient solution sufficient for expressing the recombinant protein provided herein including embodiments thereof in a cell.

[0077] The term "recombinant" when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express proteins that are not found within the native (nonrecombinant) form of the cell. In embodiments, the construct provided herein including embodiments thereof is a recombinant protein. For example, in embodiments, the construct (e.g. recombinant protein) provided herein includes amino acid sequences that would not otherwise be found together in a natural protein.

[0078] The term “inhibition”, “inhibit”, “inhibiting” and the like in reference to the immune response means negatively affecting (e.g. decreasing) the immune response relative to the activity or function of the immune response in the absence of an inhibitor (e.g. construct or recombinant protein provided herein). For example, in embodiments, inhibiting the immune response refers to increasing the inhibitory signal for NK cell activation. In embodiments, inhibiting the immune response refers to decreasing NK cell killing ability. In aspects, inhibition refers to a reduction in the activity of a particular type of cell (e.g. NK cell). In embodiments, inhibition of the immune response is from an interaction of a ligand (e.g. HLE-heavy chain) to a target (e.g. NKG2A / CD94 protein). In aspects, inhibition refers to a reduction of activity or function of a cell (e.g. NK cell) from a direct interaction (e.g. an inhibitor (e.g. construct, recombinant protein) binds to an inhibitory receptor (e.g. NKG2A / CD94) on the cell (e.g. NK cell)). In embodiments, inhibition refers to decreased binding of a ligand to an activating receptor on a cell. For example, in embodiments, inhibiting the immune response refers to decreased binding of aligand to an activating receptor (e.g. NKG2C / CD94) on an NK cell.

[0079] The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance (e.g. construct, recombinant protein) capable of detectab ly decreasing the function or activity of an immune cell. The antagonist can decrease function (e.g. cytotoxicity) or activity (e.g. cell killing, induction of apoptosis) 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 90% or more in comparison to a control in the absence of the antagonist. In certain instances, function or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the function or activity in the absence of the antagonist.

[0080] ‘ ‘Control” or “control experiment” is used in accordance with its plain ordinary meaning and refers to an experiment in which the subjects or reagents of the experiment are treated as in a parallel experiment except for omission of a procedure, reagent, or variable of the experiment. In some instances, the control is used as a standard of comparison in evaluating experimental effects. In some embodiments, a control is the measurement of the activity (e.g. cytotoxicity) of an immune cell (e.g. NK cell) in the absence of a compound as described herein (including embodiments and examples).

[0081] A “control” or “standard control” refers to a sample, measurement, or value that serves as a reference, usually a known reference, for comparison to a test sample, measurement, or value. For example, a test sample can be taken from a patient suspected of having a given disease (e.g. multiple sclerosis) and compared to a known normal (nondiseased) individual (e.g. a standard control subject). A standard control can also represent an average measurement or value gathered from a population of similar individuals (e.g. standard control subjects) that do not have a given disease (i.e. standard control population), e.g., healthy individuals with a similar medical background, same age, weight, etc. A standard control value can also be obtained from the same individual, e.g. from an earlier-obtained sample from the patient prior to disease onset. For example, a control can be devised to compare therapeutic benefit based on pharmacological data (e.g., half-life) or therapeutic measures (e.g, comparison of side effects). Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant. One of skill will recognize that standard controls can be designed for assessment of any number of parameters (e.g. RNA levels, protein levels, specific cell types, specific bodily fluids, specific tissues, etc).

[0082] One of skill in the art will understand which standard controls are most appropriate in a given situation and be able to analyze data based on comparisons to standard control values. Standard controls are also valuable for determining the significance (e.g. statistical significance) of data. For example, if values for a given parameter are widely variant in standard controls, variation in test samples will not be considered as significant.RECOMBINANT PROTEIN COMPOSITIONS

[0083] Provided herein, in an aspect, is a recombinant protein, also referred to herein as a construct, wherein the recombinant protein comprises one or more MHC regions, referred to as “human leukocyte antigen (HLA)” in humans. In embodiments, the one or more MHC regions is a HLA-E heavy chain sequence. In embodiments, the one or more MHC regions is a HLA-G heavy chain sequence. In some embodiments the one or more MHC regions comprise a polypeptide based on a non-classical HLA class I heavy chain, such as a polypeptide based on a naturally occurring allele of HLA-E or -G, or any modified polypeptide expressed therefrom (including isoforms) (each an “MHC region,” a “HLA heavy chain polypeptide”, a “HLA class I heavy chain polypeptide” or “HLA”, as in “HLA-E” or “HLA-G”). The HLA may be covalently bound to one or more of: a peptide, a B2M segment and one or more peptide linkers to generate the present recombinant proteins or constructs. In embodiments, the recombinant protein provided herein is referred to as “synHLA” or “synHLA construct”. In embodiments, the amino acid sequences of any one or more of the HLA heavy chain, peptide, B2M and the peptide linkers may be further modified to enhance the NK cell inhibitory activity (e.g., inhibiting the killing of cellular therapeutics), decrease NK cell activating signals, increase protein complex stability, increased cellular half-life in vitro or in vivo or both, reduced endocytic trafficking of the protein, increased cell surface expression of the protein complex, reduced immune cell activation when interrogated by CD8+ T cells, reduced binding to at least a subset of CD8+ T cells’ TCRs, and increased binding affinity to NKG2A and / or reduced binding affinity to NKG2C.

[0084] In an aspect is provided a recombinant protein including from the N-terminus to the C-terminus direction: a human leukocyte antigen-E (HLA-E) heavy chain binding peptide, wherein the HLA-E heavy chain binding peptide is 9 amino acids in length, and wherein the HLA-E heavy chain binding peptide includes a cysteine relative to position 5 or position 9 from the N-terminus of SEQ ID NO: 124- 126 or SEQ ID NO: 151-208; b) a first peptide linker; c) a beta-2 microglobulin (B2M) sequence; d) a second peptide linker;and e) an HLA-E heavy chain sequence, wherein said HLA-E heavy chain sequence includes a cysteine in a position equivalent to position 77 or position 155 of an HLA-E heavy chain.

[0085] As used herein “HLA-E heavy chain binding peptide” refers to a peptide 9 amino acids in length capable of binding an HLA-E heavy chain provided herein including embodiments thereof. In embodiments, the HLA-E heavy chain binding peptide increases the binding affinity of an HLA-E heavy chain to a NKG2A / CD94 receptor. In embodiments, the HLA-E heavy chain binding peptide decreases the binding affinity of an HLA-E heavy chain to a NKG2C / CD94 receptor. In embodiments, the HLA-E heavy chain binding peptide includes a cysteine relative to position 5 from the N-terminus of SEQ ID NO:124-126 or SEQ ID NO: 151-208. In embodiments, the HLA-E heavy chain binding peptide includes a cysteine relative to position 9 from the N-terminus of SEQ ID NO: 124-126 or SEQ ID NO: 151-208. In embodiments, the HLA-E heavy chain binding peptide includes any one of SEQ ID NO: 124-128 or SEQ ID NO:151-208 as set forth in Table 4.

[0086] In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 124, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 125, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 126, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 151, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 152, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 153, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 154, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 154, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 156, wherein the HLA-E heavy chain bindingpeptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 157, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:158, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 159, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 160, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 161, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 162, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 163, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 164, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 165, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 166, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 167, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 168, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 169, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 170, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 171, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence ofSEQ ID NO: 172, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 173, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 174, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 175, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 176, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 177, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 178, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 179, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 180, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 181, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 182, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 183, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 184, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 185, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 186, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 187, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, theHLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 188, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 189, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 190, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 191, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 192, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 193, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 194, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 195, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 196, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 197, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 198, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 199, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:200, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:201, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:202, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:203, wherein the HLA-E heavy chain binding peptide includes acysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:204, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:205, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:206, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:207, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 5.

[0087] In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 124, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 125, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 126, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 151, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 152, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 153, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 154, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 154, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 156, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 157, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:158, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. Inembodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 159, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 160, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 161, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 162, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 163, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 164, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 165, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 166, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 167, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 168, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 169, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 170, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 171, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 172, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 173, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 174, wherein the HLA-E heavy chain bindingpeptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 175, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 176, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 177, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 178, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 179, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 180, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 181, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 182, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 183, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 184, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 185, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 186, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 187, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 188, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 189, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence ofSEQ ID NO: 190, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 191, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 192, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 193, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 194, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ IDNO: 195, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 196, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 197, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 198, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 199, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:200, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:201, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:202, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:203, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:204, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:205, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, theHLA-E heavy chain binding peptide is the sequence of SEQ ID NO:206, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO:207, wherein the HLA-E heavy chain binding peptide includes a cysteine substitution at position 9.

[0088] In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 127. In embodiments, the HLA-E heavy chain binding peptide is the sequence of SEQ ID NO: 128.

[0089] In embodiments, the HLA-E heavy chain binding peptide includes 1-3 conservative amino acid substitutions. In embodiments, the HLA-E heavy chain binding peptide includes 1 conservative amino acid substitution. In embodiments, the HLA-E heavy chain binding peptide includes 2 conservative amino acid substitutions. In embodiments, the HLA-E heavy chain binding peptide includes 3 conservative amino acid substitutions. Thus, in embodiments, the HLA-E heavy chain binding peptide is any one of SEQ ID NO:124-128 or SEQ ID NO: 151-208, wherein the HLA-E heavy chain binding peptide includes 1-3 conservative amino acid substitutions.

[0090] For the recombinant protein provided herein, in embodiments, the B2M sequence has at least 90% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 91% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 92% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 93% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 94% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 95% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 96% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 97% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 98% identity to SEQ ID NO: 145. In embodiments, the B2M sequence has at least 99% identity to SEQ ID NO: 145. In embodiments, the B2M sequence includes SEQ ID NO: 145. In embodiments, the B2M sequence is SEQ ID NO: 145.

[0091] In embodiments, the B2M sequence has at least 90% identity to SEQ ID NO:136. In embodiments, the B2M sequence has at least 91% identity to SEQ ID NO: 136. In embodiments, the B2M sequence has at least 92% identity to SEQ ID NO: 136. In embodiments, the B2M sequence has at least 93% identity to SEQ ID NO: 136. In embodiments, the B2M sequence has at least 94% identity to SEQ ID NO: 136. In embodiments, the B2M sequence has at least 95% identity to SEQ ID NO: 136. Inembodiments, the B2M sequence has at least 96% identity to SEQ ID NO: 136. In embodiments, the B2M sequence has at least 97% identity to SEQ ID NO: 136. In embodiments, the B2M sequence has at least 98% identity to SEQ ID NO: 136. In embodiments, the B2M sequence has at least 99% identity to SEQ ID NO: 136. In embodiments, the B2M sequence includes SEQ ID NO: 136. In embodiments, the B2M sequence is SEQ ID NO: 136.

[0092] For the recombinant protein provided herein, in embodiments, the HLA-E heavy chain sequence includes a cysteine substitution mutation. In embodiments, the cysteine substitution mutation allows the HLA-E heavy chain sequence to form a disulfide bond with the HLA-E heavy chain binding peptide provided herein including embodiments thereof. In embodiments, the HLA-E heavy chain sequence includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. As described throughout the specification, the sequence of the HLA- E heavy chain to which position 77 and position 155 correspond is the sequence of SEQ ID NO: 146 or SEQ ID NO: 147.

[0093] In embodiments, the HLA-E heavy chain sequence has at least 90% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 91% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 92% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA- E heavy chain sequence has at least 93% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 94% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 95% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 96% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 97% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. Inembodiments, the HLA-E heavy chain sequence has at least 98% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 90% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes SEQ ID NO:130 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence is SEQ ID NO: 130 and includes a cysteine substitution in a position equivalent to position 77 of an HLA-E heavy chain.

[0094] ‘ ‘HLA-E heavy chain sequence” refers to a polypeptide derived from an HLA-E heavy chain protein. In embodiments, an HLA-E heavy chain sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HLA-E heavy chain protein. In embodiments, an HLA-E heavy chain sequence lacks the HLA-E tail (SEQ ID NO: 137) of an HLA-E heavy chain protein. In embodiments, the HLA-E heavy chain sequence includes one or more mutations that decreases binding affinity to a CD8 protein relative to an HLA-E heavy chain sequence lacking the mutations. In embodiments, the HLA-E heavy chain sequence has at least 90% identity to SEQ ID NO: 130 or SEQ ID NO: 131. In embodiments, the HLA-E heavy chain sequence has at least 90% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 91% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 92% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 93% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 94% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 95% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 96% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 97%sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 98% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 90% sequence identity to SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes SEQ ID NO: 130 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence is SEQ ID NO: 130 and includes a cysteine substitution in a position equivalent to position 155 of an HLA-E heavy chain.

[0095] In embodiments, the HLA-E heavy chain sequence has at least 90% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 91% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 92% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA- E heavy chain sequence has at least 93% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 94% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 95% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 96% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 97% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 98% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 99% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes SEQ ID NO : 131 and includes a cysteine substitution in a position equivalent toposition 77 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence is SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 77 of an HLA-E heavy chain.

[0096] In embodiments, the HLA-E heavy chain sequence has at least 90% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 91% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 92% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 93% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 94% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 95% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 96% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 97% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 98% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence has at least 99% sequence identity to SEQ ID NO: 131 and includes a cysteine in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes SEQ ID NO: 131 and includes a cysteine substitution in a position equivalent to position 155 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence is SEQ ID NO: 131 and includes a cysteine substitution in a position equivalent to position 155 of an HLA-E heavy chain.

[0097] For the recombinant protein provided herein, in embodiments, the HLA-E heavy chain binding peptide is an HLA-B leader peptide, an HLA-C leader peptide or an HLA-G leader peptide. In embodiments, the HLA-E heavy chain binding peptide is an HLA-B leader peptide. In embodiments, the HLA-E heavy chain binding peptide is an HLA-Cleader peptide. In embodiments, the HLA-E heavy chain binding peptide is an HLA-G leader peptide. In embodiments, the HLA-B leader peptide is the amino acid sequence of SEQ ID NO: 124. In embodiments, the HLA-C leader peptide is the amino acid sequence of SEQ ID NO: 125. In embodiments, the HLA-C leader peptide is the amino acid sequence of SEQ ID NO: 126. In embodiments, the HLA-E heavy chain binding peptide is the amino acid sequence of any one of SEQ ID NO: 151-208.

[0098] In embodiments, the HLA-E heavy chain sequence includes a cysteine in a position equivalent to position 155 of the HLA-E heavy chain and the HLA-E heavy chain binding peptide includes a cysteine at a position relative to position 9 from the N-terminus of any one of SEQ ID NO:124-126 or SEQ ID NO:151-208. In embodiments, the HLA-E heavy chain is covalently bound to the HLA-E heavy chain binding peptide though a disulfide linkage between the cysteine in a position equivalent to position 155 of said HLA-E heavy chain and said cysteine at a position relative to position 9 from the N- terminus of any one of SEQ ID NO: 124-126 or SEQ ID NO: 151-208.

