Engineered major histocompatibility complex molecules and uses thereof

Abstract

Provided herein are engineered MHC molecules compositions for treatment of a disease or disorder. The compositions described herein include an engineered MHC heavy chain; an engineered beta 2-microglobulin (B2m) protein; and / or an MHC class I molecule complex that have been modified to have increased binding affinity to cell coreceptors, such as CD8, and also have increased internalization in immune cells relative to an otherwise comparable MHC class I heavy chain, B2m protein, or MHC class I molecule complex. Various protein constructs and pharmaceutical compositions of the engineered MHC molecules are provided. Methods of utilizing the protein constructs and pharmaceutical compositions of the modified MHC molecules are also provided.

Description

ENGINEERED MAJOR HISTOCOMPATIBILITY COMPLEX MOLECULES AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 574,619, filed April 4, 2024, the contents of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Biochemical interactions between peptide epitope specific membrane molecules encoded by the Major Histocompatibility Complex (MHC, in humans HLA) and T-cell receptors (TCR) elicit specific immune responses in the mammalian adaptive immune system. Therapeutic applications of MHC molecules have been hampered by the difficult production and peptide loading of the MHC monomer protein. The peptide / MHC complex (pMHC) is generally unstable and the dissociation constant (Kd) for peptide binding is highly variable. Moreover, the interactions between the peptide and MHC complex and its binding partners such as T cell receptors and CD8 T cell coreceptors are considered weak which hinders the use of peptide and MHC complexes for therapeutic purposes. Thus, there is a need for improved MHC complex development for therapeutic purposes.A BRIEF SUMMARY

[0003] The compositions and methods provided herein include engineered MHC class I molecules with enhanced stability and potency for the therapeutic treatment of autoimmune diseases, cancer, and other disorders.

[0004] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1 or any MHC class I heavy chain reference amino acid sequence provided herein; and a P2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1. Provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Furtherprovided herein are methods, wherein the disease is an autoimmune disease or a cancer. Provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0005] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117; and a p2- microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0006] Provided herein are compositions comprising an engineered major histocompatibility complex (MHC) class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution in an F pocket in an antigen-binding groove, wherein the at least one amino acid substitution provides for increased stability of the engineered MHC class I heavy chain compared to the stability of an otherwise equivalent molecule comprising a Y84A substitution according to SEQ ID NO: 1, and wherein the at least one amino acid substitution is at an amino acid residue selected from positions 77 to 87, 95, and 135 to 145 according to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Further provided herein are methods,wherein the disease is an autoimmune disease or a cancer. Provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0007] Provided herein are compositions comprising an engineered MHC class I complex, wherein the MHC class I complex comprises an MHC class I heavy chain and a P2-microglobulin (B2m) protein, wherein the composition comprises at least one amino acid substitution, wherein the at least one amino acid substitution provides for increased binding affinity of the engineered MHC class I complex to CD8 on an immune cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, wherein the at least one amino acid substitution is within a CD8 binding interface of the engineered MHC class I complex. Further provided herein are compositions, wherein the CD8 binding interface of the engineered MHC class I complex comprises amino acid substitutions at positions 31, 32, 57, 58, 63, 64, 71, 72, 83, and 84 relative to an amino acid sequence of SEQ ID NO: 2 and amino acid residues at positions 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, 265 relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the compositions further comprise an MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0008] Provided herein are compositions, wherein the compositions comprise: an engineered major histocompatibility complex (MHC) class I heavy chain comprising: at least one amino acid substitution in an F pocket in an antigen-binding groove, wherein the at least one amino acid substitution is at an amino acid residue selected from any one of positions 77 to 87, 95, or 135 to 145 relative to SEQ ID NO: 1, and wherein the at least one amino acid substitution provides for increased stability of the engineered MHC class I heavy chain as compared to the stability of an otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ IDNO: 1. Further provided herein are compositions, wherein the compositions further comprise an MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0009] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain; and an engineered P2-microglobulin (B2m) protein, wherein the engineered B2m protein comprises at least one amino acid substitution relative to a corresponding B2m protein comprising an amino acid sequence of SEQ ID NO: 2, wherein the at least one amino acid substitution in the B2m protein provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the compositions further comprise an MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0010] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I heavy chain comprising an amino acid sequence that is at least about 85% identical to any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the compositions further comprise an MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Further provided hereinare methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0011] Provided herein are compositions, wherein the compositions comprise: any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein; and an antibody or an antibody fragment that specifically binds to a target cell. Further provided herein are compositions, wherein the compositions further comprise an MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0012] Provided herein are compositions, wherein the compositions comprise: a nucleic acid encoding any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein. Further provided herein are compositions, wherein the compositions further comprise a nucleic acid encoding for a MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject the composition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0013] Provided herein are compositions, wherein the compositions comprise: a vector comprising a nucleic acid encoding any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein. Further provided hereinare compositions, wherein the compositions further comprise a nucleic acid encoding for a MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject the composition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0014] Provided herein are compositions comprising a population of cells, wherein the population of cells comprise: any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition, or any nucleic acid provided herein. Further provided herein are compositions, wherein the population of cells further comprise: a MHC class I ligand peptide or a nucleic acid encoding the MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject the population of cells provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject the population of cells provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject the population of cells provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0015] Provided herein are compositions, wherein the compositions comprise: any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition; and a nucleic acid. Further provided herein are compositions, wherein the composition is linked to the nucleic acid. Further provided herein are compositions, wherein the composition is conjugated to the nucleic acid. Further provided herein are compositions, wherein the nucleic acid comprises a therapeutic nucleic acid. Further provided herein are compositions, wherein the compositions further comprise an MHC class I ligand peptide. Further provided herein are methods of delivering a therapeutic nucleic acid to a cell, wherein the methods comprise: contacting the cell with the composition provided herein, thereby delivering the therapeutic nucleic acid to the cell via internalization of the composition. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject thecomposition provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0016] Provided herein are pharmaceutical compositions, wherein the pharmaceutical compositions comprise: (a) any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein; and (b) a pharmaceutically acceptable excipient, diluent, or carrier.

[0017] Provided herein are methods of treating an autoimmune disorder, comprising administering to a subject in need thereof the compositions provided herein or the pharmaceutical compositions provided herein. Further provided herein are methods of treating an autoimmune disorder, wherein the autoimmune disorder is Type 1 diabetes, Celiac disease, rheumatoid arthritis, multiple sclerosis, axial spondylarthritis, birdshot uveitis, psoriasis, ankylosing spondylitis, lupus erythematosus, psoriatic arthritis, scleroderma, inflammatory bowel disease, Sjogren syndrome, or Addison disease.

[0018] Provided herein are methods of treating cancer, comprising administering to a subject in need thereof the compositions provided herein or the pharmaceutical compositions provided herein.

[0019] Provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.

[0020] Provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject a composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0022] FIGURE 1 is a graph illustrating the architecture of an MHC class 1 molecule (shown as a ribbon diagram) bound with a peptide (shown as a stick model). The al - and a2 -helices close off end of the peptide binding groove, fixing the N and C termini of the peptide in the A and F pockets, respectively.

[0023] FIGURES 2A-2B illustrate different tertiary regions of MHC class 1 molecules. FIG. 2A is a ribbon diagram showing the positioning of an MHC class I heavy chain composed of three a helices and a P2-microglobulin (B2m) protein. FIG. 2B is a diagram illustrating the architecture of a peptide-MHC class 1 complex composed of a transmembrane region, a p2-microglobulin (P2m) protein, a heavy chain region, and a peptide / antigen.

[0024] FIGURE 3 shows a schematic representation of the generation of engineered peptide- major histocompatibility complexes (pMHCs). Engineered pMHCs are produced, by introducing function-enhancing mutations in either the P2-microglobulin (P2m), the alpha-3 (u3) domain of the MHC allele, or specific residues within the peptide-binding region (F pocket) of the MHC. These modifications, optionally combined with linkers, result in enhanced properties compared to the natural pMHCs.

[0025] FIGURES 4A-4C show enrichment maps for individual protein variants measured in coreceptor screening. FIG. 4A shows an enrichment map of positive hits for improved CD8 binding (highlighted). FIG. 4B - FIG. 4C show graphs of the enrichment scores from two sequential rounds (round 3 vs. round 4) of selection. FIG. 4B shows an enrichment map of positive hits of MHC Class I variants with improved CD8 binding. FIG. 4C shows an enrichment map of positive hits of B2m variants with improved CD8 binding. Y-axis represents enrichment values from round 4 of recombinant protein screening. X-axis represents enrichment values from round 3 of recombinant protein screening.