[0099] In embodiments, the HLA-E heavy chain sequence includes a cysteine in a position equivalent to position 77 of the HLA-E heavy chain and the HLA-E heavy chain binding peptide includes a cysteine at a position relative to position 5 from the N-terminus of any one of SEQ ID NO:124-126 or SEQ ID NO:151-208. In embodiments, the HLA-E heavy chain is covalently bound to the HLA-E heavy chain binding peptide though a disulfide linkage between the cysteine in a position equivalent to position 77 of said HLA- E heavy chain and said cysteine at a position relative to position 5 from the N-terminus of any one of SEQ ID NO: 124-126 or SEQ ID NO: 151-208.

[0100] For the recombinant protein provided herein, in embodiments, the HLA-E heavy chain includes a mutation, wherein the mutation decreases the binding affinity of the recombinant protein to a CD 8 protein relative to a recombinant protein lacking said mutation. In embodiments, binding affinity is decreased by at least 2-fold, 3 -fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold. In embodiments, the mutation is a substitution mutation or a deletion mutation. Thus, in embodiments, the HLA-E heavy chain sequence includes a glutamine to alanine substitution at a position equivalent to position 226 of an HLA-E heavy chain, an aspartic acid to alanine substitution at a position equivalent to position 227 of said HLA-E heavy chain, or a combination thereof. In embodiments, the HLA-E heavy chain sequence includes a glutamine to alanine substitution at a position equivalent to position 226 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes anaspartic acid to alanine substitution at a position equivalent to position 227 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes a glutamine to alanine substitution and an aspartic acid to alanine substitution at positions equivalent to positions 226 and 227 of said HLA-E heavy chain.

[0101] For the recombinant protein provided herein, in embodiments, the HLA-E heavy chain sequence includes a mutation at the C-terminus. In embodiments, the mutation is a substitution or a deletion of amino acid residues.

[0102] In embodiments, the HLA-E heavy chain sequence includes a mutation at a position equivalent to position 310 or position 311 of an HLA-E heavy chain. In embodiments, the HLA-E heavy chain sequence includes mutations at positions equivalent to position 310 and position 311 of an HLA-E heavy chain. In embodiments, the mutation is an alanine substitution. In embodiments, the HLA-E heavy chain sequence is a truncated an HLA-E heavy chain lacking positions equivalent to positions 315-337 of an HLA-E heavy chain.

[0103] For the recombinant protein provided herein, in embodiments, the C-terminus of the HLA-E heavy chain binding peptide is covalently attached to the N-terminus of the B2M sequence by a first peptide linker, and the C-terminus of the B2M sequence is covalently attached to the N-terminus of the HLA-E heavy chain sequence by a second peptide linker. The first and second peptide linkers may be rich in glycine for flexibility, as well as serine or threonine for solubility. In embodiments, the first and second peptide linkers independently are a structured linker. For example, in embodiments, a structured linker may include amino acid residues other than glycine, serine, and threonine. In embodiments, a structured linker may adopt a secondary structure. In embodiments a recombinant protein having a structured linker has increased stability relative to a recombinant protein lacking the structured linker. In embodiments, the Tm of a recombinant protein 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, relative to a recombinant protein lacking the structured linker. In embodiments, the structured linker includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO: 122, 123, 209 or 210.

[0104] In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 8 amino acids to about 60 amino acids in length. In embodiments, first peptidelinker and said second peptide linker are independently from about 12 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 16 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 20 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 24 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 28 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 32 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 36 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 40 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 44 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 48 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 52 amino acids to about 60 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 56 amino acids to about 60 amino acids in length.

[0105] In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 56 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 52 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 48 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 44 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 40 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 36 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 32 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently fromabout 4 amino acids to about 28 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 24 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 20 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 16 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 12 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4 amino acids to about 8 amino acids in length. In embodiments, first peptide linker and said second peptide linker are independently from about 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, or 60 amino acids amino acids in length.

[0106] In embodiments, the first peptide linker and said second peptide linker independently include any one of SEQ ID NO: 138-144 or SEQ ID NO: 122, 123, 209, or 210. In embodiments, the first peptide linker includes the sequence of any one of SEQ ID NO: 138- 144 or SEQ ID NO: 122, 123, 209, or 210. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 138. In embodiments, the first peptide linker includes the sequence of SEQ ID NO:139. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 140. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 141. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 142. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 143. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 144. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 122. In embodiments, the first peptide linker includes the sequence of SEQ ID NO: 123. In embodiments, the first peptide linker includes the sequence of SEQ ID NO:209. In embodiments, the first peptide linker includes the sequence of SEQ ID NO:210.

[0107] In embodiments, the second peptide linker and said second peptide linker independently include any one of SEQ ID NO: 138-144 or SEQ ID NO: 122, 123, 209, or 210. In embodiments, the second peptide linker includes the sequence of any one of SEQ ID NO: 138-144 or SEQ ID NO: 122, 123, 209, or 210. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 138. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 139. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 140. In embodiments, the second peptidelinker includes the sequence of SEQ ID NO: 141. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 142. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 143. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 144. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 122. In embodiments, the second peptide linker includes the sequence of SEQ ID NO: 123. In embodiments, the second peptide linker includes the sequence of SEQ ID NO:209. In embodiments, the second peptide linker includes the sequence of SEQ ID NO:210.

[0108] For the recombinant protein provided herein, in embodiments, the recombinant protein further includes a B2M signal peptide covalently attached to the N-terminus of said HLA-E heavy chain binding peptide. In embodiments, the B2M signal peptide includes a sequence having at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 135. In embodiments, the B2M signal peptide includes SEQ ID NO:135. In embodiments, the B2M signal peptide is SEQ ID NO:135.

[0109] In embodiments, the recombinant protein includes a sequence having at least 90% identity to any one of SEQ ID NO: 1-116. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 1-116. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:1. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:4. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:5. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:6. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8. In embodiments, the recombinant proteinincludes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:9. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 19. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:20. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:21. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:22. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:23. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:24. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:25. In embodiments, the recombinant proteinincludes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:26. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:27. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:28. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:29. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:30. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:31. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:32. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:33. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:34. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:35. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:36. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:37. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:38. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:39. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:40. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:41. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:42. In embodiments, the recombinant proteinincludes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:43. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:44. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:45. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:46. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:47. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:48. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:49. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:50. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:51. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:52. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:53. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:54. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:55. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:56. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:57. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:58. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:59. In embodiments, the recombinant proteinincludes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:60. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:61. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:62. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:63. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:64. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:65. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:66. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:67. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:68. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:69. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:70. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:71. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:72. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:73. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:74. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:75. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:76. In embodiments, the recombinant proteinincludes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:77. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:78. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:79. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 80. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 81. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 82. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 83. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 84. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:85. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:86. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 87. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:88. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 89. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:90. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:91. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:92. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:93. In embodiments, the recombinant proteinincludes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:94. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:95. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:96. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:97. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:98. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:99. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 100. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 101. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 102. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 103. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 104. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 105. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 106. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 107. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 108. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 109. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 110. In embodiments, the recombinant proteinincludes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 111. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 112. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 113. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 114. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 115. In embodiments, the recombinant protein includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 116.

[0110] In embodiments, the recombinant protein includes a sequence having at least 95% identity to any one of SEQ ID NO: 1-14. In embodiments, the recombinant protein includes a sequence having at least 98% identity to any one of SEQ ID NO: 1-14. In embodiments, the recombinant protein includes a sequence having at least 99% identity to any one of SEQ ID NO: 1-14. In embodiments, the recombinant protein includes any one of SEQ ID NO: 1-14. In embodiments, the recombinant protein is any one of SEQ ID NO: 1-14.

[0111] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO: 1. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 1. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO: 1. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO: 1. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 1. In embodiments, the recombinant protein includes includes SEQ ID NO:1. In embodiments, the recombinant protein is SEQ ID NO: 1.

[0112] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:2. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO:2. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO:2. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:2. In embodiments, the recombinant protein includes a sequence having atleast 99% identity to SEQ ID NO:2. In embodiments, the recombinant protein includes SEQ ID NO:2. In embodiments, the recombinant protein is SEQ ID NO:2.

[0113] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:3. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 3. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO:3. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:3. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 3. In embodiments, the recombinant protein includes SEQ ID NO:3. In embodiments, the recombinant protein is SEQ ID NO:3.

[0114] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:4. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO:4. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO:4. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:4. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO:4. In embodiments, the recombinant protein includes SEQ ID NO:4. In embodiments, the recombinant protein is SEQ ID NO:4.In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:5. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 5. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO: 5. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:5. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO:5. In embodiments, the recombinant protein includes SEQ ID NO:5. In embodiments, the recombinant protein includes is SEQ ID NO:5.

[0115] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:6. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO:6. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO:6. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:6. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO:6. In embodiments, the recombinant protein includes SEQ ID NO:6. In embodiments, the recombinant protein is SEQ ID NO:6.

[0116] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:7. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO:7. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO:7. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:7. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO:7. In embodiments, the recombinant protein includes SEQ ID NO:7. In embodiments, the recombinant protein is SEQ ID NO:7.

[0117] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:8. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 8. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO:8. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO: 8. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 8. In embodiments, the recombinant protein includes SEQ ID NO:8. In embodiments, the recombinant protein is SEQ ID NO: 8.

[0118] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO:9. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO:9. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO:9. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:9. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO:9. In embodiments, the recombinant protein includes SEQ ID NO:9. In embodiments, the recombinant protein is SEQ ID NO:9.

[0119] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO: 10. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 10. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO: 10. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:10. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 10. In embodiments, the recombinant protein includes SEQ ID NO: 10. In embodiments, the recombinant protein is SEQ ID NO: 10.

[0120] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO: 11. In embodiments, the recombinant protein includes asequence having at least 96% identity to SEQ ID NO: 11. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO: 11. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO:11. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 11. In embodiments, the recombinant protein includes SEQ ID NO: 11. In embodiments, the recombinant protein is SEQ ID NO: 11.

[0121] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO: 12. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 12. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO: 12. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO: 12. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 12. In embodiments, the recombinant protein includes SEQ ID NO: 12. In embodiments, the recombinant protein is SEQ ID NO: 12.

[0122] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO: 13. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 13. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO: 13. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO: 13. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 13. In embodiments, the recombinant protein includes SEQ ID NO:13. In embodiments, the recombinant protein is SEQ ID NO: 13.

[0123] In embodiments, the recombinant protein includes a sequence having at least 95% identity to SEQ ID NO: 14. In embodiments, the recombinant protein includes a sequence having at least 96% identity to SEQ ID NO: 14. In embodiments, the recombinant protein includes a sequence having at least 97% identity to SEQ ID NO: 14. In embodiments, the recombinant protein includes a sequence having at least 98% identity to SEQ ID NO: 14. In embodiments, the recombinant protein includes a sequence having at least 99% identity to SEQ ID NO: 14. In embodiments, the recombinant protein includes SEQ ID NO: 14. In embodiments, the recombinant protein is SEQ ID NO: 14.

[0124] In some embodiments, the construct comprises, in N-terminus to C-terminus order, a segment comprising a peptide and a segment comprising the one or more major histocompatibility complex (MHC) regions. In some embodiments, the construct comprises, in N-terminus to C-terminus order, a segment comprising a peptide, a segmentcomprising a B2M sequence, and one or more synHLA polypeptides. In embodiments, the terms “construct” and “recombinant protein” are used interchangeably.

[0125] In some embodiments, the one or more synHLA polypeptides comprises one or more mutated HLA class I heavy chain sequences. In some embodiments, the one or more HLA class I heavy chain sequences comprises an HLA-E or a fragment thereof, HLA-G or a fragment thereof, or any combination thereof. Either the HLA-E or the HLA-G or both may be mutated in comparison to a respective reference HLA-E or HLA-G sequence. In some embodiments, the HLA class I heavy chain sequence comprises HLA-E or a fragment thereof and HLA-G or a fragment thereof. In some embodiments, the present constructs comprising a synHLA polypeptide may be used in combination with one or more HLA class I heavy chain sequences obtained from other class I MHCs, such as HLA-A, HLA-B, HLA-C, HLA-E, HLA-E, or HLA-G. These additional HLA regions may be an unmodified HLA class I heavy chain sequence or may be a modified HLA class I heavy chain sequence.

[0126] In some embodiments, at least one of the HLA-E or the fragment thereof, or HLA-G or the fragment thereof, or any combination thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, HLA-E or the fragment thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, HLA-G or the fragment thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, HLA-E or the fragment thereof and HLA-G or the fragment thereof are inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, HLA-E or the fragment thereof and HLA-G or the fragment thereof are inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells.

[0127] In some embodiments, the one or more major histocompatibility construct (MHC) regions comprise an HLA-E region. In some embodiments, the HLA-E region is an HLA-E*01 :01 region. In some embodiments, the HLA-E region is an HLA-E*01:03 region.

[0128] In some embodiments the HLA-E region comprises one or more modifications, substitutions, or mutations. In some embodiments, the one or more mutations comprise a substitution at Serine in position 42 of HLA-E. In some embodiments, the one or more mutations comprise a substitution at the Serine in position 147. In some embodiments, theone or more mutations comprise substitutions at S42 and S147. In some embodiments, the mutations comprise S42C, S147C, or serine to cysteine mutations at both positions. In some embodiments, one or more of these serine to cysteine mutations allow the constructs of the present invention to dimerize or trimerize. In some embodiments the dimers may be homodimers of two synHLA constructs described herein. In some embodiments the dimer or trimer may be a dimer or trimer comprised of a mixture of HLA constructs described herein and wild-type HLAs, such as HLA-G. In some embodiments the dimer or trimer may be a heterodimer or heterotrimer having a mixture comprising at least one HLA (e.g., a modified HLA-E) as described herein and one or more other HLAs, such as HLA-G, or another class I HLA. In some embodiments, the dimers and trimers comprise single chain HLA constructs of the invention. In some embodiments, the HLA-E based construct may be membrane bound. In some embodiments the HLA-E based construct may be secreted (soluble).

[0129] In some embodiments, the synHLA construct further comprises one or more CD8 binding site. The CD8 binding site may comprise a mutation at any residue to any other amino acid or a deletion at any residue. In some embodiments, the CD8 binding site comprises one or more mutations. In some embodiments, the mutations in the CD8 binding sites significantly reduce CD8 binding to the presently claimed constructs.

[0130] In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the Q226 residue of an HLA-E heavy chain. In some embodiments, the CD8 binding site comprises a mutation to the residue corresponding to the D227 residue of an HLA-E heavy chain. In some embodiments the mutation is Q226A. In some embodiments the mutation is D227K. In some embodiments the amino acids at both Q226 and D227 are mutated. In some embodiments the CD8 binding site comprises one or more of the following mutations: a mutation at Q226 such as Q226A, and a mutation at D227 such as D227K.

[0131] In some embodiments, the one or more MHC regions comprise a truncated HLA heavy chain sequence. In some embodiments, the truncated HLA heavy chain sequence further comprises one or more C-terminus modifications. In some embodiments, the truncated HLA heavy chain sequence further comprises C-terminus modification. In some embodiments, the truncated HLA-E comprises removal of two amino acids from the C- terminus. In some embodiments, the truncated HLA-E comprises removal of at least one and up to 5 amino acids in from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of at least one and up to 10 amino acids from the C-terminus.In some embodiments, the truncated HLA-E comprises removal of 10-15 amino acids in positions from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 10-16 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 17 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 18 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 19 amino acids from the C- terminus. In some embodiments, the truncated HLA-E comprises removal of 20 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 21 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 22 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 23 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 24 amino acids from the C- terminus. In some embodiments, the truncated HLA-E comprises removal of 25 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 26 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 27 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 28 amino acids from the C-terminus. In some embodiments, the truncated HLA-E comprises removal of 29 amino acids from the C- terminus.

[0132] In some embodiments, the one or more C-terminus modifications comprise substituting one or more lysines with one or more other amino acid residues at the C- terminus of either the full length or truncated HLA-E. In some embodiments, the one or more C-terminus modifications comprise substituting one of the lysines in the final five amino acids with another amino acid. In some embodiments, the one or more C-terminus modifications comprise substituting two of the lysines in the final five amino acids with another amino acid. In some embodiments, the one or more C-terminus modifications comprise substituting three of the final five amino acids with another amino acid.

[0133] In some embodiments, the one or more MHC regions comprise a truncated HLA- E. In some embodiments, truncated HLA-E comprises removal amino acids in positions equivalent to positions 315-337 of an HLA-E heavy chain. In some embodiments, the truncated HLA-E comprises removal of all amino acids C-terminal of the amino acid equivalent to position 314 of an HLA-E heavy chain. One or more amino acids may be removed, including all amino acids C-terminal of one of the following: amino acid 315, amino acid 316, amino acid 317, amino acid 318, amino acid 319, amino acid 320, aminoacid 321, amino acid 322, amino acid 323, amino acid 324, amino acid 325, amino acid 326, amino acid 327, amino acid 328, amino acid 329, amino acid 330, amino acid 331, amino acid 332, amino acid 333, amino acid 334, amino acid 335, amino acid 336, and amino acid 337 equivalent to positions of an HLA-E heavy chain.

[0134] In some embodiments, the truncated HLA-E heavy chain or full-length HLA-G heavy chain comprises a C-terminus modification. In some embodiments, the C-terminus modification comprises a mutation of the K310 residue. In some embodiments, the C- terminus modification comprises a mutation of the K311 residue. In some embodiments, the C-terminus modification comprises mutations of the K310 and K311 residues. In some embodiments, the C-terminus modification comprises a K310A substitution. In some embodiments, the C-terminus modification comprises a K311 A substitution. In some embodiments, the C-terminus modification comprises K310A and K311 A substitutions. In some embodiments, the C-terminus modification comprises a mutation of K311 residue and a K310A substitution. In some embodiments, the C-terminus modification comprises a substitution of K310 residue and a mutation K311 A.

[0135] In some embodiments, the one or more MHC regions comprise an HLA-G region. In some embodiments, the HLA-G region comprises an HLA-G*01 :01 sequence. In some embodiments, the HLA-G region comprises an HLA-G*01:04 sequence. In some embodiments, the HLA-G region comprises one or more mutations. In some embodiments, the HLA-G based construct may be membrane bound. In some embodiments the HLA-G based construct may be secreted (soluble).

[0136] In some embodiments the HLA-G region comprises one or more modifications, substitutions, or mutations. In some embodiments, the one or more mutations comprise a substitution in position 42 of HLA-G to serine. In some embodiments, the one or more mutations comprise a substitution in position 147 to Serine. Lor example, in embodiments, the one or more mutations include substitution of a cysteine residue to a serine residue. In some embodiments, the one or more mutations comprise substitutions at C42S or C147S. In some embodiments, one or more of these cysteine to serine mutations prevent the constructs of the present invention to dimerize or trimerize. In some embodiments the dimers may be homodimers of two HLA constructs described herein. In some embodiments the dimers may be a dimer or trimer comprised of a mixture of HLA constructs described herein and wild-type HLAs, such as HLA-E. In some embodiments the dimer or trimer may be a heterodimer or heterotrimer having a mixture comprising at least one HLA as described herein and one or more other HLAs, such as HLA-G, oranother class I HLA. In some embodiments, the dimers and trimers comprise single chain HLA constructs of the invention.

[0137] In some embodiments, the one or more HLAs are inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the one or more HLAs are inhibited from eliciting an 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., peptides); one or more linker regions; one or more major histocompatibility complex (MHC) regions (such as an HLA class I heavy chain sequence), wherein at least one of the one or more MHC regions comprises a cluster of differentiation 8 (CD8) binding site and wherein the CD8 binding site comprises one or more mutations to reduce CD8 binding; and one or more disulfide staple pairs.

[0138] 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 sequence is derived from (or based upon) the wild-type sequence of HLA- A, HLA-B, HLA-C, HLA- E, HLA-F, HLA-G, or any combination thereof. In some cases, the human HLA class I heavy chain sequence 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 NO:130-134 or SEQ ID: 146-148.

[0139] In some embodiments, the 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 a sequence listed in Table 1 or Table 2.HLA LEADER SEQUENCES

[0140] In a wild-type HLA, HLA-E recognizes a 9-mer HLA-E heavy chain binding peptide processed from leader sequences of class I molecules HLA-A, -B, and -G. A wild-type HLA-E + peptide complex binding to CD94 / NKG2A would inhibit NK cells. In one aspect, the recombinant protein (e.g. construct) of the present invention encodes a peptide sequence (e.g. an HLA heavy chain binding sequence) covalently bound to a sequence encoding the HLA based heavy chain. Alternatively, the HLA-E heavy chain binding peptide may be introduced to cells expressing the HLA based heavy chain of thepresent invention. Further provided herein, in one aspect, are constructs that comprise a peptide (e.g. HLA-E heavy chain binding peptide). In some embodiments, the peptide (e.g. HLA-E heavy chain binding peptide) is configured to weaken or inhibit HLA activity. In some embodiments, the weakened or inhibited HLA activity may be hypoimmunogenic or immune -protective. In some embodiments, the peptide (e.g. HLA-E heavy chain binding peptide) is configured to weaken or inhibit T cell activity. In some embodiments, the peptide (e.g. HLA-E heavy chain binding peptide) is configured to weaken or inhibit NK cell activity. In some embodiments, the peptide (e.g. HLA-E heavy chain binding peptide) is configured to inhibit NK cell lysis.

[0141] In some embodiments, the peptide does not elicit an immune cell response when the peptide (or a complex comprising the peptide) is interrogated by one or more immune cells. In some embodiments, the peptide is incapable of activating the one or more T cells. In some embodiments, the peptide is capable of binding to a receptor of the one or more T cells, and wherein the binding is insufficient to activate the 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 incapable of activating the one or more NK cells. In some embodiments, the peptide is capable of binding to a receptor of the one or more NK cells, and wherein the binding is insufficient to activate the one or more NK cells. In some embodiments, the peptide is capable of preferentially binding to a receptor of the one or more NK cells, and wherein the binding is sufficient modulate the activity the one or more NK cells. In embodiments, the peptide is an HLA-E heavy chain binding peptide.

[0142] In some embodiments, a peptide is configured to covalently bind to HLA. In some embodiments, the peptide is configured to bind to the N-terminus of the heavy chain of HLA class I. In some embodiments, the peptide is configured to be specific for the MHC binding groove but does not comprise the correct TCR-facing / solvent exposed amino acids required for recognition by the T-cell receptor (TCR). The presence of specific “anchor residues” in the MHC binding groove of HLA act to anchor the bound peptide and stabilize the protein complex. In embodiments, the peptide is an HLA-E heavy chain binding peptide.

[0143] The peptides of the current HLA based structures may differ from a wild-type peptide. In some embodiments, residues in the peptide can be altered in order to configure the peptide so to alter the peptide -bound MHC complex’s (pMHC’s) (e.g., a peptide bound to a human HLA class I heavy chain) ability to signal through a NKG2C / CD94 receptorcomplex vs a NKG2A / CD94 receptor complex and thus inhibit NK cell lysis rather than activating NK cell lysis. In addition, use of a human-derived peptide may also decrease the immunogenicity of cells expressing the pMHCs on their surface. In embodiments, the peptide is an HLA-E heavy chain binding peptide.

[0144] In some embodiments, the peptide binds to one or more HLA binding groove domain residues of the human HLA class I heavy chain sequence. In some embodiments, the peptide modulates a conformation of the human HLA class I heavy chain sequence. In some embodiments, the conformation prevents the immune cell from binding to the human HLA class I heavy chain sequence. In embodiments, the peptide is an HLA-E heavy chain binding peptide.

[0145] In some embodiments, the sequence of the bound peptide can affect the intrinsic flexibility of the pMHC. In some embodiments, the conformational flexibility of the pMHC facilitates TCR interaction. In some embodiments, the conformational flexibility of the pMHC facilitates NK cell receptor interaction. In some embodiments, the peptide configured to bind to HLA is further configured to increase the conformational variability of pMHC and to prevent TCR engagement. In some embodiments, the peptide configured to bind to HLA is further configured to increase the conformational variability of pMHC and to selectively modify NK cell receptor engagement. In embodiments, the peptide is an HLA-E heavy chain binding peptide.

[0146] In some embodiments, the peptide is about 8 amino acids in length to about 15 amino acids in length. In some embodiments, the peptide is about 8 amino acids in length to about 9 amino acids in length, about 8 amino acids in length to about 10 amino acids in length, about 8 amino acids in length to about 11 amino acids in length, about 8 amino acids in length to about 12 amino acids in length, about 8 amino acids in length to about 13 amino acids in length, about 8 amino acids in length to about 14 amino acids in length, about 8 amino acids in length to about 15 amino acids in length, about 9 amino acids in length to about 10 amino acids in length, about 9 amino acids in length to about 11 amino acids in length, about 9 amino acids in length to about 12 amino acids in length, about 9 amino acids in length to about 13 amino acids in length, about 9 amino acids in length to about 14 amino acids in length, about 9 amino acids in length to about 15 amino acids in length, about 10 amino acids in length to about 11 amino acids in length, about 10 amino acids in length to about 12 amino acids in length, about 10 amino acids in length to about 13 amino acids in length, about 10 amino acids in length to about 14 amino acids in length, about 10 amino acids in length to about 15 amino acids in length, about 11 aminoacids in length to about 12 amino acids in length, about 11 amino acids in length to about13 amino acids in length, about 11 amino acids in length to about 14 amino acids in length, about 11 amino acids in length to about 15 amino acids in length, about 12 amino acids in length to about 13 amino acids in length, about 12 amino acids in length to about14 amino acids in length, about 12 amino acids in length to about 15 amino acids in length, about 13 amino acids in length to about 14 amino acids in length, about 13 amino acids in length to about 15 amino acids in length, or about 14 amino acids in length 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 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, or about 14 amino acids in length. In some embodiments, the peptide is at most 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 comprises greater than 15 amino acids.

[0147] In some embodiments, the HLA-E heavy chain binding peptide is covalently attached to the HLA-E heavy chain sequence (e.g. construct) by a disulfide bond. In embodiments, the disulfide bond is referred to herein as a “cysteine staple” or “disulfide staple”. In embodiments, the disulfide staple is a covalent bond between a cysteine residue within the HLA-E heavy chain sequence and a cysteine residue within the HLA-heavy chain binding peptide. In embodiments, the disulfide staple is a covalent bond between a cysteine residue within the HLA-E heavy chain sequence and a cysteine residue within the first peptide linker. In embodiments, the disulfide staple is a covalent bond between a cysteine residue within the HLA-E heavy chain sequence and a cysteine residue within the second peptide linker. In embodiments, the disulfide staple is a covalent bond between a cysteine residue within the HLA-E heavy chain binding peptide and a cysteine residue within the first peptide linker. In embodiments, the disulfide staple is a covalent bond between a cysteine residue within the HLA-E heavy chain binding peptide and a cysteine residue within the second peptide linker.

[0148] In some embodiments, the peptide comprising a sequence at least about 50%, about 52%, about 55%, about 57%, about 60%, about 61%, about 70%, about 71%, about76%, about 77%, 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 4.

[0149] In some embodiments, the peptide is derived from an HLA-G leader sequence. In some embodiment, the peptide is derived from an HLA-Cw*03:02 leader sequence. In some embodiment, the peptide is derived from an HLA-B*0702 leader sequence.

[0150] In some embodiments, the peptide may be derived from any protein in the human proteome. In some embodiments, the peptide may be identified as having or constructed to have certain properties, for example peptide size, expected binding strength to MHC class I molecules, or the presence of cysteines in specific locations of the peptide.PEPTIDE LINKER AND B2M SEQUENCES

[0151] In some embodiments, the construct comprises one or more linkers between the targeting moiety (e.g., peptide, HLA-E heavy chain binding peptide) and an MHC region (e.g., a sequence derived from a human HLA class I heavy chain sequence, an HLA-E 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 an amino acid sequence configured to not block one or more killer-cell immunoglobulin-like receptor (KIR) binding sites on the human HLA class I heavy chain sequence. In some embodiments, the one or more linkers comprise a conformation configured to not block one or more NKG2A / CD94 or NKG2C / CD94 binding sites on the human HLA class I heavy chain sequence. In some embodiments, the one or more linkers are structurally stable. In some embodiments, one or more of the one or more linkers possess limited flexibility. In some embodiments, the structural stability and limited flexibility of the one or more linkers increase resistance to proteolytic degradation. In some embodiments, the one or more linkers comprise a cysteine which is part of a disulfide staple pair as described herein. In embodiments, the one or more linkers include a first peptide linker and a second peptide linker. In embodiments, the one or more linkers is a first peptide linker and a second peptide linker.

[0152] In some embodiments, a linker (e.g. first peptide linker, second peptide linker) of the one or more linkers is disposed between the targeting moiety (e.g., peptide) and a sequence derived from the human beta-2 microglobulin (B2M) sequence, between the human B2M sequence and the human HLA class I heavy chain sequence, or both. In some embodiments, a linker of the one or more linkers is disposed between the peptide and thehuman 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 I 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 I heavy chain sequence. In some embodiments, the second linker comprises a conformation configured to resist proteolytic cleavage.