[0026] FIGURE 5 shows an analysis of amino acid frequencies in mutated regions of MHC. Frequency of amino acids are shown before selection and after four rounds of selection in HLA- A*02:01. Amino acid mutations that contribute to selection criteria (including binding to CD8) are marked by read frequency that deviates from wild-type. X-axis: amino acid position. Y-axis: amino acid residue.

[0027] FIGURE 6 shows CD8 affinity enhancing mutations identified screens are aligned to a representative set of class I MHC allele sequences. Wild-type residues for the beta-2-microglobulin for each HLA allele are represented by single-letter amino acid symbols with conserved positions across all alleles indicated by a dot. The amino acid mutations identified at each position are shown as a bar graph, showing these mutations fall in highly conserved regions of a pMHC. The figure includes SEQ ID NO: 80 to SEQ ID NO: 95. X-axis: amino acid residue. Y-axis: number of mutations.

[0028] FIGURE 7 shows CD8 affinity enhancing mutations identified screens are aligned to a representative set of class I MHC allele sequences. Wild-type residues for each HLA allele are represented by single-letter amino acid symbols with conserved positions across all alleles indicated by a dot. The amino acid mutations identified at each position are shown as a bar graph, showing these mutations fall in highly conserved regions of a pMHC. The figure includes SEQ ID NO: 80 to SEQ ID NO: 95 X-axis: amino acid residue. Y-axis: number of mutations.

[0029] FIGURE 8 shows screens performed on HLA-A*02:01 (A02) as well as two additional HLA alleles, HLA-B*27:05 (B27) and HLA-C*06:02 (C06). This approach targeted specific positions to identify mutations that function in the specific HLA allele. Shown are the amino acid frequencies for each selected library, with higher frequency corresponding to mutations that contribute to CD8 affinity. X-axis: amino acid positions. Y-axis (left): amino acid. Y-axis (right): frequency read percentage (%).

[0030] FIGURES 9A-9B show scatter plots of amino acid frequency in the A02 screen. FIG. 9A shows amino acid frequency in the A02 screen correlated against B27 amino acid frequencies. X- axis: HLA-A*02:01 library percentage frequency. Y- axis: HLA-B*27:05 library percentage frequency. FIG. 9B shows amino acid frequency in the A02 screen correlated against C06 amino acid frequencies. X-axis: HLA-A*02:01 library percentage frequency. Y- axis: HLA-C*06:02 library percentage frequency.

[0031] FIGURE 10 shows surface plasmon resonance (SPR) analysis demonstrating enhanced CD8 binding affinity of soluble NLV-A02 monomer variants that incorporate different combinations of mutations identified in screen that includes: A02.var3 (84A / 115E substitutions in MHC class I heavy chain, SEQ ID NO: 108); A02.var5 (84A / 115E / 199L / 200A / 215V / 216M substitutions, SEQ ID NO: 109); A02.var6 (84A / 115E / 1891 / 197R / 198W / 215 V / 216M substitutions, SEQ ID NO: 97); A02.var7 (84A / 115E / 216M substitutions, SEQ ID NO: 110); and A02.var8 (84A / 115E / 1891 / 197R / 198W substitutions, SEQ ID NO: 111). This is compared against A02.Q115E (SEQ ID NO: 96) which is the only existing mutation that contributes to overall CD8 affinity in MHC class I, as measured by equilibrium dissociation constant (KD). X-axis: Time. Y- axis: binding response.

[0032] FIGURE 11 shows biolayer interferometry (BLI) analysis demonstrating enhanced CD8 binding affinity of soluble A02 monomer variants (var6 (SEQ ID NO: 97) and varl2 (SEQ ID NO: 98) and B27 fusion variants (var6 and varl2, SEQ ID NO: 123). The affinity for both of these variants greatly exceeds the natural affinity between MHC and CD8 (estimated at 200 pM), demonstrating mutations identified in screen lead to improved engagement of CD8.

[0033] FIGURE 12A-12B show graphs of flow cytometry-based internalization assays illustrating enhanced uptake of engineered soluble pMHC variants A02.Var6 (SEQ ID NO: 97),A02.Varl2 (SEQ ID NO: 98), B27.Var6 (SEQ ID NO: 123), and B27.Varl2 by CD8+ T cells over time, compared to A02.Q115E (SEQ ID NO: 96) and A02 wild-type controls (SEQ ID NO: 117).

[0034] FIGURE 13 shows screens performed on HLA-A*02:01 (A02) to identify stabilizing mutations in the F pocket for two different peptides. This approach introduces mutations at key residues in this region. Shown are the amino acid frequencies for each selected library, with higher frequency corresponding to mutations that contribute to overall protein stability. X-axis: amino acid positions. Y-axis (left): amino acid. Y-axis (right): frequency read percentage (%).

[0035] FIGURE 14A-14F show graphs of thermal stability analysis (melting temperature, Tm) of engineered soluble pMHC variants bearing stabilization mutations.

[0036] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.DETAILED DESCRIPTION OF THE INVENTION

[0037] The following description and examples illustrate embodiments of the invention in detail. It is to be understood that this invention is not limited to the particular embodiments described herein and as such can vary. Briefly, provided herein are (1) engineered major histocompatibility complex (MHC) compositions; (2) fusion proteins and conjugates; (3) nucleic acids; (4) delivery systems; (5) pharmaceutical compositions, dosing, and administration; (6) cells expressing an engineered MHC molecule; and (7) therapeutic applications for the engineered MHC compositions in the treatment of a disease or a disorder.Definitions

[0038] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0039] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as“either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0040] The terms "about" or “approximately” and their grammatical equivalents in relation to a reference numerical value and its grammatical equivalents as used herein can include a range of values plus or minus 10% from that value. For example, the amount "about 10" includes amounts from 9 to 11. The term "about" in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0041] The term "antigen" and its grammatical equivalents as used herein refer to a molecule that contains one or more epitopes or binding sites capable of being bound by one or more receptors or antibodies. For example, an antigen can stimulate a host's immune system to elicit a cellular antigen-specific immune response or a humoral antibody response when the antigen is presented. An antigen can also have the ability to elicit a cellular and / or humoral response by itself or when present in combination with another molecule or other molecules.

[0042] The term "construct" and its grammatical equivalents as used herein refer to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo.

[0043] The term "vector" and its grammatical equivalents as used herein refer to any nucleic acid construct capable of directing the delivery or transfer of a foreign genetic material to target cells, where it can be replicated and / or expressed. The term "vector" as used herein comprises the construct to be delivered. A vector can be a linear or a circular molecule. A vector can be integrating or nonintegrating.

[0044] The term "sequence" and its grammatical equivalents as used herein refer to a nucleotide or amino acid sequence; can be linear, circular, or branched; and can be either single stranded or double stranded. A sequence can be mutated. A sequence can be of any length, for example, between 2 and 1,000 or more amino acids in length (or any integer value there between or there above), e.g., between about 100 and about 10,000 nucleotides or between about 200 and about 500 nucleotides.

[0045] The terms, “bind,” “binding,” “interact” and “interacting,” as used herein, refer to a non- covalent interaction between macromolecules (e.g., between two polypeptides, between a polypeptide and a nucleic acid; between a polypeptide / guide nucleic acid complex and a target nucleic acid; and the like). While in a state of noncovalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Nonlimiting examples of non-covalent interactions are ionic bonds, hydrogen bonds, van der Waals and hydrophobic interactions. Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific.

[0046] The term “nucleotide,” as used herein, can refer to a base-sugar-phosphate combination. The nucleotide can be composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate. The four nucleobases in DNA can include guanine, adenine, cytosine, and thymine; in RNA, uracil can be used in place of thymine. Where DNA sequences are included herein, the corresponding RNA sequences, wherein at least one, two, three, four, five, or all T are replaced with U, are contemplated. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g. , deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)).

[0047] As used herein, the term "treating" or "treatment” refers to clinical intervention in an attempt to alter the disease course of the individual or subject or subject in need thereof or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of the progression of a disease or health condition, amelioration, or palliation of the disease state. By preventing progression of a disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or subject in need thereof or a subject or subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder.