[0153] A linker (e.g. first peptide linker, second peptide linker) may impart a certain secondary, tertiary, or quaternary structure when the construct sequence is arranged in three-dimensional space, such as when expressed in a host cell. The secondary, tertiary, or quaternary structure may be determined from experimental structural biology data (e.g., X-ray crystallographic data, cryogenic electron microscopy data, nuclear magnetic resonance data), biochemical data (e.g., mass spectrometry data, chromatographic data, electrophoretic data), or computer simulation or modeling data (e.g., molecular dynamics simulations, de novo or ab initio prediction, homology modeling, fragment assembly, secondary structure prediction). Alternatively, or additionally, the linker may be configured to impart a certain functional consequence on the expressed construct. In some embodiments, the linker enhances expression (e.g., cell surface expression) of the construct, enhance stability of the construct, prevent exchange of the peptide, or some combination thereof. In some embodiments, the linker enhances expression of the construct.

[0154] In some embodiments, the linker (e.g. first peptide linker, second peptide linker) may increase expression in a host cell (e.g., relative to a wild-type or other construct lacking the linker). In some embodiments, the expression is cell-surface expression. The expression may be measured by, for example, flow cytometry, fluorescence microscopy, mass spectrometry, or any other suitable quantification method. In some embodiments, the presence of a linker increases expression of the construct in a host cell relative 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 increased by about 1% to about 10%, about 1% to about 30%, about 1% to about 50%, about 1% to about 70%, about 1% to about 90%, about 1% to about 100%, at least about 5% at least about 10% at least about 20% at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%. In some embodiments, expression is increased by at most about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0155] In some embodiments, the linker (e.g. first peptide linker, second peptide linker) may increase stability of the construct (e.g., as compared to a construct not comprising the linker, such as a wild-type construct). The presence of the linker may increase stability by restricting the conformational flexibility of a construct to help the construct fold into and retain a particular secondary, tertiary, and / or quaternary structure. The stability of a construct may be measured by differential scanning calorimetry (DSC), pulse-chase assays (such as 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 a construct is expressed in relative terms (e.g., percent or fold change in stability with respect to a reference, such as a wild-type construct). In some embodiments, the stability of a construct is expressed in absolute terms (e.g., in terms of thermodynamic coordinates such as melting or other phase transition temperatures or a free energy of folding).

[0156] In some embodiments, the presence of the one or more linkers (e.g. first peptide linker, second peptide linker) increases the stability of a construct 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, relative to a reference (e.g., construct not comprising the linker, such as a wild-type construct). In some embodiments, stability is increased by about 1 % to about 100%. In some embodiments, stability is increased by at least aboutlO, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%.

[0157] In some embodiments, the presence of a linker (e.g. first peptide linker, second peptide linker) increases the stability of a construct by about 2-fold, about 3 -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, relative to a refence (e.g., construct not comprising the linker, such as a wild-type construct). In some embodiments, stability is increased by about 1-fold to about 1,000-fold. In some embodiments, stability is increased by about 1-fold to about 2-fold, about 1-fold to about 3-fold, about 1-fold toabout 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, 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, 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, stability is increased by at most about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000-fold.

[0158] In some embodiments, a linker (e.g. first peptide linker, second peptide linker) of the one or more linkers 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 NO:138-144 or SEQ ID NO:122, 123, 209, or 210.DISULFIDE STAPLE PAIRS

[0159] In some embodiments, the recombinant protein (e.g. construct) provided herein may comprise one or more disulfide staple pairs. A disulfide staple pair may comprise two cysteine residues that are configured to form a disulfide bond under suitable conditions (e.g., oxidizing conditions, such as in a cellular compartment). The two cysteine residues may be located at any suitable position on the construct (e.g. recombinant protein). In some embodiments, the disulfide staple pair is distributed across two parts of the construct, such as across the peptide (e.g. HLA-E heavy chain binding peptide) and the MHC region (e.g., HLA class I heavy chain, HLA-E heavy chain sequence, HLA-G heavy chain sequence) or across the peptide and a linker, or across the peptide and the HLA class I heavy chain. In some embodiments, the disulfide staple pair is wholly contained within one region of the construct, such as wholly within a linker, wholly within the HLA class I heavy chain, or wholly within the peptide. In some embodiments, the one or moredisulfide staple pairs are introduced to the construct by engineering (e.g., mutated, such as by site-directed mutagenesis). Alternatively, or additionally, the one or more disulfide staple pairs are wholly or partially present in a wild-type or unmutated sequence.

[0160] In embodiments, the HLA-E heavy chain sequence is covalently bound to the HLA-E heavy chain binding peptide though a disulfide linkage between said cysteine in a position equivalent to position 155 of a HLA-E heavy chain and a cysteine at a position relative to position 9 from the N-terminus of any one of SEQ ID NO:124-126 or SEQ ID NO: 151 -208. Thus, in this instance, the disulfide staple is formed between a cysteine in a position equivalent to position 155 of a HLA-E heavy chain and a cysteine at a position relative to position 9 from the N-terminus of any one of SEQ ID NO: 124- 126 or SEQ ID NO: 151-208.

[0161] In embodiments, the HLA-E heavy chain sequence is covalently bound to the HLA-E heavy chain binding peptide though a disulfide linkage between a cysteine in a position equivalent to position 77 of a HLA-E heavy chain and a cysteine at a position relative to position 5 of any one of SEQ ID NO: 124-126 or SEQ ID NO: 151-208. Thus, in this instance, the disulfide staple is formed between a cysteine in a position equivalent to position 77 of said HLA-E heavy chain and a cysteine at a position relative to position 5 from the N-terminus of any one of SEQ ID NO: 124- 126 or SEQ ID NO: 151-208.

[0162] The disulfide staple pairs may impart a certain secondary, tertiary, or quaternary structure when the construct sequence is arranged in three-dimensional space, such as when expressed in a host cell. The secondary, tertiary, or quaternary structure may be determined from experimental structural biology data (e.g., X-ray crystallographic data, cryogenic electron microscopy data, nuclear magnetic resonance data), biochemical data (e.g., mass spectrometry data, chromatographic data, electrophoretic data), or computer simulation or modeling data (e.g., molecular dynamics simulations, de novo or ab initio prediction, homology modeling, fragment assembly, secondary structure prediction). Alternatively, or additionally, the one or more disulfide staple pairs may be configured to impart a certain functional consequence on the expressed construct. In some embodiments, the one or more disulfide staple pairs enhances expression (e.g., cell surface expression) of the contrast, enhance stability of the construct, prevent exchange of the peptide, or some combination thereof. In some embodiments, the one or more disulfide staple pairs enhance expression of the construct. In some embodiments, the one or more disulfide staple pairs enhance stability of the construct. In some embodiments, the one or more disulfide staple pairs prevent exchange of the peptide.

[0163] In some embodiments, the disulfide staple pair may increase expression in a host cell (e.g., relative to a wild-type or other construct lacking the disulfide staple pair). In some embodiments, the expression is cell-surface expression. The expression may be measured by, for example, flow cytometry, fluorescence microscopy, mass spectrometry, or any other suitable quantification method. In some embodiments, the presence of a disulfide staple pair increases expression of the construct in a host cell relative to a reference or control (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.

[0164] In some embodiments, the disulfide staple pair may be configured to increase stability of the construct (e.g., as compared to a construct not comprising the disulfide staple pair, such as a wild-type construct). The presence of the disulfide staple pair may increase stability by allowing for the formation of an additional disulfide bond to help the construct retain a particular secondary, tertiary, and / or quaternary structure even in the presence of forces or conditions (e.g., chaotropic agents, increased temperature) that tend to unfold proteins. The stability of a construct may be measured by differential scanning calorimetry (DSC), pulse-chase assays (such as 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 a construct is expressed in relative terms (e.g., percent or fold change in stability with respect to a reference, such as a wild-type construct). In some embodiments, the stability of a construct is expressed in absolute terms (e.g., in terms of thermodynamic coordinates such as melting or other phase transition temperatures or a free energy of folding).

[0165] In some embodiments, the presence of a disulfide staple pair increases the stability of a construct 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, relative to a reference (e.g., construct not comprising the disulfide pair, such as a wild-type construct).

[0166] In some embodiments, the presence of a disulfide staple pair increases the stability of a construct by about 2-fold, about 3 -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, or more, relative to a refence (e.g., construct not comprising the disulfide staple pair, such as a wild-type construct). In some embodiments, stability is increased byabout 1-fold to about 1,000-fold. In some embodiments, stability is increased by 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, about2-fold to about 1,000-fold, about 3-fold to about 4-fold, about 3-fold to about 5-fold, about3 -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, 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, 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, stability is increased by at most about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 100-fold, or about 1,000- fold.

[0167] In some embodiments, a construct as described herein comprising a disulfide staple pair may show a higher melting temperature (Tm) (e.g., as determined by a thermodynamic technique, such as DSC) relative to a reference (e.g., a construct that does not comprise the disulfide staple pair, such as a wild-type construct). In some embodiments, the Tm 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, relative to the reference. In some embodiments, the Tm is increased by about 1 °C to about 30 °C.

[0168] In some embodiments, the construct may not comprise one or more disulfide staple pairs. In some embodiments, the construct may be engineered (e.g., mutated, such as by site-directed mutagenesis) to ablate one or more disulfide staple pairs. Such ablation may be performed to reduce the likelihood of alternative or undesired arrangements of construct component from forming under certain conditions (e.g., when expressed in a host cell).

[0169] In some aspects, the construct further comprises one or more disulfide staple pairs formed between one or more MHC regions (e.g. HLA-E heavy chain sequence) and a peptide (e.g. HLA-E heavy chain binding peptide). In some embodiments, the one ormore disulfide staple pairs form between a residue of the one or more MHC regions corresponding to the N77 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide). In some embodiments, the one or more disulfide staple pairs form between a residue of the one or more MHC regions corresponding to the Hl 55 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide). In some embodiments, the one or more disulfide staple pairs form between one or more MHC regions (e.g. HLA-E heavy chain sequence) and a peptide (e.g. HLA-E heavy chain binding peptide) and between a residue of the one or more MHC regions (e.g. HLA-E heavy chain sequence) corresponding to the N77 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide). In some embodiments, the one or more disulfide staple pairs form between one or more MHC regions and a peptide (e.g. HLA-E heavy chain binding peptide) and between a residue of the one or more MHC regions corresponding to the H155 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide). In some embodiments, the one or more disulfide staple pairs form between a residue of the one or more MHC regions corresponding to the N77 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide) and between a residue of the one or more MHC regions corresponding to the H155 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide). In some embodiments, the one or more disulfide staple pairs form between one or more MHC regions and a peptide, between a residue of the one or more MHC regions corresponding to the N77 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide), and between a residue of the one or more MHC regions corresponding to the Hl 55 residue of an HLA-E heavy chain and a residue in the peptide (e.g. HLA-E heavy chain binding peptide).N- AND C-TERMINAL MODIFICATIONS

[0170] In some embodiments, the recombinant protein (e.g. construct) as described herein comprises one or more N- or C-terminal modifications. The N- or C-terminal modifications may be added, for example, for purposes of purification or targeting to a particular cellular component (e.g., nucleus). In some embodiments, the construct comprises an N-terminal nuclear localization signal (NLS). In some embodiments, the construct comprises a purification tag, such as a hexa-his tag. The purification tag may be located at the N-terminus or the C-terminus of the construct. In some embodiments, the N- or C-terminal addition comprises a Tobacco Etch Virus (TEV) protease cleavage site. Insome embodiments, the tag comprises a peptide sequence comprising one part of a cognate binding pair. In some embodiments, the cognate binding pair comprise an avidinbiotin binding pair or a streptavidin-biotin binding pair. In such embodiments, the tag may comprise a sequence configured to be biotinylated such that the biotinylated construct may be bound to a streptavidin or avidin moiety. In some embodiments, the N- or C-terminal addition comprises 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, P2A peptide, E2A peptide, or F2A peptide. In some embodiments, an Gly-Ser linker is disposed between the 2A peptide and the rest of the construct (e.g., the HLA class I heavy chain domain). In some embodiments, the Gly-Ser linker has a sequence comprising SGSG. In some embodiments, the signal peptide (sometimes referred to as signal sequence, targeting signal, localization signal, leader sequence or leader peptide) region in SEQ ID N0: l-121 may be replaced with a signal peptide from a different protein. Examples of signal peptides include, without limitation, the native signal peptide of wild-type HLA-E, the native signal peptide of HLA-G, or the Ig kappa leader sequence.NUCLEIC ACID COMPOSITIONS

[0171] In an aspect is provided a nucleic acid encoding the recombinant protein (e.g. construct) provided herein including embodiments thereof. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human HLA class I heavy chain sequence. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises an HLA-E sequence or an HLA-G sequence or any combination thereof. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises an HLA-E sequence. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises an HLA-G sequence.

[0172] In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises multiple alleles of an HLA-E sequence or an HLA-G sequence or any combination thereof. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises multiple alleles of an HLA-E sequence. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises multiple alleles of an HLA-G sequence.

[0173] In some embodiments, the alleles of an HLA-E sequence are selected from among HLA-E*01 :03 and HLA-E*01:01. In embodiments, the sequence may be a mature HLA-E*01 :03 which amino acid sequence can be found in Uniprot reference numberP13747. In embodiments, the sequence is a mature HLA-E*01 :01 which comprises a G107R mutation relative to Uniprot reference number P13747. In embodiments, the sequence includes the sequence of SEQ ID NO: 146. In embodiments, the sequence includes the sequence SEQ ID NO: 147.

[0174] In some embodiments, the alleles of an HLA-G sequence are selected from among HLA-G *01 :01 and HLA-G*01 :04. In embodiments, the sequence may be a mature HLA-G*01 :01 which amino acid sequence can be found in Uniprot designation Pl 7693-1. In embodiments, the sequence is a mature HLA-G*01 :04 which comprises an LI 101 mutation relative to Uniprot reference number Pl 7693-1. In embodiments, the sequence includes the sequence SEQ ID NO: 148. In embodiments, the sequence includes the sequence SEQ ID NO:149.

[0175] In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1700 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1600 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1500 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1400 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1300 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1200 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1100 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1000 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 900 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 800 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 700 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 600 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 500 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 450 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 400 bp. In some embodiments, the sequence encoding a human HLAclass I heavy chain sequence comprises fewer than 350 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 300 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 250 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 200 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 150 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 100 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 50 bp.