[0048] The term “subject,” as used herein, generally refers to an animal, such as a mammal (e.g., human) or avian (e.g, bird), or other organism, such as a plant. For example, the subject can be a vertebrate, a mammal, a rodent (e.g, a mouse), a primate, a simian or a human. Animals may include, but are not limited to, farm animals, sport animals, and pets. A subject can be a healthy or asymptomatic individual, an individual that has or is suspected of having a disease (e.g., cancer) or a pre-disposition to the disease, and / or an individual that is in need of therapy or suspected of needing therapy. A subject can be a patient.

[0049] As used herein, the terms “protein”, “peptide” and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, “peptide” and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides,whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein”, “peptide” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Amino acid codes provided here are as follows (name / three letter code / single letter code): alanine / ala / A; arginine / arg / R; asparagine / asn / N; aspartic acid / asp / D; asparagine or aspartic acid / asx / B; cysteine / cys / C; glutamic acid / glu / E; glutamine / gin / Q; glutamine or glutamic acid / glx / Z; glycine / gly / G; histidine / his / H; isoleucine / ile / 1; leucine / leu / L; lysine / lys / K; methionine / met / M; phenylalanine / phe / F; proline / pro / P; serine / ser / S; threonine / thr / T; tryptophan / trp / W; tyrosine / tyr / Y; valine / val / V.

[0050] As used herein, the term “affinity” refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen) and is expressed as a dissociation constant (Kd). As used herein, the term “avidity” refers to the resistance of a complex of two or more agents to dissociate after dilution. The terms “immunoreactive” and “preferentially binds” are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0051] As used herein, the term “MHC Class I molecules” or “MHC Class I complex” refers to major histocompatibility complex Class I molecules found on the cell surface of all nucleated cells in the bodies of vertebrates that transport antigenic peptides to the cell surface and present them to cytotoxic T cells. Structurally, MHC Class I molecules or an MHC Class I complex consist of three components: (i) a heavy chain with a molecular weight of about 50 kilodaltons (MHC Class I heavy chain), (ii) a light, non-polymorphic chain, referred to as beta-2 microglobulin, “beta2M”, “P2M”, or “B2m” with a molecular weight of about 10 kilodaltons, and (iii) a peptide which generally consists of 8-10 amino acids which lies in a specific binding groove made by the heavy chain N terminal domain of MHC (FIG. 2B). The first item, i.e., the heavy chain, exhibits genetic polymorphism at its extracellular N-terminus, and a non-polymorphic, partially intracellular C- terminus. The third item, i.e., the peptide, varies, depending upon the nature of the polymorphism in (i).

[0052] As used herein, the term the term “sequence similarity” or “sequence identity” in all their grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and does not necessarily relate to a common evolutionary origin. In a specific embodiment, two amino acid sequences are “substantially homologous” or “substantially identical” when at least about 80%, and most preferably at least about 90 or at least about 95%) of the nucleotides matchover the defined length of the amino acid sequences, as determined by sequence comparison algorithms. Sequence comparison algorithms include, but are not limited to BLAST and FASTA.(1) Engineered MHC Class I Compositions

[0053] Provided herein are engineered MHC class I molecules with enhanced stability and / or potency and compositions comprising engineered MHC class I molecules or MHC class I complexes.F Pocket

[0054] Provided herein are compositions comprising engineered MHC class I molecules or complexes. In some embodiments, an engineered MHC Class I complex comprises an engineered MHC Class I heavy chain. In some embodiments, the engineered MHC class I heavy chain can be an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, or an HLA-G class I heavy chain.

[0055] Provided herein are compositions comprising an engineered MHC class I heavy chain, wherein the MHC class I heavy chain comprises at least one amino acid substitution relative to SEQ ID NO: 1 In some embodiments, the engineered MHC class I heavy chain comprises at least one amino acid substitution in an F pocket. Residues that can be modified in the F pocket are shown in SEQ ID NO: 80 and SEQ ID NO: 1 in bold and underlined font below.

[0056] SEQ ID NO: 80 (wild-type sequence without a Y84A stability amino acid substitution) - F pocket amino acid residues that increase MHC class I stability are shown in bold / underlined font.5 10 15 20 25 30 35 40 45 50GSHSM RYFFT SVSRP GRGEP RFIAV GYVDD TQFVR FDSDA ASQRM EPRAP55 60 65 70 75 80 85 90 95 100WIEQE GPEYW DGETR KVKAH SQTHR VDLGT LRGYY NQSEA GSHTV QRMYG105 110 115 120 125 130 135 140 145 150CDVGS DWRFL RGYHQ YAYDG KDYIA LKEDL RSWTA ADMAA QTTKH KWEAA155 160 165 170 175 180 185 190 195 200HVAEQ LRAYL EGTCV EWLRR YLENG KETLQ RTDAP KTHMT HHAVS DHEAT205 210 215 220 225 230 235 240 245 250LRCWA LSFYP AEITL TWQRD GEDQT QDTEL VETRP AGDGT FQKWA AWVP255 260 265 270 275 278SGQEQ RYTCH VQHEG LPKPL TLRWE PSS

[0057] SEQ ID NO: 1 (Y84A) - F pocket amino acid residues that increase MHC class I stability are shown in bold / underlined font.5 10 15 20 25 30 35 40 45 50GSHSM RYFFT SVSRP GRGEP RFIAV GYVDD TQFVR FDSDA ASQRM EPRAP55 60 65 70 75 80 85 90 95 100WIEQE GPEYW DGETR KVKAH SQTHR VDLGT LRGAY NQSEA GSHTV QRMYG105 110 115 120 125 130 135 140 145 150CDVGS DWRFL RGYHQ YAYDG KDYIA LKEDL RSWTA ADMAA QTTKH KWEAA155 160 165 170 175 180 185 190 195 200HVAEQ LRAYL EGTCV EWLRR YLENG KETLQ RTDAP KTHMT HHAVS DHEAT205 210 215 220 225 230 235 240 245 250LRCWA LSFYP AEITL TWQRD GEDQT QDTEL VETRP AGDGT FQKWA AWVP255 260 265 270 275 278SGQEQ RYTCH VQHEG LPKPL TLRWE PSS

[0058] The sequence of a peptide that can bind to class I MHC molecules is determined by interactions with the amino acid side chains of the peptide-binding groove (also referred to herein as the antigen-binding groove), within which peptides can contact six different binding pockets (A, B, C, D, E, and F). As illustrated in FIG. 1, the al and a2 helices close off the ends of the groove, fixing the N and C termini of peptides in the A and F pockets, respectively. In the case of HLA-I, the F pocket is composed of residues 77, 80, 81, 84, 95, 116, 123, 143, 146, and 147. In some embodiments, the engineered MHC class I heavy chain comprises at least one amino acid substitution in an F pocket within an antigen-binding groove (as highlighted in FIG. 3). In some embodiments, the at least one substitution provides for increased stability of the engineered MHC class I heavy chain compared to the stability of an otherwise equivalent molecule. In some embodiments, the at least one substitution provides for increased stability of the engineered MHC class I heavy chain compared to the stability of an otherwise equivalent molecule comprising a Y84A substitution according to SEQ ID NO: 1 or relative to SEQ ID NO: 80.

[0059] In some embodiments, the amino acid substitutions described above are made at equivalent positions in other MHC Class I heavy chains. “Other MHC class I heavy chains” refers to other MHC class I heavy chains encoded by other MHC class I alleles and have between 20 and 100% amino acid sequence identity to the MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1 or relative to SEQ ID NO: 80. The sequence identity can be determined by sequence alignment algorithm such as BLAST. In some embodiments, an engineered MHC class I heavy chain described herein comprises 1, 2, 3, 4, or 5 substitutions compared to an engineered MHC class I heavy chain encoded by an HLA-A allele, an HLA-B allele, an HLA-C allele, or an HLA-E allele. Exemplary HLA alleles include, but not limited to, HLA-A*01:01, HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06,HLA-A*03:01, HLA-A*24:01, HLA-A*24:02, HLA-A*26:01, HLA-B*27:05, HLA-B*57, HLA- B*15:02, HLA-B *35:01, HLA-B *44:02, HLA-B *51 :01, HLA-B *57:01, HLA-C *06:02, HLA- C*07:02, HLA-C*12:02, HLA-C*15:02, HLA-E*01 :01. In some embodiments, the at least one amino acid substitution is in amino acid residue positions 80 to 87 or 135 to 145 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117. In some embodiments, 2, 3, 4, or 5 amino acid substitutions are made in the engineered MHC class I heavy chain. In some embodiments, the at least one amino acid substitution is at the amino acid positions selected from the group consisting of G83, Y84, Y85, M138, and A139 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117. Exemplary mutations on G83, Y84, Y85, M138, and A139 are listed in Table 1. An MHC composition provided herein can comprises any number or any combination of the amino acid substitutions provided in Table 1 below. In some embodiments, the at least one amino acid substitution is at the amino acid position G83, relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117 In some embodiments, the at least one amino acid substitution is at the amino acid position Y84 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117 In some embodiments, the at least one amino acid substitution is at the amino acid position Y85 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117. In some embodiments, the at least one amino acid substitution is at the amino acid position M138 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117 In some embodiments, the at least one amino acid substitution is at the amino acid position M139 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117

[0060] In some embodiments, an engineered MHC class I heavy chain comprises an amino acid substitution G83A, G83D, G83E, G83H, G83K, G83L, G83M, G83N, G83Q, G83R, G83S, or G83T. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution Y84C, Y84L, Y84M, Y84Q, or Y84S, relative to the sequence of SEQ ID NO: 80. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution A84C, A84L, A84M, A84Q, or A84S, relative to the sequence of SEQ ID NO: 1, SEQ ID NO: 86, or SEQ ID NO: 91. In some embodiments, an engineered MHC class I heavy chain comprises an amino acid substitution Y85F relative to the sequence of SEQ ID NO: 1, SEQ ID NO: 80, SEQ ID NO: 86, or SEQ ID NO: 91 In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution N138A, N138E, N138G, N138K, N138N, N138P, N138Q, N138S, orN138, relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution A139C, A139I, or A139V, relative tothe amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 86, or SEQ ID NO: 91. In some embodiments, the at least one amino acid substitution is G83K, Y84M, M138G, A139V, or any combination thereof relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 86, or SEQ ID NO: 91 In some embodiments, an engineered MHC class I heavy chain comprises two, three or four amino acid substitutions selected from G83K, Y84M, M138G, and A139V relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 86, or SEQ ID NO: 91. In some embodiments, an engineered MHC class I molecule comprises G83K, Y84M, M138G, and A139V relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 86, or SEQ ID NO: 91.Table 1. Exemplary Mutations in the F pocket.

[0061] Protein stability can be assessed in many ways. In some embodiments, the increased stability is assessed by increased thermostability. In some embodiments, the increased stability is assessed by increased shelf-life. Increased thermostability and increased shelf-life can be assessed by monitoring conformational change over a range of temperatures (thermostability) and / or time periods (shelf-life) and / or after exposure to stressful handling situations.

[0062] In some embodiments, the increased stability is assessed by monitoring the protein aggregation rate. In some embodiments, the amount of protein aggregation can be measured by visual observation of turbidity, by measuring absorbance at a specific wavelength, by size exclusion chromatography, HPLC, or other chromatographic methods. In some embodiments, the increased thermal stability is determined by increased melting temperature (Tm) as measured by a spectroscopic method. In some embodiments, the spectroscopic method is circular dichroism or differential scanning calorimetry. In some embodiments, Tm is defined as the temperature at the inflection point of the fluorescence ratio measured at 350 nm over 330 nm.

[0063] In some embodiments, the at least one amino acid substitution provides for increased Tm. In some embodiments, the melting temperature of an engineered MHC class I heavy chain described herein is increased by about 1 degree Celsius to about 15 degrees Celsius as compared to that of an otherwise equivalent molecule. In some embodiments, the melting temperature of the engineered MHC class I heavy chain is increased by about 2 degrees Celsius, 3 degrees Celsius, 4degrees Celsius, 5 degrees Celsius, 6 degree Celsius, 7 degrees Celsius, 8 degrees Celsius, 9 degrees Celsius, 10 degrees Celsius, 11 degrees Celsius, 12 degrees Celsius, 13 degrees Celsius, 14 degrees Celsius, or 15 degrees Celsius, as compared to that of an otherwise equivalent molecule. In some embodiments, the melting temperature of an engineered MHC class I heavy chain described herein is increased by 16 degrees Celsius or higher.

[0064] In some embodiments, the at least one amino acid substitution provides for increased expression yield of the engineered MHC class I molecule as compared to that of an otherwise equivalent MHC class I molecule. In some embodiments, the increased yield is about 1.1 fold, 1.2 fold, about 1.3 fold, about 1.4 fold, about 1.5 fold, about 1.6 fold, about 1.7 fold, about 1.8 fold, about 1.9 fold, 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 11 fold, about 12 fold, about 13 fold, about 14 fold, or about 15 fold.

[0065] In some embodiments, the at least one amino acid substitution provides for increased resistance to pepsin digestion of the engineered MHC class I molecule as compared to that of an otherwise equivalent MHC class I molecule. In some embodiments, the increased resistance is about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 22%, about 24%, about 26%, about 28%, or about 30%.Coreceptor binding regions

[0066] Provided herein are engineered MHC class I heavy chain molecules, engineered P2- microglobulin (B2m) proteins, and engineered MHC class I molecule complexes comprising at least one amino acid substitution that enhances binding to a coreceptor relative to otherwise comparable MHC class I molecules, p2-microglobulin (B2m) proteins, or MHC class I molecule complexes that do not comprise the at least one amino acid substitution. Receptor binding to an MHC molecule is complemented by additional interaction events prior to T cell or natural killer (NK) cell activation. Coreceptors CD4 and CD8 bind to mostly conserved regions on the side of MHC. In the case of MHC class I molecules, based on homology modeling and structure information, the coreceptor CD8 binds to the underside of the ala2 platform and a3 domain of pMHCs (ala2 platform and a3 domain are illustrated in FIG. 2A, and the CD8 binding interface is highlighted in FIG. 3). CD8 is expressed on the surface of cytotoxic T cells (CTLs) as an aP heterodimer or an aa homodimer, where it improves recognition of antigen. CD8 binds MHC-I via two Ig-like ectodomains, one from each CD8 subunit and predominantly contacts the conserved a3-domain (alpha3 domain) and the P2-microglobulin (B2m) domain of MHC class I molecules. In some embodiments, the binding of CD8 to MHC class I molecules recruits the Src family kinaseLck to the TCR signaling complex. This amplifies the signal required for T cell activation, leading to a robust immune response.

[0067] Further provided herein are engineered MHC class I heavy chain molecules, engineered B2m proteins, and engineered MHC class I molecule complexes with increased internalization by a cell relative to an otherwise comparable reference MHC class I molecule complexes. MHC class I molecules are internalized from cell surfaces through endocytosis, where they are transported into endosome and lysosomes. In the lysosomal compartments, the processed peptides (e.g., antigens) are loaded onto the MHC class I molecules, which is crucial for the presentation of antigens to CD8 T cells.

[0068] Provided herein are compositions comprising: at least one engineered MHC class I molecule complex with enhanced binding to CD8. In some embodiments, the engineered MHC class I complex comprises an MHC class I heavy chain; and a P2-microglobulin (B2m) protein. In some embodiments, an engineered MHC class I complex described herein comprises at least one amino acid substitution. In some embodiments, the at least one amino acid substitution provides for increased binding affinity of the engineered MHC class I complex to CD8 on an immune cell surface as compared to an MHC molecule comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is within a CD8 binding interface. In some embodiments, the at least one amino acid substitution is within an engineered MHC class I heavy chain. In some embodiments, the at least one amino acid substitution is within an engineered B2m protein.

[0069] Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is within a CD8 binding interface. In some embodiments, the at least one amino acid substitution is within an engineered MHC class I heavy chain. In some embodiments, the at least one amino acid substitution is within an engineered B2m protein.

[0070] In some embodiments, an engineered MHC class I complex provided herein comprises an engineered B2m protein. In some embodiments, the engineered B2m protein comprises at least one amino acid substitution relative to a B2m protein reference sequence. In some embodiments, the engineered B2m protein comprises at least one amino acid substitution relative to SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is within the B2m protein of an engineered MHC class I complex described herein. The amino acid substitutions may be made at equivalent positions in other B2m proteins. “Other B2m protein” refers to other MHC Class I heavy chains encoded by other B2m proteins and have between 20 and 100% amino acid sequenceidentity to the B2m protein comprising an amino acid sequence of SEQ ID NO: 2. The sequence identity to a reference sequence for B2m can be determined by sequence alignment algorithm such as BLAST.