[0176] In some embodiments, the HLA-E sequence, HLA-G sequence, or combination thereof comprises one or more flanking sequences. In some embodiments, the HLA-E sequence comprises one or more flanking sequences. In some embodiments, the HLA-G sequence comprises one or more flanking sequences.

[0177] 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 to one or more promoters. In some embodiments, the promoters comprise an HLA-E promoter, HLA-G promoter, or combination thereof. In some embodiments, the HLA-E sequence comprises an endogenous HLA-E promoter. In some embodiments, the HLA-G sequence comprises an endogenous HLA-G promoter.

[0178] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human beta-2 microglobulin (B2M) sequence. In some embodiments, the nucleic acid molecule further comprises a sequence encoding an endogenous human beta- 2 microglobulin sequence. In some embodiments, the nucleic acid molecule further comprises a sequence encoding a peptide (e.g. HLA-E heavy chain binding peptide). The peptide may comprise any sequence or feature disclosed herein.

[0179] In some embodiments, the sequence encoding the human HLA class I 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 I heavy chain sequence comprises an HLA-E sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class I heavy chain sequence comprises an HLA-F sequence or a fragment thereof. In some embodiments, the sequence encoding the human HLA class I heavy chain sequence comprises an HLA-G sequence or a fragment thereof.In some embodiments, the sequence encoding the human HLA class I 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 I 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 I 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 I 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.

[0180] In some embodiments, at least one of the HLA-E sequence or the fragment thereof, HLA-F sequence or the fragment thereof, HLA-G sequence or the fragment thereof, or any combination thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the HLA-E sequence or the fragment thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the HLA-F sequence or the fragment thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the HLA-G sequence or the fragment thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the HLA-E sequence or the fragment thereof and the HLA-F sequence or the fragment thereof are inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the HLA-E sequence or the fragment thereof and the HLA-G sequence or the fragment thereof are inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the HLA-F sequence or the fragment thereof and the HLA-G sequence or the fragment thereof are inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells. In some embodiments, the HLA-E sequence or the fragment thereof, the HLA-F sequence or the fragment thereof, and the HLA-G sequence or the fragment thereof are inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells.

[0181] In some embodiments, the nucleic acid molecule further comprises a sequence encoding one or more knocked out proteins corresponding to the class II, majorhistocompatibility complex, transactivator (CUT A). In some embodiments, the entire class II, major histocompatibility complex, transactivator (CUT A) locus is knocked out.

[0182] In some embodiments, the nucleic acid molecule further comprises a sequence encoding an epitope configured to allow for detection of the construct.

[0183] In some embodiments, the nucleic acid molecule comprises, a) the sequence encoding the peptide; b) a first sequence encoding a first linker of the one or more sequences encoding one or more linkers; c) the sequence encoding the human beta-2 microglobulin sequence; d) a second sequence encoding a second linker of the one or more sequences encoding one or more linkers; and e) the sequence encoding the human HLA class I heavy chain sequence.

[0184] Provided herein, in another aspect, is a nucleic acid molecule comprising a sequence encoding a construct comprising one or more class I human leukocyte antigen (HLA) proteins, wherein the one or more class I HLA proteins are inhibited from eliciting an immune response when the construct is interrogated by one or more immune cells, and wherein the nucleic acid molecule comprises, a) a sequence encoding a peptide, wherein the peptide is incapable of activating the one or more T cells; b) a first sequence encoding a first linker; and c) a sequence encoding one or more class I HLA proteins; wherein 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 I heavy chain sequence, potentially has a higher affinity for NKG2A / CD94 than NKG2C / CD94, and wherein the conformation is further configured to resist proteolytic cleavage.

[0185] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human beta-2 microglobulin sequence between the sequence encoding the linker and the sequence encoding the human HLA class I heavy chain sequence. In some embodiments, the nucleic acid molecule further comprises a sequence encoding an endogenous human beta-2 microglobulin sequence between the sequence encoding the linker and the sequence encoding the human HLA class I heavy chain sequence.

[0186] In some embodiments, the nucleic acid molecule further comprises a sequence encoding one or more proteins or fragments thereof that inhibit an immune response by 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 is CD48. In some embodiments, the one or more proteins or fragments thereof is CD59. In some embodiments, the one or more proteins or fragments thereof are CD48 and CD59.

[0187] Provided herein, in another aspect, is a method for generating the nucleic acid molecule provided herein, comprising displacing a sequence encoding a region configured to receive a sequence comprising the deletion in the HLA locus, a sequence encoding the human HLA class I heavy chain sequence, or any combination thereof. In some embodiments, the method comprises displacing a sequence encoding a region configured to receive a sequence comprising the deletion in the HLA locus. In some embodiments, the method comprises displacing a sequence encoding a region configured to receive a sequence encoding the human HLA class I heavy chain sequence. In some embodiments, the method comprises displacing a sequence encoding a region configured to receive a sequence comprising the deletion in the HLA locus and a sequence encoding the human HLA class I heavy chain sequence.HYPOIMMUNOGEN IC OR NON-IMMUNOGENIC CELLS

[0188] In an aspect is provided a method of making a hypoimmunogenic cell, including delivering to a cell the nucleic acid provided herein including embodiments thereof, and culturing said cell under conditions to express the recombinant protein. In another aspect is provided a cell including the recombinant protein provided herein including embodiments thereof or the nucleic acid provided herein including embodiments thereof. In embodiments, the cell includes the recombinant protein provided herein including embodiments thereof. In embodiments, the cell includes the nucleic acid provided herein including embodiments thereof.

[0189] Provided herein, in another aspect, is a method of making a hypoimmunogenic cell, comprising administering the construct provided herein or the nucleic acid molecule provided herein to a cell. In some embodiments, the nucleic acid molecule is delivered to the cell’s genome. In some embodiments, the cell is contacted with the construct.

[0190] 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. In some embodiments, the cell is an engineered T-cell Receptor (TCR) T cell. In some embodiments, the cell is a TCR macrophage (TCR-M) cell. In some embodiments, the cell is a TCR natural killer (TCR-NK) cell.

[0191] In some embodiments, the hypoimmunogenic cells are suitable for use in cellular therapy. In some embodiments, the hypoimmunogenic cells are suitable for administration to a subject without causing a major adverse immune response (e.g., grades III-IV acute GvHD, cytokine storm) or are suitable for administration to a subject with causing only a minor adverse immune response. An adverse immune response is a response not desired from a clinical or patient care standpoint.

[0192] In some embodiments, the synHLA constructs of the invention preferably engage the NKG2A / CD94 complex on the surface of NK cells to send an inhibitory signal. In some embodiments the synHLA constructs decrease signaling via the NKG2C / CD94 complex. In some embodiments the synHLA constructs bind the inhibitory molecules KIR2DL4, ILT-2 (LILRB1) and ILT-4 (LILRB2), so it can inhibit NK cell killing through multiple pathways. In one embodiment, when expressed on a cell, the present synHLA constructs recapitulate and potentially improve on the NK cell inhibitory activity found in the wild-type expressed HLA structures while potentially providing the additional advantages including one or more of: increased protein complex stability, increased cellular half-life in vitro or in vivo or both, reduced endocytic trafficking of the protein, increased cell surface expression of the protein, reduced immune cell activation when interrogated by CD8+ T cells, reduced binding to at least a subset of CD8+ T cells’ TCRs, and increased binding signaling via NKG2A and / or reduced signaling via NKG2C. In some embodiments the synHLA constructs have a higher binding affinity to NKG2A than a reference HLA. Further, in another embodiment the present synHLA constructs are versatile and may be expressed as a free protein and potentially have increased stability and solubility as free proteins when compared to wild-type HLA structures.

[0193] In some embodiments, cells expressing the synHLA constructs are inhibited from eliciting an immune cell response when the construct is interrogated by one or more other immune cells, whether they be other immune cells to be administered, or host immune cells upon administration. In some embodiments, the one or more synHLA constructs 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 synHLA constructs are inhibited from eliciting an immune cell response when interrogated by one or more immune cells (including T cells) and from eliciting an NK cell response when interrogated by one or more immune cells (including NK cells).

[0194] In some embodiments, the cells may be further modified. In some embodiments, the further modification may be modification to the ability of the cell to express proteinsor express function versions of a protein. In some embodiments, the cells may be unable to express B2M.In some embodiments, the cells may be modified to express one or more synthetic constructs. In some embodiments, the cell may comprise more than one synHLA constructs. At least one or more of these synHLA constructs may be the constructs of the present disclosure or other synthetic HLA constructs.NUCLEIC ACID VECTORS

[0195] A variety of nucleic acids may be introduced into cells, for knockout purposes, or to obtain expression of a gene for other purposes. Nucleic acid constructs that can be used to produce transgenic cells including a target nucleic acid sequence. As used herein, the term nucleic acid or “nucleic acid molecule” includes DNA, RNA, and nucleic acid analogs, and nucleic acids that are double-stranded or single- stranded (i.e., a sense or an antisense single strand). Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, 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'-doxy cytidine for deoxycytidine. Modifications of 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, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the 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. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphoro dithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.

[0196] The target nucleic acid sequence can be operably linked to a regulatory region such as a promoter. Regulatory regions can be from any species. As used herein, operably linked refers to positioning of a regulatory region relative to a nucleic acid sequence in such a way as to permit or facilitate transcription of the target nucleic acid.

[0197] Any type of promoter can be operably linked to a target nucleic acid sequence. Examples of promoters include, without limitation, tissue-specific promoters, constitutive promoters, and promoters responsive or unresponsive to a particular stimulus. Suitable tissue specific promoters can result in preferential expression of a nucleic acid transcript in beta cells and include, for example, the human insulin promoter. Other tissue specificpromoters can result in preferential expression in, for example, hepatocytes or heart tissue and can include the albumin or alpha-myosin heavy chain promoters, respectively. In other embodiments, a promoter that facilitates the expression of a nucleic acid molecule without significant tissue- or temporal-specificity can be used (i.e., a constitutive promoter). For example, a beta-actin promoter such as the chicken beta-actin gene promoter, ubiquitin promoter, miniCAGs promoter, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) promoter, or 3 -phosphoglycerate kinase (PGK) promoter can be used, as well as viral promoters such as the herpes simplex virus thymidine kinase (HSV-TK) promoter, the SV40 promoter, or a cytomegalovirus (CMV) promoter. In some embodiments, a fusion of the chicken beta actin gene promoter and the CMV enhancer is used as a promoter. See, for example, Xu et al. (2001) Hum. Gene Ther. 12:563; and Kiwaki et al. (1996) Hum. Gene Ther. 7:821.

[0198] An example of an inducible promoter is the tetracycline (tet)-on promoter system, which can be used to regulate transcription of the nucleic acid. In this system, a mutated Tet repressor (TetR) is fused to the activation domain of herpes simplex virus VP 16 trans-activator protein to create a tetracycline-controlled transcriptional activator (tTA), which is regulated by tet or doxycycline (dox). In the absence of antibiotic, transcription is minimal, while in the presence of tet or dox, transcription is induced. Alternative inducible systems include the ecdysone or rapamycin systems. 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 such as muristerone A. The agent that is administered to the subject to trigger the inducible system is referred to as an induction agent.

[0199] Additional regulatory regions that may be useful in nucleic acid constructs, include, but are not limited to, polyadenylation sequences, translation control sequences (e.g., an internal ribosome entry segment, IRES), enhancers, inducible elements, or introns. Such regulatory regions may not be necessary, although they may increase expression by affecting transcription, stability of the mRNA, translational efficiency, or the like. Such regulatory regions can be included in a nucleic acid construct as desired to obtain optimal expression of the nucleic acids in the cell(s). Sufficient expression, however, can sometimes be obtained without such additional elements.

[0200] A nucleic acid construct may be used that encodes signal peptides or selectable markers. Signal peptides can be used such that an encoded polypeptide is directed to a particular cellular location (e.g., the cell surface). Non-limiting examples of selectablemarkers include puromycin, ganciclovir, adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH), dihydro folate reductase (DHFR), hygromycin-B- phosphotransferase, thymidine kinase (TK), and xanthin-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.

[0201] In some embodiments, a sequence encoding a selectable marker can be flanked by recognition sequences for a recombinase such as, e.g., Cre or Flp. For example, the selectable marker can be flanked by loxP recognition sites (34-bp recognition sites recognized by the Cre recombinase) or FRT recognition sites such 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. A transposon containing a Cre- or Flp-activatable transgene interrupted by a selectable marker gene also can be used to obtain transgenic cells with conditional expression of a transgene.

[0202] In some embodiments, the target nucleic acid encodes a polypeptide and potentially one or more isoforms of a polypeptide. A nucleic acid sequence encoding a polypeptide can include a tag sequence that encodes a “tag” designed to facilitate subsequent manipulation of the encoded polypeptide (e.g., to facilitate localization or detection). Tag sequences can be inserted in 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™ tag (Kodak, New Haven, Conn.).

[0203] Nucleic acid constructs can be introduced into embryonic, fetal, or adult cells of any type, including, for example, germ cells such as an oocyte or an egg, a progenitor cell, an adult or embryonic stem cell, a hematopoietic stem cell, a mesenchymal stem cell, a primordial germ cell, a kidney cell such as a PK- 15 cell, an islet cell, a beta cell, a liver cell, or a fibroblast such as a dermal fibroblast, using a variety of techniques. Non- limiting examples of techniques include the use of transposon systems, recombinant viruses that can infect cells, or liposomes or other non- viral methods such as electroporation, microinjection, or calcium phosphate precipitation, that are capable of delivering nucleic acids to cells.

[0204] In transposon systems, the transcriptional unit of a nucleic acid construct, i.e., the regulatory region operably linked to a target nucleic acid sequence, is flanked by aninverted repeat of a transposon. Several transposon systems, including, for example, Sleeping Beauty (see, U.S. Pat. No. 6,613,752 and U.S. Publication No. 2005 / 0003542); Frog Prince (Miskey et al. (2003) Nucleic Acids Res. 31 :6873); Tol2 (Kawakami (2007) Genome Biology 8(Suppl. l):S7; Minos (Pavlopoulos et al. (2007) Genome Biology 8(Suppl.l):S2); Hsmarl (Miskey et al. (2007)) Mol Cell Biol. 27:4589); and Passport have been developed to introduce nucleic acids into cells. The Sleeping Beauty and Passport transposon is particularly useful. A 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 provided as an mRNA (e.g., an in vitro-transcribed and capped mRNA).