[0071] In some embodiments, one amino acid substitution is made at least one amino acid residue position selected from: 31, 32, 57, 58, 63, 64, 71, 72, 83, or 84, relative to the sequence of SEQ ID NO: 2. In some embodiments, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions are made at the amino acid positions selected from the group consisting of 31, 32, 57, 58, 63, 64, 71, 72, 83, or 84, relative to the sequence of SEQ ID NO: 2. Exemplary mutations on 31, 32, 57, 58, 63, 64, 71, 72, 83, or 84 are listed in Table 2. In some embodiments, the at least one amino acid substitution is at the amino acid position H31, relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position S32 relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position S57 relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position K58 relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position N63 relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position E64 relative to the sequence of SEQ ID NO: 2 In some embodiments, the at least one amino acid substitution is at the amino acid position T71 relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position P72 relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position N83 relative to the sequence of SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution is at the amino acid position H84 relative to the sequence of SEQ ID NO: 2.

[0072] In some embodiments, the at least one amino acid substitution in the B2m protein comprises a H3 IV substitution relative to the sequence of SEQ ID NO: 2. In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in a B2m protein. In some embodiments, the B2m protein comprises at least one amino acid substitution at any one of positions 21, 31, 32, 57, 58, 63, 64, 71, 72, or 83 relative to SEQ ID NO: 2. In some embodiments, the at least one amino acid substitution in the B2m protein comprises: a S32G substitution, a S32K substitution, or S32A, relative to the sequence of SEQ ID NO: 2. In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the amino acid substitution comprises a S57G substitution, a S57Q substitution, a S57L substitution, a S57N substitution, a S57T substitution, a S57R substitution, or a S57V substitution relative to SEQ ID NO: 2 In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the aminoacid substitution comprises a K58F substitution, a K58T substitution, a K58G substitution, or a K58M substitution relative to SEQ ID NO: 2 In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the amino acid substitution comprises aN63S substitution relative to SEQ ID NO: 2. In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the amino acid substitution comprises a E64T substitution relative to SEQ ID NO: 2. In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the amino acid substitution comprises a T71I substitution relative to SEQ ID NO: 2 In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the amino acid substitution comprises a P72S substitution relative to SEQ ID NO: 2. In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the amino acid substitution comprises a N83R substitution relative to SEQ ID NO: 2. In some embodiments, an engineered MHC class I molecule complex comprises an amino acid substitution in the B2m protein, wherein the amino acid substitution comprises a H84T substitution relative to SEQ ID NO: 2

[0073] In some embodiments, provided herein is a composition comprising an engineered MHC class I molecule complex comprising: an amino acid sequence about 90% identical to about 100% identical that set forth in any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148 In some embodiments, provided herein is a composition comprising an engineered MHC class I molecule complex comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% and 100% identical to that set forth in SEQ ID NO: 13 to SEQ ID NO: 47. In some embodiments, provided herein is a composition comprising an engineered MHC class I molecule complex comprising an amino acid sequence of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148Table 2. B2m Mutations.

[0074] In some embodiments, the at least one amino acid substitution is within an a3 (alpha3) domain of an MHC class I heavy chain of an MHC class I complex described herein. In some embodiments, a MHC I class I heavy chain of an MHC class I complex comprises an amino acid sequence of SEQ ID NO: 1 (or comprises an Y84A mutation relative to SEQ ID NO: 80) as provided in Table 4. The amino acid substitutions may be made at equivalent positions in other MHC class I heavy chains. “Other MHC Class I heavy chains” refers to other MHC Class I heavy chains encoded by other MHC Class I alleles and have between 20 and 100% amino acid sequence identity to the MHC Class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1. The sequence identity can be determined by sequence alignment algorithm such as BLAST. In some embodiments, an engineered MHC class I heavy chain described herein comprises 1, 2, 3, 4, or 5 substitutions compared to an engineered MHC class I heavy chain encoded by an HLA-A allele, an HLA-B allele, an HLA-C allele, or an HLA-E allele. Exemplary HLA alleles include, but not limited to, HLA-A*01:01, HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*03:01, HLA-A*24:01, HLA-A*24:02, HLA-A*26:01, HLA-B*27:05, HLA-B*57, HLA-B*15:02, HLA-B *35:01, HLA-B *44:02, HLA-B *51 :01, HLA-B *57:01, HLA-C *06:02, HLA-C*07:02, HLA-C*12:02, HLA-C*15:02, HLA-E*01 :01. Non-limiting examples of HLA reference sequences that can be used herein are provided in Table 4 and Table 9 (SEQ ID NO: 80 to SEQ ID NO: 95)

[0075] In some embodiments, one amino acid substitution is made in amino acid residue position 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, or 265 relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 In some embodiments, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions are made at the aminoacid positions selected from the group consisting of: 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, 265 relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Exemplary mutations on 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, and 265 are listed in Table 3.

[0076] In some embodiments, the at least one amino acid substitution is at the amino acid position Ml 89, relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position Hl 91 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position Hl 92 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position Hl 97 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position E198 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position A199 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position T200 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position L215 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position T216 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position Q224 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position V231 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position A245 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position V247 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position V248 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position Q255 relative to the sequence of SEQ ID NO: 1. In some embodiments, the at least one amino acid substitution is at the amino acid position G265 relative to the sequence of SEQ ID NO: 1.

[0077] In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution M189I or M189L relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution H191K relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution H192P or Hl 92V, relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an H197E amino acid substitution or a H197R amino acid substitution, relative to the sequence of SEQ ID NO: 1. Insome embodiments, an engineered MHC class I molecule comprises an amino acid substitution E198Y relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution A199L relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution T200A relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution L215V relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution T216M relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution Q224L relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises a V231G amino acid substitution relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution A245D relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution V247S relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution V248D relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises a Q255D amino acid substitution relative to the sequence of SEQ ID NO: 1. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution G265A relative to the sequence of SEQ ID NO: 1.Table 3. Alpha3 Mutations.In some embodiments, an engineered MHC composition, complex, fusion protein, or conjugate provided herein can comprise any F pocket mutation in Table 1, B2m mutation in Table 2, Alpha3 mutation in Table 3, or any sequence listed in Table 4, and / or Tables 8-12, in any combination. For example, an engineered MHC class I heavy chain provided herein can comprise two or more amino acid substitutions provided herein. In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84 and 115 relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95 In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84, 115, and 216 relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95. In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84, 115, 199, 200, 215, and 216 relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95 In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84, 115, 189, 197, and 198 relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95 In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84, 115, 189, 197, 198, 215, and 216 relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95 In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84, 115, 189, 197, 198, 215, 216, and 224 relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95. In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84, 115, 189, 197, 198, 215, 216, 224, and 231 relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95. In some embodiments, an engineered MHC class I heavy chain provided herein comprises amino acid substitutions at positions 84, 115, 189, 197, 198, 215, 216, 224, 231, relative to SEQ ID NO: 1, SEQ ID NO: 80, or SEQ ID NO: 81 - SEQ ID NO: 95 In some embodiments, an engineered B2m protein provided herein comprises an amino acid substitution at position 57relative to SEQ ID NO: 2 In some embodiments, an engineered B2m protein provided herein comprises an amino acid substitution at position 57 and 58 relative to SEQ ID NO: 2.

[0078] The engineered MHC compositions, complexes, fusion proteins, and conjugates described herein have increased binding affinity to cluster of differentiation molecules, for example CD8 and increased stability relative to an MHC molecule that does not comprise a mutation or a combination of mutations provided herein. The engineered MHC compositions, complexes, fusion proteins, and conjugates described herein can also be internalized by an immune cell. In some embodiments, the immune cell that internalizes the compositions provided herein comprise a T cell or a natural killer (NK) cell. In some embodiments, an engineered MHC composition, complex, fusion protein, or conjugate provided herein further comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitution comprises amino acid substitution of QI 15E, relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117. The QI 15E mutation increases CD8 binding affinity in the MHC class I heavy chain. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by 1% to about 100%. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by at least about 10%, 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%, or 100%. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or 10%.

[0079] In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by about 1.1-fold to about 10-fold. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 3-fold, about 4-fold, about 5- fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold.

[0080] In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by about 10-fold to about 1000-fold. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by about 10-fold to about 100-fold.

[0081] In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is increased by about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60- fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about95-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, or about 1000-fold.

[0082] In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 100 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 90 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 80 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 70 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 60 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 50 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 40 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 30 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 20 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 15 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 10 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 5 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 1 gM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 500 nM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 400 nM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 300 nM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 200 nM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 100 nM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8is with a dissociation constant (Kd) of less than 50 nM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 20 nM. In some embodiments, the binding affinity of an engineered MHC class I molecule described herein to CD8 is with a dissociation constant (Kd) of less than 10 nM.

[0083] In some embodiments, provided herein is a composition comprising an engineered MHC class I heavy chain comprising an amino acid sequence about 90% identical to about 100% identical that set forth in SEQ ID NO: 7 to SEQ ID NO: 45, SEQ ID NO: 57 to SEQ ID NO: 79. In some embodiments, provided herein is a composition comprising an engineered MHC class I heavy chain comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% and 100% identical to a sequence set forth in any one of SEQ ID NO: 7 to SEQ ID NO: 45, SEQ ID NO: 57 to SEQ ID NO: 79 or a combination thereof. In some embodiments, provided herein is a composition comprising an engineered MHC class I molecule comprising an amino acid sequence of any one of SEQ ID NO: 7 to SEQ ID NO: 45, SEQ ID NO: 57 to SEQ ID NO: 79 or a combination thereof.(2) Fusion Proteins and Conjugates

[0084] Provided herein are fusion proteins and protein conjugates comprising engineered MHC molecules described herein. A fusion protein includes, for example, a protein comprising at least two heterologous polypeptides. The fusion protein can comprise one or more effector proteins and effector partners. In some instances, an effector protein and effector partner are not found connected to one another as a native protein or complex that occurs together in nature.MHC / Peptide Complexes

[0085] Provided herein are compositions comprising MHC class I molecules described herein and an MHC ligand peptide (“MHC / peptide complex” or “pMHC”). In some embodiments, a composition described herein comprises an MHC heavy chain, a P2-microglobulin (B2m) protein, and / or an MHC ligand peptide. In some embodiments, the MHC ligand peptide is an MHC antigen peptide. In some embodiments, an antigen peptide sequence can be that of a peptide which can be presented by an MHC class I molecule, in various configurations, an antigen peptide sequence can comprise from about 8 to about 15 contiguous amino acids. In some embodiments, the antigen peptide sequence can comprise 9 contiguous amino acids. In various aspects, a peptide sequence can be that of a protein fragment, wherein the protein is a pathogen protein or a cellular protein. In some embodiments, the pathogen protein or the cellular protein is a protein expressed by a cancercell. In some embodiments, the pathogen protein or the cellular protein is a protein expressed by a virally infected cell. In some embodiments, an antigen can comprise an antigen peptide such as that of an HLA-A restricted peptide or HLA-B restricted peptide, such as an HLA-A*0201- restricted peptide. In some embodiments, an antigen peptide can comprise a sequence as set forth in any one of SEQ ID NO: 46 - SEQ ID NO: 51, TRLALIAPK (SEQ ID NO: 118), or VRSRRCLRL (SEQ ID NO: 150).

[0086] In some embodiments, the peptide-MHC complexes are engineered as single chain trimers (SCTs). In some embodiments, the single chain trimers described herein comprises an antigenic peptide followed by a first flexible linker that connects the C terminus of the peptide to the N terminus of P2-microglobulin (P2m), and a second flexible linker, which connects the C terminus of P2m to the N terminus of the heavy chain. In some embodiments, a SCT comprises, in amino- to-carboxy terminal order, the MHC class I ligand peptide, a first flexible linker, the P2- microglobulin protein, a second flexible linker sequence and the MHC class I heavy chain sequence. In some embodiments, the first flexible linker has an amino acid sequence comprising GGGGSGGGGSGGGGS (SEQ ID NO: 53). In some embodiments, the second linker has an amino acid sequence comprising GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 54). In some embodiments, the linker has an amino acid sequence comprising GCGGSGGGGSGGGGS (SEQ ID NO: 119) In some embodiments, the linker has an amino acid sequence comprising GSGGGGSGGGGS (SEQ ID NO: 120). In some embodiments, the linker has an amino acid sequence comprising GSGGGGSGGGGS (SEQ ID NO: 121).

[0087] Provided herein are compositions comprising an engineered MHC class I heavy chain provided in Table 4 or an amino acid sequence having at least 85% identity to any one of the amino acid sequences selected from: SEQ ID NO: 7 to SEQ ID NO: 45, SEQ ID NO: 57 to SEQ ID NO: 79, or a functional fragment thereof. Provided herein are compositions comprising an engineered B2m protein provided in Table 4 or an amino acid sequence having at least 85% identity to any one of the amino acid sequences selected from: SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148, or a functional fragment thereof. Provided herein are compositions comprising any combination of engineered MHC class I heavy chains, engineered B2m proteins, and MHC ligand peptides provided herein (for example any amino acid sequence corresponding to any one of SEQ ID NO: 46 to SEQ ID NO: 51, SEQ ID NO: 118, or SEQ ID NO: 150)Table 4. Amino Acid Sequences of MHC Molecules and Peptides.Conjugates

[0088] Provided herein are engineered MHC molecules conjugated to a functional moiety. In some embodiments, the function moiety is a small molecule, a protein, a nucleic acid, an siRNA, an antibody, an antibody fragment, or an oligonucleotide. In some embodiments, conjugation of an engineered MHC molecule can occur by providing a nucleic acid that encodes for an engineered MHC molecule, an amino acid linker, and the functional moiety (for example, a chemical or enzyme moiety) or, conjugation of the functional moiety (for example, a cytokine) via chemical conjugation. Conjugating engineered MHC molecules result in enhanced biological properties and other activity profile measures including: i) targeted cytotoxicity, ii) half-life, iii) biological activity, iv) specificity, v) stability, and / or vi) targeted delivery. In some embodiments, the engineered cytokine is conjugated to at least one of: i) a toxin, ii) a fusion protein, iii) an antibody or an antibody fragment, or iv) another chemical, protein, or polymer. Fusion proteins include, for example, Fc fusion proteins and albumin fusion proteins. MHC-albumin fusion proteins can exhibit increased biological activity and half-life properties. In some embodiments are MHC-Fc fusion protein provided herein exhibits targeted cytotoxicity properties. In some embodiments, the antibody or the antibody fragment provided herein comprises a single domain antibody, a heavychain only antibody (HCAb), a single chain antigen-binding fragment (ScFab), a fragment antigenbinding (e.g., Fab, Fab', Fab'-SH, F(ab')2) domain, a fragment crystallizable (Fc) domain, a single chain variable fragment (e.g. scFv), single-chain antibody molecules, a minibody, an antibody, diabodies, or linear antibodies. In some embodiments, the Fc domain comprises a sequence that is at least 85% identical to:EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALGAPI EKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGS (SEQ ID NO: 149). In some embodiments, the antibody or antibody fragment is selected from the group consisting of: abagovomab, abciximab, abituzumab, abrilumab, actinium Ac-225 lintuzumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anetumab ravtansine, anifrolumab, anrukinzumab, apolizumab, sevacizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atorolimumab, avelumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, 177Lu-tetraxetan-tetulomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bivatuzumab mertansine, blinatumomab, blosozumab, bococizumab,brentuximab vedotin, BrevaRex, briakinumab, brodalumab, brolucizumab, brontictuzumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, CBR96-doxorubicin immunoconjugate, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine, conatumumab, concizumab, cR6261, crenezumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, darleukin, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab, depatuxizumab mafodotin, derlotuximab biotin, detumomab, dinutuximab, diridavumab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, durvalumab, dusigitumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, FGFR2 Antibody-Drug Conjugate, Fibromun, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab tiuxetan, icrucumab, idarucizumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, indusatumab vedotin, inebilizumab, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, labetuzumab govitecan, lambrolizumab, lampalizumab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, leukotuximab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, milatuzumab-SN-38, minretumomab, mirvetuximab soravtansine, mitazalimab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab, pasudotox, MU1053, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab,PankoMab-GEX, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranibizumab, raxibacumab, refanezumab, regavirumab, reslizumab, rilotumumab, rinucumab, risankizumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, sacituzumab govitecan, samalizumab, sarilumab, satumomab pendetide, secukinumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talizumab, tanezumab, tanibirumab, taplitumomab paptox, tarextumab, tefibazumab, teleukin, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tetulomab, Thorium-227-Epratuzumab Conjugate, ticilimumab, tigatuzumab, tildrakizumab, tisotumab vedotin, tocilizumab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab deruxtecan, trastuzumab emtansine, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, utomilumab, ustekinumab, vadastuximab talirine, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, zolimomab aritox, any fragments thereof, or any biosimilars thereof.