[0205] Insulator elements also can be included in a nucleic acid construct to maintain expression of the target nucleic acid and to inhibit the unwanted transcription of host genes. See, for example, U.S. Publication No. 2004 / 0203158. Typically, an insulator element flanks each side of the transcriptional unit and is internal to the inverted repeat of the transposon. Non- limiting examples of insulator elements include the matrix attachment region-(MAR) type insulator elements and border-type insulator elements. See, for example, U.S. Pat. Nos. 6,395,549, 5,731,178, 6,100,448 and 5,610,053, and U.S. Publication No. 2004 / 0203158.

[0206] Nucleic acids can be incorporated into vectors. A vector is a broad term that includes any specific DNA segment that is designed to move from a carrier into a target DNA. A vector may be referred to as an expression vector, or a vector system, which is a set of components needed to bring about DNA insertion into a genome or other targeted DNA sequence such as an episome, plasmid, or even virus / phage DNA segment. Vector systems such as viral vectors (e.g., retroviruses, adeno-associated virus and integrating phage viruses), and non- viral vectors (e.g., transposons) used for gene delivery in subjects have two basic components: 1) a vector comprised of DNA (or RNA that is reverse transcribed into a cDNA) and 2) a transposase, recombinase, or other integrase enzyme that recognizes both the vector and a DNA target sequence and inserts the vector into the target DNA sequence. Vectors most often contain one or more expression cassettes that comprise one or more expression control sequences, wherein an expression control sequence is a DNA sequence that controls and regulates 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, e.g., retroviral vectors, are known. Mammalian expression plasmids typicallyhave an origin of replication, a suitable promoter and optional enhancer, and also any necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences. Examples of vectors include: plasmids (which may also be a carrier of another type of vector), adenovirus, adeno-associated virus (AAV), lentivirus (e.g., HIV-1, SIV or FIV), retrovirus (e.g., ASV, ALV or MoMLV), herpes simplex virus (HSV), and transposons (e.g., Sleeping Beauty, P-elements, Tol-2, Frog Prince, piggyBac).METHODS OF DELIVERING A NUCLEIC ACID

[0208] Provided herein, in other aspects, are additional methods of delivering a nucleic acid molecule encoding one or more recombinant proteins provided herein including embodiments thereof. In some embodiments, the method comprises delivery of a nucleic acid molecule encoding one or more HLAs via a viral vector. In some embodiments, the method comprises delivery of a nucleic acid molecule encoding one or more HLAs 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 the endogenous HLA locus. In some embodiments, the deletion comprises a deletion in the endogenous HLA-E, HLA-G, or any combination thereof. In some embodiments, the deletion is complete deletion of the endogenous HLA locus.

[0210] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human HLA class I heavy chain sequence. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises an HLA-E sequence or an HLA-G sequence or any combination thereof. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises multiple alleles of an HLA-E sequence or an HLA-G sequence or any combination thereof.

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

[0212] In some embodiments, the HLA-E sequence, HLA-G sequence, or combination thereof comprises one or more flanking sequences. 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 to one or more promoters. In someembodiments, the promoters comprise an HLA-E promoter, HLA-G promoter, or combination thereof.

[0213] In some embodiments, the nucleic acid molecule encoding the human HLA class I 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 the fragment thereof, HLA-F sequence or the fragment thereof, HLA-G sequence or the fragment thereof, or any combination thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells.

[0214] In some embodiments, the nucleic acid molecule encoding a human HLA class I heavy chain sequence comprises one or more mutations, wherein a cell comprising the mutated human HLA class I heavy chain sequence comprising the one or more mutations does not elicit an immune response when the cell is interrogated by one or more CD8+ T cells. In some embodiments, the mutated human HLA class I 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.TEMPLATED AND NON-TEMPLATED REPAIRS

[0215] Targeted endonuclease technologies, such as zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats / CRISPR associated endonuclease Cas9 (CRISPR / Cas9) can be utilized to disrupt gene function by introducing insertions and / or deletions (indels) into genomes of species, such as by non-homologous end-joining (NHEJ). However, indels introduced by NHEJ are variable in size and sequence which makes screening for functionally disrupted clones arduous and does not enable precise alterations. TALEN or CRISPR / Cas9 mediated homology-directed repair (HDR) supports the introduction of defined nucleotide changes in eukaryotic cells.

[0216] A subject may be modified using TALENs, zinc finger nucleases, or other genetic engineering tools, including various vectors that are known. A genetic modification made by such tools may comprise inactivation of a gene. The term inactivation of a gene refers to preventing the formation of a functional gene product. A gene product is functional only if it fulfills its normal (wild-type) functions. Materials and methods of genetically modifying subjects are further detailed in U.S. Ser. No. 13 / 404,662 filed Feb. 24, 2012, Ser. No. 13 / 467,588 filed May 9, 2012, and Ser. No. 12 / 622,886 filedNov. 10, 2009 which are hereby incorporated herein by reference for all purposes; in case of conflict, the instant specification is controlling. The term trans-acting refers to processes acting on a target gene from a different molecule (i.e., intermolecular). A transacting element is usually a DNA sequence that contains a gene. This gene codes for a protein (or microRNA or other diffusible molecule) that is used in the regulation of the target gene. The trans-acting gene may be on the same chromosome as the target gene, but the activity is via the intermediary protein or RNA that it encodes. Inactivation of a gene using a dominant negative generally involves a trans-acting element. The term cis- regulatory or cis-acting means an action without coding for protein or RNA; in the context of gene inactivation, this generally means inactivation of the coding portion of a gene, or a promoter and / or operator that is necessary for expression of the functional gene.

[0217] Various techniques known in the art can be used to introduce nucleic acid constructs into non-humans and humans to produce founder lines, in which the nucleic acid construct is integrated into the genome. Such techniques include, without limitation, pronuclear microinjection (U.S. Pat. No. 4,873,191), retrovirus mediated gene transfer into germ lines (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. (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 transplantation (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 well as cytoplasmic injection, primordial germ cell transplantation (Brinster), and blastocyst chimera production whereby a germ cell is propagated in an embryo.

[0218] TALENs, zinc finger nucleases, CRISPR nuclease (e.g., CRISPR / Cas9) and recombinase fusion proteins may be used with or without a template.

[0219] A genetic modification made by TALENs or other tools may be, for example, chosen from the list consisting of an insertion, a deletion, insertion of an exogenous nucleic acid fragment, and a substitution. The term “insertion” is used broadly to mean either literal insertion into the chromosome or use of the exogenous sequence as a template for repair

[0220] The term exogenous nucleic acid means a nucleic acid that is added to the cell or embryo, regardless of whether the nucleic acid is the same or distinct from nucleic acid sequences naturally in the cell. In some cases, the exogenous nucleic acid differs in sequence from any nucleic acid sequence that occurs naturally within the cell. The term nucleic acid fragment is broad and includes a chromosome, expression cassette, gene, DNA, RNA, mRNA, or portion thereof.

[0221] Provided herein, in other aspects, are additional methods of delivering a nucleic acid molecule encoding one or more human leukocyte antigens (HLAs) to a cell. In some embodiments, the method comprises delivery of a nucleic acid molecule encoding one or more HLAs via transcription activator-like effector nucleases (TALENs). In some embodiments, the method comprises delivery of a nucleic acid molecule encoding one or more HLAs via zinc finger nucleases (ZFNs). In some embodiments, the method comprises delivery of a nucleic acid molecule encoding one or more HLAs via clustered regularly interspaced short palindromic repeats / CRISPR associated endonuclease Cas9 (CRISPR / Cas9).

[0222] In some embodiments, the nucleic acid molecule comprises a deletion in the endogenous HLA locus. In some embodiments, the deletion comprises a deletion in the endogenous HLA-E, HLA-G or any combination thereof. In some embodiments, the deletion is complete deletion of the endogenous HLA locus and replacement with a nucleic acid encoding the HLA heavy chain of the invention.

[0223] In some embodiments, the nucleic acid molecule further comprises a sequence encoding a human HLA class I heavy chain sequence. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises an HLA-E sequence or an HLA-G sequence or any combination thereof. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises multiple alleles of an HLA-E sequence or an HLA-G sequence or any combination thereof.

[0224] In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 1700 base pairs (bp). In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 500 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 250 bp. In some embodiments, the sequence encoding a human HLA class I heavy chain sequence comprises fewer than 150 bp.

[0225] In some embodiments, the sequence encoding a human HLA class I heavy chain sequence does not comprise at least a portion of the HLA-A sequence, HLA-B sequence, HLA-C sequence, or combination thereof.

[0226] In some embodiments, the nucleic acid molecule encoding the human HLA class I 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 the fragment thereof, HLA-F sequence or the fragment thereof, HLA-G sequence or the fragment thereof, or any combination thereof is inhibited from eliciting an immune cell response when the construct is interrogated by one or more immune cells.

[0227] In some embodiments, the nucleic acid molecule encoding a human HLA class I heavy chain sequence comprises one or more mutations, wherein a cell comprising the mutated human HLA class I heavy chain sequence comprising the one or more mutations does not elicit an immune response when the cell is interrogated by one or more CD8+ T cells. In some embodiments, the mutated human HLA class I 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.METHODS OF TREATMENT

[0228] 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 the recombinant protein (e.g. construct) provided herein, a nucleic acid molecule encoding the recombinant protein or the hypoimmunogenic cell expressing the recombinant protein.

[0229] 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 perniciousanemia. In some embodiments, the disease is celiac disease. In some embodiments, the disease is vasculitis.

[0230] In some embodiments, the disease is a 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 stomach 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.

[0231] 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.

[0232] In some embodiments, the administration of the hypoimmunogenic cells provided herein treats the disease or disorder without provoking an adverse immune response in the patient.

[0233] When one or more constructs, nucleic acid molecules, or hypoimmunogenic cells as provided herein are administered into a human subject, the dosage will normally be determined by a physician with the dosage generally varying according to the age, weight, and response of the individual subject, as well as the severity of the subject’s symptoms.

[0234] The actual dosage employed may be varied depending upon the requirements of the subject and the severity of the condition being treated. Determination of the proper dosage for a particular situation is within the skill of the art. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the one or more constructs, nucleic acid molecules, or hypoimmunogenic cells as provided herein.Thereafter, the dosage is increased by small amounts until the optimum effect under the circumstances is reached.

[0235] The amount and frequency of administration of the one or more constructs, nucleic acid molecules, or hypoimmunogenic cells as provided herein, and if applicable other chemotherapeutic agents and / or radiation therapy, will be regulated according to the judgment of the attending clinician (physician) considering such factors as age, condition and size of the subject as well as severity of the disease being treated.

[0236] The chemotherapeutic agent and / or radiation therapy can be administered according to therapeutic protocols well known in the art. It will be apparent to those skilled in the art that the administration of the chemotherapeutic agent and / or radiation therapy can be varied depending on the disease being treated and the known effects of the chemotherapeutic agent and / or radiation therapy on that disease. Also, in accordance with the knowledge of the skilled clinician, the therapeutic protocols (e.g., dosage amounts and times of administration) can be varied in view of the observed effects of the administered therapeutic agents (i.e., antineoplastic agent or radiation) on the subject, and in view of the observed responses of the disease to the administered therapeutic agents.

[0237] Also, in general, the one or more constructs, nucleic acid molecules, or hypoimmunogenic cells as provided herein need not be administered in the same pharmaceutical composition as a chemotherapeutic agent, and may, because of different physical and chemical characteristics, be administered by a different route. The determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is well within the knowledge of the skilled clinician. The initial administration can be made according to established protocols known in the art, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the skilled clinician.

[0238] The particular choice of the one or more constructs, nucleic acid molecules, or hypoimmunogenic cells as provided herein (and where appropriate, chemotherapeutic agent and / or radiation) will depend upon the diagnosis of the attending physicians and their judgment of the condition of the subject and the appropriate treatment protocol.

[0239] The one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein (and where appropriate chemotherapeutic agent and / or radiation) may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the proliferative disease, the condition of the subject, and the actual choice ofchemotherapeutic agent and / or radiation to be administered in conjunction (i.e., within a single treatment protocol) with the one or more constructs, nucleic acid molecules, or hypoimmunogenic cells as provided herein.

[0240] In combinational applications and uses, the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein and the chemotherapeutic agent and / or radiation need not be administered simultaneously or essentially simultaneously, and the initial order of administration of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein, and the chemotherapeutic agent and / or radiation, may not be important. Thus, the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein may be administered first followed by the administration of the chemotherapeutic agent and / or radiation; or the chemotherapeutic agent and / or radiation may be administered first followed by the administration of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein. This alternate administration may be repeated during a single treatment protocol. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is well within the knowledge of the skilled physician after evaluation of the disease being treated and the condition of the subject. For example, the chemotherapeutic agent and / or radiation may be administered first, and then the treatment continued with the administration of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein followed, where determined advantageous, by the administration of the chemotherapeutic agent and / or radiation, and so on until the treatment protocol is complete.

[0241] Thus, in accordance with experience and knowledge, the practicing physician can modify each protocol for the administration of one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein for treatment according to the individual subject 's needs, as the treatment proceeds.

[0242] The attending clinician, in judging whether treatment is effective at the dosage administered, will consider the general well-being of the subject as well as more definite signs such as relief of disease-related symptoms, inhibition of tumor growth, actual shrinkage of the tumor, or inhibition of metastasis. Size of the tumor can be measured by standard methods such as radiological studies, e.g., CAT or MRI scan, and successive measurements can be used to judge whether or not growth of the tumor has been retarded(stabilized) or even reversed. Relief of disease-related symptoms such as pain, and improvement in overall condition can also be used to help judge effectiveness of treatment.

[0243] In some embodiments, therapeutic efficacy is measured based on an effect of treating a proliferative disorder, such as cancer. In general, therapeutic efficacy of the methods and compositions of the invention, with regard to the treatment of a proliferative disorder (e.g. cancer, whether benign or malignant), may be measured by the degree to which the methods and compositions promote inhibition of tumor cell proliferation, the inhibition of tumor vascularization, the eradication of tumor cells, the reduction in the rate of growth of a tumor, and / or a reduction in the size of at least one tumor. Several parameters to be considered in the determination of therapeutic efficacy are discussed herein. The proper combination of parameters for a particular situation can be established by the clinician. The progress of the inventive method in treating cancer (e.g., reducing tumor size or eradicating cancerous cells) can be ascertained using any suitable method, such as those methods currently used in the clinic to track tumor size and cancer progress. The primary efficacy parameter used to evaluate the treatment of cancer by the inventive method and compositions preferably is a reduction in the size of a tumor. Tumor size can be figured using any suitable technique, such as measurement of dimensions, or estimation of tumor volume using available computer software.