[0089] Provided herein compositions comprising a homodimer of an MHC class I heavy chain and a fragment crystallizable (Fc) region of an antibody. In some embodiments, the homodimer comprises an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 100 to SEQ ID NO: 104

[0090] Targeted delivery properties of the compositions provided herein can also be tailored using immunocytokines. Immunocytokines are molecules that combine a tumor directed antibody, a cytotoxic drug, and an engineered cytokine described herein. In some embodiments, the compositions provided herein comprise multivalent MHC fusions with enhanced specificity and potency through avidity. In some cases, the multivalent MHC fusion comprises multiple binding domains resulting in enhanced avidity. In some cases, a first MHC class I heavy chain fragment is linked to a second MHC class I heavy chain fragment. In some embodiments, the MHC fusion is a chimeric MHC class I heavy chain.

[0091] Also described herein are compositions comprising MHC-cell conjugates. In some embodiments, an engineered MHC class I molecule or an engineered MHC class I complexdescribed herein is present on the surface of a cell. MHC-cell conjugates include T-cell fusion moieties which allows for local, concentrated activity of otherwise toxic anti-tumor MHC.

[0092] Also described herein are MHC-drug conjugates. An MHC-drug conjugate can comprise an engineered MHC class I heavy chain provided herein; an engineered B2m protein provided herein; or a combination thereof; and a therapeutic agent. The therapeutic agent can include but is not limited to compounds, small molecules, proteins, biologies, antibodies, anti-cancer drugs, antitumor drugs, anti-inflammatory drugs, pro-inflammatory drugs, and therapeutic nucleic acids. In some embodiments, the therapeutic agent comprises an siRNA, an oligonucleotide, a gene editing system (for example, a CRISPR / Cas system and a guide nucleic acid), an RNA therapeutic, a base editor, a TALEN, a zinc finger, a nanoparticle, or any combination thereof. The MHC class I heavy chains and complexes provided herein when in contact with a cell are internalized by the cell, which permits the targeted delivery of nucleic acids into a cell.(3) Nucleic Acids

[0093] Engineered MHC class I molecules, complexes or fusion proteins described herein are made using recombinant DNA techniques available to one skilled in the art. Nucleic acid sequences which encode for the selected peptides of the disclosure may be incorporated in a known manner into appropriate expression vectors (i.e., recombinant expression vectors). In some embodiments, appropriate expression vectors are recombinant expression vectors. Exemplary expression vectors include, but are not limited to, cosmids, bacmids, plasmids, or modified viruses, so long as the vector is compatible with the host cell used. In some embodiments, modified viruses are replication defective retroviruses, adenoviruses and adeno-associated viruses, lentiviruses; herpes viruses, poxviruses. In some embodiments, a nucleic acid provided herein is linked to at least one regulatory sequence. The regulatory sequence can be linked in a manner which allows for expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacteria), fungal, or viral genes. Selection of appropriate regulatory sequence(s) is dependent on the host cell(s) chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include the following: a transcriptional promoter and enhancer, RNA polymerase binding sequence, or a ribosomal binding sequence (including a translation initiation signal). Depending on the host cell chosen and the expression vector employed, other additional sequences (such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription) may be incorporated into the expression vector.

[0094] Also provided here are vectors that comprise nucleic acids coding for at least one engineered MHC molecule in the present disclosure. In some embodiments, the polypeptidescomprising the engineered MHC molecules or complexes provided herein may also be produced using cell free protein expression systems.

[0095] In some embodiments, the engineered MHC molecules, complexes, or fusion proteins of the present disclosure are provided with a cell membrane penetrating peptide, such as a TAT protein transduction domain. TAT-fusions have been shown to cross cell membranes and, in some instances, blood barriers.

[0096] In some embodiments, the engineered MHC molecules, complexes, or fusion proteins described herein are labelled to facilitate their detection in a variety of assays as is understood by one of skill in the art. Such labels may include but are not limited to radioactive label, biotin, a magnetic label, a paramagnetic label, a radiodense label, an enzyme, a hapten, a cytotoxic label, a luminescent label, a fluorescent label and nucleic acid labels. In some embodiments, the engineered MHC molecules, complexes, or fusion proteins described herein couple to bovine serum albumin (BSA) or keyhole limpet hemocyanin. The peptides may be covalently or non- covalently coupled to a solid carrier such as a microsphere of gold or polystyrene, a slide, chip or to a wall of a microtiter plate. The peptide may be labelled directly or indirectly with a label selected from but not limited to biotin, fluorescein and an enzyme such as horseradish peroxidase alkaline phosphatase, or luciferase. In some embodiments, the engineered MHC molecules, complexes, or fusion proteins are preceded by a Biotin N-terminal sequence that may facilitate peptide concentration determination by OD280 (of Tyr or Y) measurement.(4) Delivery Systems

[0097] Provided herein are compositions comprising an engineered MHC molecule, a fusion protein or a conjugate comprising an engineered MHC and a delivery vehicle. In some embodiments, an MHC class I molecule that is internalized by a cell can serve as a delivery vehicle for a nucleic acid (e.g., an RNA or a DNA). The compositions provided herein can be delivered to a target cell, tissue, organ, or subject by any suitable means. In some embodiments, the engineered MHC molecule, fusion protein or the conjugate and delivery vehicle can be delivered to a target cell, tissue, organ, or subject by any suitable means. Provided herein are methods of delivering a nucleic acid to a cell, wherein the methods comprise: contacting the cell with a composition provided herein and the nucleic acid, thereby delivering the nucleic acid to the cell via internalization of the composition.

[0098] The compositions as described herein can be admixed with a delivery vehicle that permits delivery of the system to the target nucleic acid sequence. In some embodiments, the delivery vehicle comprises a vector, a lipid, a nanoparticle, a plasmid, a virus, a liposome, an extracellular vesicle, or a combination thereof. Additional non-limiting examples of delivery vehicles includean emulsion, a suspension, a liposome, a micelle, an exosome, an endosome, a virus, a vector, a particle, a nanoparticle, a polymer, microcapsules, recombinant cells, cell culture medium, blood, or serum. Specific types of delivery vehicles that can be used in a composition provided herein are further described below.

[0099] In some embodiments, the delivery vehicle is a liposome. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms containing an aqueous solution in the core. Liposomes interact with cells via different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one can operate at the same time. Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkylcyanoacrylate nanoparticles can also be used as a delivery vehicle.

[0100] In some embodiments, the delivery vehicle is a phospholipid. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios, the liposomes form. Physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a tightly packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars, and drugs.

[0101] In some embodiments, the delivery vehicle is a nanoparticle. Nanoparticle carriers that specifically target a tissue provided herein may also be used as a pharmaceutically acceptable carrier. In some embodiments, the nanoparticle is a gold nanoparticle, a platinum nanoparticle, an iron-oxide nanoparticle, a lipid nanoparticle, a selenium nanoparticle, a tumor-targeting glycolchitosan nanoparticle (CNP), a cathepsin B sensitive nanoparticle, a hyaluronic acid nanoparticle, a paramagnetic nanoparticle, or a polymeric nanoparticle.

[0102] Compositions comprising nucleic acids coding for engineered MHC molecules or fusion proteins provided herein can be delivered to a cell system using vectors, for example containing polynucleotide sequences encoding a system, a guide polynucleotide, an engineered protein, or a composition provided herein. In some embodiments, a system as described herein can be delivered absent a viral vector. Any vector systems can be used including, but not limited to, plasmid vectors, viral vectors, and oncolytic viral vectors. Furthermore, any of these vectors can comprise one or more transcription factor, transgene, or molecular tag.

[0103] In some embodiments, the vectors provided herein are viral vectors. Exemplary viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adeno-associated viral vectors (AAV), adenoviral vectors, herpes simplex viral vectors, alpha viral vectors, flaviviral vectors, rhabdoviral vectors, measles viral vectors, Newcastle disease viral vectors, poxviral vectors, picornaviral vectors, and oncolytic viral vectors.