[0244] In some desirable embodiments, the growth of a tumor is stabilized (i.e., one or more tumors do not increase more than 1%, 5%, 10%, 15%, or 20% in size, and / or do not metastasize) as a result of the inventive method and compositions. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments, a tumor is stabilized for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Preferably, the inventive method reduces the size of a tumor at least about 5% (e.g., at least about 10%, 15%, 20%, or 25%). More preferably, tumor size is reduced at least about 30% (e.g., at least about 35%, 40%, 45%, 50%, 55%, 60%, or 65%). Even more preferably, tumor size is reduced at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, or 95%). Most preferably, the tumor is completely eliminated, or reduced below a level of detection. In some embodiments, a subject remains tumor free (e.g. in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or more weeks following treatment. In some embodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months following treatment. In someembodiments, a subject remains tumor free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years after treatment.

[0245] In some embodiments, the efficacy of the inventive method in reducing tumor size can be determined by measuring the percentage of necrotic (i.e., dead) tissue of a surgically resected tumor following completion of the therapeutic period. In some further embodiments, a treatment is therapeutically effective if the necrosis 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 necrosis percentage of the resected tissue is 100%, that is, no tumor tissue is present or detectable.

[0246] The efficacy of the inventive method can be determined by a number of 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., conversion of 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 the Clinical Benefit Response Assessment, and the like, all of which can point to the overall progression (or regression) of cancer in a human. Biopsy is particularly useful in detecting the eradication of cancerous cells within a tissue. Radioimmunodetection (RAID) is used to locate and stage tumors using serum levels of markers (antigens) produced by and / or associated with tumors (“tumor markers” or “tumor-associated antigens”), and can be useful as a pretreatment diagnostic predicate, a post-treatment diagnostic indicator of recurrence, and a post-treatment 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, carcinembryonic antigen (CEA), prostate-specific antigen (PSA), CA- 125, CAI 9-9, 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 gp 100. Other tumor-associated antigens are known in the art. RAID technology in combination with endoscopic detection systems also can efficiently distinguish small tumors from surrounding tissue (see, for example, U.S. Pat. No. 4,932,412).

[0247] In additional desirable embodiments, the treatment of cancer in a human patient in accordance with the inventive method is evidenced by one or more of the following results: (a) the complete disappearance of a tumor (i.e., a complete response), (b) about a25% to about a 50% reduction in the size of a tumor for at least four weeks after completion of the therapeutic period as compared to the size of the tumor before treatment, (c) at least about a 50% reduction in the size of a tumor for at least four weeks after completion of the therapeutic period as compared to the size of the tumor before the therapeutic 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 a specific tumor-associated antigen level at about 4-12 weeks after completion of the therapeutic period as compared to the tumor- associated antigen level before the therapeutic period. While at least a 2% decrease in a tumor-associated antigen level is preferred, any decrease in the tumor-associated antigen level is evidence of treatment of a cancer in a patient by the inventive method. For example, with respect to unresectable, locally advanced pancreatic cancer, treatment can be evidenced by at least a 10% decrease in the CAI 9-9 tumor-associated antigen level at 4-12 weeks after completion of the therapeutic period as compared to the CA19-9 level before the therapeutic period. Similarly, with respect to locally advanced rectal cancer, treatment can be evidenced by at least a 10% decrease in the CEA tumor-associated antigen level at 4-12 weeks after completion of the therapeutic period as compared to the CEA level before the therapeutic period.

[0248] With respect to quality of life assessments, such as the Clinical Benefit Response Criteria, the therapeutic benefit of the treatment in accordance with the invention can be evidenced in terms of pain intensity, analgesic consumption, and / or the Kamofsky Performance Scale score. The treatment of cancer in a human patient alternatively, or in addition, is evidenced by (a) at least a 50% decrease (e.g., at least a 60%, 70%, 80%, 90%, or 100% decrease) in pain intensity reported by a patient, such as for any consecutive four week period in the 12 weeks after completion of treatment, as compared to the pain intensity reported by the patient before treatment, (b) at least a 50% decrease (e.g., at least a 60%, 70%, 80%, 90%, or 100% decrease) in analgesic consumption reported by a patient, such as for any consecutive four week period in the 12 weeks after completion of treatment as compared to the analgesic consumption reported by the patient before 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 Kamofsky Performance Scale score reported by a patient, such as for any consecutive four week period in the 12 weeks after completion of the therapeutic period as compared to the Kamofsky Performance Scale score reported by the patient before the therapeutic period.

[0249] The treatment of a proliferative disorder (e.g. cancer, whether benign or malignant) in a human patient desirably is evidenced by one or more (in any combination) of the foregoing results, although alternative or additional results of the referenced tests and / or other tests can evidence treatment efficacy.

[0250] In some embodiments, tumor size is reduced as a result of the inventive method preferably without significant adverse events in the subject. Adverse events are categorized 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 inventive method is associated with minimal adverse events, e.g. Grade 0, Grade 1, or Grade 2 adverse events, as graded by the CTEP / NCI. However, as discussed herein, reduction of tumor size, although preferred, is not required in that the actual size of tumor may not shrink despite the eradication of tumor cells. Eradication of cancerous cells is sufficient to realize a therapeutic effect. Likewise, any reduction in tumor size is sufficient to realize a therapeutic effect.

[0251] Detection, monitoring and rating of various cancers in a human are further described in Cancer Facts and Figures 2001, American Cancer Society, New York, N.Y., and International Patent Application WO 01 / 24684. Accordingly, a clinician can use standard tests to determine the efficacy of the various embodiments of the inventive method in treating cancer. However, in addition to tumor size and spread, the clinician also may consider quality of life and survival of the patient in evaluating efficacy of treatment.

[0252] In some embodiments, administration of one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein provides improved therapeutic efficacy. Improved efficacy may be measured using any method known in the art, including but not limited to those described herein. In some embodiments, the improved therapeutic efficacy is an improvement of 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, using an appropriate measure (e.g. tumor size reduction, duration of tumor size stability, duration of time free from metastatic events, duration of disease-free survival). Improved efficacy may also be expressed as fold improvement, such as at least about 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 more, using an appropriate measure (e.g.tumor size reduction, duration of tumor size stability, duration of time free from metastatic events, duration of disease-free survival).METHODS OF USE INCLUDING HLA HEAVY CHAIN BINDING PEPTIDES

[0253] Provided herein, in another aspect, is a method of inhibiting the activity of function of ahuman leukocyte antigen (HLA) comprising contacting the HLA with a peptide that does not comprise T-cell receptor-binding residues or fragments. In some embodiments, the peptide binds to one or more HLA binding groove domain residues of the HLA. In some embodiments, the peptide modulates a conformation of the HLA. In some embodiments, the conformation prevents a T cell from binding the HLA. In some embodiments, the HLA is synthetic.PHARMACEUTICAL COMPOSITIONS

[0254] The disclosure provides compositions, including pharmaceutical compositions, comprising one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein. In some embodiments, the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are formulated into pharmaceutical compositions. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are used as suitable 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, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999).

[0255] Provided herein are pharmaceutical compositions comprising one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein and a pharmaceutically acceptable diluent(s), excipient(s), or carrier(s). In certain embodiments, the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are administered as pharmaceutical compositions in which the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are mixed with other active ingredients, as in combination therapy. In specific embodiments, the pharmaceutical compositions include one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein.

[0256] A pharmaceutical composition, as used herein, refers to a mixture of one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein to an organism. In some embodiments, in practicing the methods of treatment or use provided herein, therapeutically effective amounts of one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are 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, therapeutically effective amounts vary depending on the severity of the disease, the age and relative health of the subject and other factors. The one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are used singly or in combination with one or more therapeutic agents as components of mixtures.

[0257] In one embodiment, one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are formulated in an aqueous solution. In specific embodiments, the aqueous solution is selected from, by way of example only, a physiologically compatible buffer, such as Hank’s solution, Ringer’s solution, or physiological saline buffer. In other embodiments, one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are formulated for transmucosal administration. In specific embodiments, transmucosal formulations include penetrants that are appropriate to the barrier to be permeated. In still other embodiments wherein the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are formulated for other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions. In specific embodiments, such solutions include physiologically compatible buffers and / or excipients.

[0258] In still other embodiments, the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are formulated for parental injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, formulations for injection are presented in unit dosage form (e.g., in ampoules) or in multi-dose containers. Preservatives are,optionally, added to the injection formulations. In still other embodiments, the pharmaceutical composition of one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein is formulated in a form suitable for parenteral injection as sterile suspension, solution or emulsion in oily or aqueous vehicles. Parenteral injection formulations optionally contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In specific embodiments, pharmaceutical formulations for parenteral administration include aqueous solutions of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein in water-soluble form. In additional embodiments, suspensions of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are prepared as appropriate oily injection suspensions. 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 which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension contains suitable stabilizers or agents which increase the solubility of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein to allow for the preparation of highly concentrated solutions. Alternatively, in other embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- free water, before use.

[0259] In certain embodiments, pharmaceutical compositions are formulated in any conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are optionally used as suitable. Pharmaceutical compositions comprising the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0260] In some embodiments, a pharmaceutical composition comprising one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein illustratively takes the form of a liquid where the agents are present in solution, in suspension or both. Typically when the composition is administered as a solution or suspension a first portion of the agent is present in solution and a second portion of the agent is present in particulate form, in suspension in a liquid matrix. In some embodiments, a liquid composition includes a gel formulation. In other embodiments, the liquid composition is aqueous.

[0261] In certain embodiments, useful aqueous suspension contain one or more polymers as suspending agents. Useful polymers include water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein comprise a mucoadhesive polymer, selected for example from carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran.

[0262] Useful pharmaceutical compositions also, optionally, include solubilizing agents to aid in the solubility of one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein. The term “solubilizing agent” generally includes agents that result in formation of a micellar solution or a true solution of the agent. Certain acceptable nonionic surfactants, for example polysorbate 80, are useful as solubilizing agents, as can ophthalmically acceptable glycols, polyglycols, e.g., polyethylene glycol 400, and glycol ethers.

[0263] Furthermore, useful pharmaceutical compositions optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; 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 an amount required to maintain pH of the composition in an acceptable range.

[0264] Additionally, useful compositions also, optionally, include one or more salts in an amount required to bring 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; suitablesalts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0265] Other useful pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury- containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0266] Still other useful compositions include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

[0267] Still other useful compositions include one or more antioxidants to enhance chemical stability where required. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0268] In certain embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case it is typical to include a preservative in the composition.

[0269] In alternative embodiments, other delivery systems are employed. Liposomes and emulsions are examples of delivery vehicles or carriers useful herein. In additional embodiments, the one or more recombinant proteins (e.g. constructs), nucleic acid molecules, or hypoimmunogenic cells as provided herein are delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials are useful herein. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization are employed.

[0270] In certain embodiments, the formulations described herein comprise one or more antioxidants, metal chelating agents, thiol containing compounds and / or other general stabilizing agents. Examples of such stabilizing agents, 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, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.KITS

[0271] For use in the therapeutic applications described herein, kits and articles of manufacture are also provided. In some embodiments, such kits comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers are formed from a variety of materials such as glass or plastic.

[0272] The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products Include those found in, e.g., U.S. Pat. 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 a selected formulation and intended mode of administration and treatment. For example, the container(s) includes one or more nucleic acid molecules or hypoimmunogenic cells described herein, optionally in a composition. The container(s) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise a composition with an identifying description or label or instructions relating to its use in the methods described herein.

[0273] 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) desirable from a commercial and user standpoint for use of a composition described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. A label is optionally on or associated with the container. For example, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In addition, a label is used to indicate that the contents are to be used for a specific therapeutic application. In addition,the label indicates directions for use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical composition is presented in a pack or dispenser device which contains one or more unit dosage forms comprising one or more recombinant proteins (e.g. constructs), nucleic acid molecules, hypoimmunogenic cells, or pharmaceutical compositions thereof provided herein. The pack for example contains 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 accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In some embodiments, compositions comprising hypoimmunogenic cells formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0274] All publications, patents, and patent applications mentioned in this specification and appendences 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 publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0275] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.P EMBODIMENTS

[0276] P embodiment 1. An amino acid construct comprising: a) one or more major histocompatibility complex (MHC) regions, wherein at least one of said one or more MHC regions comprise a cluster of differentiation 8 (CD8) binding site; b. one or more linker regions; c. a beta-2 microglobulin (B2M) or portion thereof; and d. a peptide.

[0277] P embodiment 2. The construct of P embodiment 1, wherein the peptide is between 3 and 15 amino acid residues in length.

[0278] P embodiment 3. The construct of P embodiment 1 or 2, wherein the peptide is bound within an antigen presenting region of the one or more MHC regions

[0279] P embodiment 4. The construct of any one of P embodiments 1-3, wherein the peptide is a 9 amino acid sequence derived from an HLA leader sequence.

[0280] P embodiment 5. The construct of any one of P embodiments 1-4, wherein the peptide further comprises a mutation.

[0281] P embodiment 6. The construct of P embodiment 5, wherein said mutation in the peptide comprises an amino acid substituted for a cysteine.

[0282] P embodiment 7. The construct of P embodiment 6, wherein said cysteine is in the fifth position in the peptide.

[0283] P embodiment 8. The construct of P embodiment 6, wherein said cysteine is in the ninth position in the peptide.

[0284] P embodiment 9. The construct of any one of P embodiments 6-9, wherein said cysteine forms a disulfide bridge within the construct.

[0285] P embodiment 10. The construct of P embodiment 9, wherein said disulfide bridge is between the peptide and one of the said one or more MHC regions of the construct.

[0286] P embodiment 11. The construct of any one of P embodiments 1-10, wherein the peptide is derived from an HLA-B, HLA-C, HLA-G, or H2A leader sequence.

[0287] P embodiment 12. The construct of any one of P embodiments 1-10, wherein the peptide is VMAPRTLFL (SEQ ID NO: 126).

[0288] P embodiment 13. The construct of any one of P embodiments 1-10, wherein the peptide is derived from an HLA-B*0702 leader sequence.

[0289] P embodiment 14. The construct of any one of P embodiments 1-10, wherein the peptide is VMAPRTVLL (SEQ ID NO: 124).

[0290] P embodiment 15. The construct of any one of P embodiments 1-10, wherein the peptide is derived from an HLA-Cw*0302 leader sequence.

[0291] P embodiment 16. The construct of any one of P embodiments 1-10, wherein the peptide is VMAPRTLIL (SEQ ID NO: 125).

[0292] P embodiment 17. The construct of any one of P embodiments 1-10, wherein the peptide is VMAPCTLFL (SEQ ID NO: 127).

[0293] P embodiment 18. The construct of any one of P embodiments 1-10, wherein the peptide is VMAPRTLFC (SEQ ID NO: 128).

[0294] P embodiment 19. The construct of any one of P embodiments 1-10, wherein the peptide is RIIPRHLQL (SEQ ID NO: 150).

[0295] P embodiment 20. The construct of any one of P embodiments 1-10, wherein the peptide is any one of SEQ ID NO: 124-208.

[0296] P embodiment 21. The construct of any one of P embodiments 1-18, wherein the construct has an increased affinity for a NKG2A / CD94 receptor or a decreased affinity for a NKG2C / CD94.

[0297] P embodiment 22. The construct of any one of P embodiments 1-19, wherein the construct inhibits NK cell lysis when interacting with NK cells.

[0298] P embodiment 23. The construct of any one of P embodiments 1-20, wherein said CD8 binding site comprises one or more mutations.

[0299] P embodiment 24. The construct of any one of P embodiments 1-21, wherein said one or more MHC regions comprise one or more human HLA class 1 heavy chain sequences.

[0300] P embodiment 25. The construct of P embodiment 22, wherein said one or more 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 some combination thereof.

[0301] P embodiment 26. The construct of any one of P embodiments 1-23, wherein the one or more major histocompatibility construct (MHC) regions comprise an HLA-E region.

[0302] P embodiment 27. The construct of P embodiment 25, wherein the HLA-E region is an HLA-E*01:03 region.

[0303] P embodiment 28. The construct of P embodiment 25, wherein the HLA-E region is an HLA-E*01:01 region.

[0304] P embodiment 29. The construct of P embodiment 1, wherein said one or more MHC regions comprise one or more mutations.

[0305] P embodiment 30. The construct of any one of P embodiments 1-28, wherein said one or more mutations are not present in said CD8 binding site.

[0306] P embodiment 31. The construct of P embodiment 1-28, wherein said CD8 binding site comprises one or more mutations.

[0307] P embodiment 32. The construct of P embodiment 28, wherein said one or more mutations comprise a substitution of a cysteine residue at the 77 amino acid position of an HLA-E.

[0308] P embodiment 33. The construct of P embodiment 29, wherein said one or more mutations comprise a substitution of a cysteine residue at the 155 amino acid position of an HLA-E.

[0309] P embodiment 34. The construct of any one of P embodiments 1-29 or 30-33, wherein said CD8 binding site comprises a mutation to the residue corresponding to the Q226 residue of HLA-E.

[0310] P embodiment 35. The construct of any one of P embodiments 1-29 or 30-33, wherein said CD8 binding site comprises a mutation to the residue corresponding to the D227K residue of HLA-E.

[0311] P embodiment 36. The construct of any one of P embodiments 1-29 or 30-33, wherein the Q226 residue of said CD8 binding site is deleted.

[0312] P embodiment 37. The construct of any one of P embodiments 1-36, wherein said construct further comprises one or more disulfide staple pairs.

[0313] P embodiment 38. The construct of P embodiment 37, wherein the one or more disulfide staple pairs create a disulfide bond between said one or more MHC regions and the peptide.

[0314] P embodiment 39. The construct of P embodiment 37 or 38, wherein said disulfide staple pair is formed between the residue of said one or more MHC regions corresponding to the N77 residue of an unmodified HLA-E and a residue in said peptide.

[0315] P embodiment 40. The construct of any one of P embodiments 37-39, wherein said disulfide staple pair is formed between the residue of said one or more MHC regions corresponding to the Hl 55 residue of an HLA-E and a residue in said peptide.

[0316] P embodiment 41. The construct of any one of P embodiments 1 -40, wherein the one or more major histocompatibility construct (MHC) regions comprise a truncated HLA heavy chain sequence.

[0317] P embodiment 42. The construct of P embodiment 41 , wherein truncated HLA heavy chain sequence further comprises one or more C-terminus modifications.

[0318] P embodiment 43. The construct of P embodiment 42, wherein the one or more C-terminus modifications comprise substituting one or more lysines.

[0319] P embodiment 44. The construct of P embodiment 41 , wherein the one or more major histocompatibility construct (MHC) regions comprise a truncated HLA-E.

[0320] P embodiment 45. The construct of P embodiment 44, wherein said truncated HLA-E comprises removal of the amino acids in positions 315-337 of HLA-E.

[0321] P embodiment 46. The construct of P embodiment 42, 44, or 45, wherein said C- terminus modification comprises mutations of one or more of the K310 and K311 residues.

[0322] P embodiment 47. The construct of P embodiment 46, wherein said modification comprises a 310A substitution.

[0323] P embodiment 48. The construct of any one of P embodiments 44 or 45, wherein said modification comprises a 311 A substitution.

[0324] P embodiment 49. The construct of any one of P embodiments 1-48, wherein the one or more major histocompatibility construct (MHC) regions comprise an HLA-G region.

[0325] P embodiment 50. The construct of any one of P embodiments 1-49, wherein the HLA-G region comprises an HLA-G*01 :01 sequence.

[0326] P embodiment 51. The construct of any one of P embodiments 1 -49, wherein the HLA-G region comprises an HLA-G*01 :04 sequence.

[0327] P embodiment 52. The construct of any one of P embodiment 49-51, wherein the HLA-G region comprises one or more mutations.

[0328] P embodiment 53. The construct of P embodiment 52, wherein the one or more mutations comprise a substitution at the Cys-42.

[0329] P embodiment 54. The construct of P embodiment 52, wherein the one or more mutations comprise a substitution at the Cys-147.

[0330] P embodiment 55. The construct of any one of P embodiments 1-54, wherein a linker region of said one or more linker regions 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 NO: 138-144 or SEQ ID NO:122, 123, 209, or 210.

[0331] P embodiment 56. The construct of any one of P embodiments 1-55, comprising a first linker region and a second linker region, wherein said first linker region 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: 192-198; and wherein said second linker region 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 NO:138-144 or SEQ ID NO: 122, 123, 209, or 210.

[0332] P embodiment 57. The construct of any one of P embodiments 1-55, wherein the construct 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 NO:1-121.

[0333] P embodiment 58. The construct of any one of P embodiments 1-57, wherein said construct is soluble.

[0334] P embodiment 59. The construct of any one of P embodiments 1-57, wherein said construct is insoluble.

[0335] P embodiment 60. The construct of any one of P embodiments 1-57, wherein said construct is a single chain trimer (SCT)

[0336] P embodiment 61. The construct of any one of P embodiments 1-57, wherein said construct comprises a single chain dimer (SCD).

[0337] P embodiment 62. The construct of any one of P embodiments 1-61, wherein said construct comprises in N-terminus to C-terminus order: a. a peptide; b. first linker of said one or more linkers; c. a beta-2 microglobulin (B2M) or portion thereof; d. a second linker of the said one or more linkers; and e. an MHC region of said one or more MHC regions.

[0338] P embodiment 63. The construct of P embodiment 63, wherein said construct further comprises a disulfide staple pair configured to associate said peptide and said MHC region.

[0339] P embodiment 64. The construct of P embodiment 63 or 64, wherein said construct further comprises one or more signal sequences.

[0340] P embodiment 65. The construct of any one of P embodiments 62-64, wherein said peptide comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 1-121.

[0341] P embodiment 66. The construct of any one of P embodiments 63-66, wherein said first linker comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 138-144 or SEQ ID NO: 122, 123, 209, or 210.

[0342] P embodiment 67. The construct of any one of P embodiments 63-68, wherein said second linker comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 138-144 or SEQ ID NO:122, 123, 209, or 210.

[0343] P embodiment 68. The construct of any one of P embodiments 63-69, wherein said MHC region comprises a sequence at least about 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to any one of SEQ ID NO: 146-149.

[0344] P embodiment 69. A nucleic acid encoding the construct of any one of any one of P embodiments 1-68.

[0345] P embodiment 70. The nucleic acid of P embodiment 69, wherein the nucleic acid further comprises a signal sequence.

[0346] P embodiment 71. The nucleic acid of P embodiment 70, wherein the signal sequence is a B2M signal sequence.

[0347] P embodiment 72. The nucleic acid of P embodiment 70, wherein the signal sequence is MSRSVALAVLALLSLSGLEA (SEQ ID NO: 135).

[0348] P embodiment 73. An engineered vector encoding the nucleic acid of P embodiment 7L

[0349] P embodiment 74. The engineered vector of P embodiment 72, wherein said vector is a plasmid, a minicircle, an adeno-associated virus (AAV) derived virion, a lentivirus, an adenovirus, or a herpes simplex virus (HSV).

[0350] P embodiment 75. A method of generating a hypoimmunogenic cell comprising administering to a cell said vector of P embodiment 72 or 73.

[0351] P embodiment 76. A method of generating an immune-protective cell comprising administering to a cell said vector of P embodiment 72 or 73.

[0352] P embodiment 77. A cell comprising a complex comprising: A. one or more major histocompatibility complex (MHC) regions, wherein at least one of said one or more MHC regions comprise a cluster of differentiation 8 (CD8) binding site; b. a peptide; and c. one or more linker regions.

[0353] P embodiment 78. The cell of P embodiments 77, wherein the peptide is between 3 and 15 amino acid sequence in length.

[0354] P embodiment 79. The cell of P embodiment 77 or 78, wherein the peptide is bound within an antigen presenting region of the one or more MHC regions.

[0355] P embodiment 80. The cell of any one of P embodiments 77-79, wherein the peptide is a 9 amino acid sequence derived from an HLA leader sequence.

[0356] P embodiment 8L The cell of any one of P embodiments 77-80, wherein the peptide further comprises a mutation.

[0357] P embodiment 82. The cell of P embodiment 81, wherein said mutation in the peptide comprises an amino acid substituted for a cysteine.

[0358] P embodiment 83. The cell of P embodiment 82, wherein said cyst...

Claims

WHAT IS CLAIMED IS:

1. A recombinant protein comprising from the N-terminus to the C- terminus direction: a) a human leukocyte antigen-E (HLA-E) heavy chain binding peptide, wherein said HLA-E heavy chain binding peptide is 9 amino acids in length, and wherein said HLA-E heavy chain binding peptide comprises a cysteine relative to position 5 or position 9 from the N-terminus of SEQ ID NO: 124-126 or SEQ ID NO:151-208; b) a first peptide linker; c) a beta-2 microglobulin (B2M) sequence; d) a second peptide linker; and e) an HLA-E heavy chain sequence, wherein said HLA-E heavy chain sequence comprises a cysteine in a position equivalent to position 77 or position 155 of an HLA-E heavy chain.

2. The recombinant protein of claim 1, wherein said HLA-E heavy chain binding peptide is an HLA-B leader peptide, an HLA-C leader peptide or an HLA-G leader peptide.

3. The recombinant protein of claim 2, wherein said HLA-B leader peptide is the amino acid sequence of SEQ ID NO: 124.

4. The recombinant protein of claim 2, wherein said HLA-C leader peptide is the amino acid sequence of SEQ ID NO: 125.

5. The recombinant protein of claim 2, wherein said HLA-C leader peptide is the amino acid sequence of SEQ ID NO: 126.

6. The recombinant protein of any claim 1, wherein said HLA-E heavy chain binding peptide is the amino acid sequence of any one of SEQ ID NO: 151-208.

7. The recombinant protein of claim 1, wherein said HLA-E heavy chain sequence comprises a cysteine in a position equivalent to position 155 of said HLA- E heavy chain and said HLA-E heavy chain binding peptide comprises a cysteine at a position relative to position 9 from the N-terminus of any one of SEQ ID NO: 124- 126 or SEQ ID NO: 151-208.

8. The recombinant protein of claim 7, wherein said HLA-E heavy chain sequence is covalently bound to said HLA-E heavy chain binding peptide though a disulfide linkage between said cysteine in a position equivalent to position 155 of said HLA-E heavy chain and said cysteine at a position relative to position 9 from the N- terminus of any one of SEQ ID NO: 124-126 or SEQ ID NO: 151-208.

9. The recombinant protein of claim 1, wherein said HLA-E heavy chain sequence comprises a cysteine in a position equivalent to position 77 of said HLA-E heavy chain and said HLA-E heavy chain binding peptide comprises a cysteine at a position relative to position 5 of any one of SEQ ID NO:124-126 or SEQ ID NO:151-208.

10. The recombinant protein of claim 9, wherein said HLA-E heavy chain sequence is covalently bound to said HLA-E heavy chain binding peptide though a disulfide linkage between said cysteine in a position equivalent to position 77 of said HLA-E heavy chain and said cysteine at a position relative to position 5 of any one of SEQ ID NO:124-126 or SEQ ID NO:151-208.

11. The recombinant protein of claim 1 , wherein said HLA-E heavy chain sequence comprises a glutamine to alanine substitution at a position equivalent to position 226 of an HLA-E heavy chain, an aspartic acid to alanine substitution at a position equivalent to position 227 of said HLA-E heavy chain, or a combination thereof.

12. The recombinant protein of claim 1, wherein said HLA-E heavy chain sequence comprises a mutation at a position equivalent to position 310 or position 311 of an HLA-E heavy chain.

13. The recombinant protein of claim 1, wherein said HLA-E heavy chain sequence comprises mutations at positions equivalent to position 310 and position 311 of an HLA-E heavy chain.

14. The recombinant protein of claim 12, wherein said mutation is an alanine substitution.

15. The recombinant protein of claim 1, wherein said HLA-E heavy chain sequence is a truncated an HLA-E heavy chain lacking positions equivalent to positions 315-337 of an HLA-E heavy chain.

16. The recombinant protein of claim 1 , wherein said first peptide linker and said second peptide linker are independently from about 4 amino acids to about 60 amino acids in length.

17. The recombinant protein of claim 16, wherein said first peptide linker and said second peptide linker independently comprise any one of SEQ ID NO: 138- 144 or SEQ ID NO: 122, 123, 209, or 210.

18. The recombinant protein of claim 1, comprising a B2M signal peptide covalently attached to the N-terminus of said HLA-E heavy chain binding peptide.

19. The recombinant protein of claim 1 , wherein said recombinant protein comprises a sequence having at least 90% identity to any one of SEQ ID NO: 1- 116.

20. The recombinant protein of claim 19, wherein said recombinant protein comprises a sequence having at least 95% identity to any one of SEQ ID NO: 1-14.

21. The recombinant protein of claim 20, wherein said recombinant protein comprises a sequence having at least 98% identity to any one of SEQ ID NO: 1-14.

22. The recombinant protein of claim 21 , wherein said recombinant protein comprises a sequence having at least 99% identity to any one of SEQ ID NO: 1-14.

23. The recombinant protein of claim 22, wherein said recombinant protein comprises any one of SEQ ID NO: 1-14.

24. A nucleic acid encoding the recombinant protein of claim 1.

25. A cell comprising the recombinant protein of claim 1.

26. A method of making a hypoimmunogenic cell, comprising delivering to a cell the nucleic acid of claim 24, and culturing said cell under conditions to express the recombinant protein.

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