[0104] In some embodiments, the viral vector comprises an AAV. AAVs can have one or more of the AAV wild-type genes deleted in whole or part, e.g, the rep and / or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication, and packaging of the AAV virion. The ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g, by the insertion, deletion, or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging. A recombinant AAV vector (rAAV) comprises an infectious, replication-defective virus composed of an AAV protein shell encapsulating a heterologous nucleotide sequence of interest that is flanked on both sides by AAV ITRs. An rAAV vector is produced in a suitable host cell comprising an AAV vector, AAV helper functions, and accessory functions. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the AAV vector (containing a recombinant nucleotide sequence of interest) into infectious recombinant virion particles for subsequent gene delivery. In some embodiments, the AAV or the rAAV provided herein comprises a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV12, AAV13, AAVrhlO, or any combination thereof.

[0105] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is selected from the group consisting of: a human immunodeficiency virus I (HIV- 1); a human immunodeficiency virus 2 (HIV-2), a visna-maedi virus (VMV) virus; a caprine arthritis-encephalitis virus (CAEV): an equine infectious anemia virus (EIAV); a feline immunodeficiency virus (FIV); a bovine immune deficiency virus (BIV); and a simian immunodeficiency vims (SIV), fragments, derivatives, or variants thereof.

[0106] Conventional viral and non-viral based gene transfer methods can be used to introduce polynucleotides encoding for a composition, system, guide polynucleotide, or an engineered protein provided herein to cells (e.g., mammalian cells) and target tissues. Exemplary non-viral vector delivery systems can include DNA plasmids, naked nucleic acid, and nucleic acids complexed with a delivery vehicle such as a liposome or poloxamer. Viral vector delivery systems can also include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.

[0107] Methods of non-viral delivery of nucleic acids include electroporation, lipofection, nucleofection, gold nanoparticle delivery, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, mRNA, artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. Additional exemplary nucleic acid delivery systems include those provided by AMAXA® Biosystems (Cologne, Germany), Life Technologies (Frederick, Md.), MAXCYTE, Inc. (Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) and Copernicus Therapeutics Inc. Lipofection reagents are sold commercially (e.g., TRANSFECTAM® and LIPOFECTIN®).

[0108] Delivery of the engineered MHC molecules, the fusion proteins, or the conjugates provided herein can be to cells (ex vivo administration) or target tissues (in vivo administration). Additional methods of delivery include the use of packaging the polynucleotides to be delivered into EnGenelC delivery vehicles (EDVs). These EDVs are specifically delivered to target tissues using bispecific antibodies where one arm of the antibody has specificity for the target tissue and the other has specificity for the EDV. The antibody brings the EDVs to the target cell surface and then the EDV is brought into the cell by endocytosis.

[0109] Vectors including viral and non-viral vectors containing nucleic acids encoding a nucleic editing system provided herein can also be administered directly to an organism for transduction of cells in vivo. Alternatively, naked DNA or mRNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells including, but not limited to, injection, infusion, topical application, and electroporation. More than one route can be used to administer a particular composition.

[0110] In some embodiments, a vector encoding for a composition provided herein can be shuttled to a cellular nucleus. For example, a vector can contain a nuclear localization sequence (NLS). A vector can also be shuttled by a protein or protein complex. In some embodiments, nucleic acids encoding engineered MHC molecules or fusion proteins described herein can be introduced to a cell or a target tissue by a minicircle vector.[OHl] In some embodiments, a vector or a polynucleotide provided herein can be pre-complexed with an engineered protein provided herein prior to electroporation into a cell. An engineered protein that can be used for shuttling can be a nickase or a catalytically dead Cas protein. A nuclease that can be used for shuttling can be a nuclease-competent protein. In some embodiments, an engineered protein herein can be pre-mixed with a guide polynucleotide provided herein an any additional elements (e.g., transgenes or other engineered proteins).

[0112] A cell can be transfected with a mutant or chimeric adeno-associated viral vector encoding at least one engineered MHC molecule or a fusion protein comprising at least one engineered MHC molecule provided herein. For example, an AAV vector concentration can be from 0.5 nanograms to 50 micrograms.

[0113] Nucleic acids encoding at least one engineered MHC molecule or a fusion protein comprising an engineered MHC molecule, or a conjugate provided herein can also be introduced to a cell via electroporation techniques. The number of polynucleotides that can be introduced into the cell by electroporation can be varied to optimize transfection efficiency and / or cell viability. In some embodiments, less than about 100 picograms of nucleic acid can be added to each cell sample (e.g., one or more cells being electroporated). In some embodiments, at least about 100 picograms, at least about 200 picograms, at least about 300 picograms, at least about 400 picograms, at least about 500 picograms, at least about 600 picograms, at least about 700 picograms, at least about 800 picograms, at least about 900 picograms, at least about 1 microgram, at least about 1.5 micrograms, at least about 2 micrograms, at least about 2.5 micrograms, at least about 3 micrograms, at least about 3.5 micrograms, at least about 4 micrograms, at least about 4.5 micrograms, at least about 5 micrograms, at least about 5.5 micrograms, at least about 6 micrograms, at least about 6.5 micrograms, at least about 7 micrograms, at least about 7.5 micrograms, at least about 8 micrograms, at least about 8.5 micrograms, at least about 9 micrograms, at least about 9.5 micrograms, at least about 10 micrograms, at least about 11 micrograms, at least about 12 micrograms, at least about 13 micrograms, at least about 14 micrograms, at least about 15 micrograms, at least about 20 micrograms, at least about 25 micrograms, at least about 30 micrograms, at least about 35 micrograms, at least about 40 micrograms, at least about 45 micrograms, or at least about 50 micrograms, of nucleic acid can be added to each cell sample (e.g., one or more cells being electroporated). For example, 1 microgram of dsDNA can be added to each cell sample for electroporation. In some embodiments, the amount of nucleic acid (e.g., dsDNA) required for optimal transfection efficiency and / or cell viability can be specific to the cell type. In some embodiments, the amount of nucleic acid (e.g., dsDNA) used for each sample can directly correspond to the transfection efficiency and / or cell viability. The transfection efficiency of cells with any of the nucleic acid delivery platforms described herein,for example, nucleofection or electroporation, can be or can be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more than 99.9%.

[0114] Viral particles, such as AAV, can be used to deliver a viral vector comprising a gene of interest or a transgene into a cell ex vivo or in vivo. In some embodiments, a mutated or chimeric adeno-associated viral vector as disclosed herein can be measured as pfu (plaque forming units). In some embodiments, the pfu of recombinant virus or mutated or chimeric adeno-associated viral vector of the compositions and methods of the disclosure can be about 108to about 5* 1010pfu. In some embodiments, recombinant viruses of this disclosure are at least about l><108, 2*108, 3*108, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xl010, 4xlO10, and 5xl010pfu. In some embodiments, recombinant viruses of this disclosure are at most about IxlO8, 2xl08, 3xl08, 4xl08, 5xl08, 6xl08, 7xl08, 8xl08, 9xl08, IxlO9, 2xl09, 3xl09, 4xl09, 5xl09, 6xl09, 7xl09, 8xl09, 9xl09, IxlO10, 2xlO10, 3xl010, 4xlO10, and 5xl010pfu. In some embodiments, a mutated or chimeric adeno-associated viral vector of the disclosure can be measured as vector genomes. In some embodiments, recombinant viruses of this disclosure are IxlO10to 3xl012vector genomes, or IxlO9to 3xl013vector genomes, or IxlO8to 3 xlO14vector genomes, or at least about IxlO1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO15, IxlO16, 1 x 1017, and IxlO18vector genomes, or are 1 x 108to 3 x 1014vector genomes, or are at most about IxlO1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO15, IxlO16, IxlO17, and IxlO18vector genomes.

[0115] In some embodiments, a mutated or chimeric adeno-associated viral vector of the disclosure can be measured using multiplicity of infection (MOI). In some embodiments, MOI can refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic can be delivered. In some embodiments, MOI can refer to the ratio, or multiple of vector or viral genomes to the cells to which the nucleic can be delivered. In some embodiments, the MOI can be IxlO6GC / mL. In some embodiments, the MOI can be IxlO5GC / mL to IxlO7GC / mL. In some embodiments, the MOI can be IxlO4GC / mL to IxlO8GC / mL. In some embodiments, recombinant viruses of the disclosure are at least about IxlO1GC / mL, IxlO2GC / mL, IxlO3GC / mL, IxlO4GC / mL, IxlO5GC / mL, IxlO6GC / mL, IxlO7GC / mL, IxlO8GC / mL, IxlO9GC / mL, IxlO10GC / mL, IxlO11GC / mL, IxlO12GC / mL, IxlO13GC / mL, IxlO14GC / mL, IxlO15GC / mL, IxlO16GC / mL, IxlO17GC / mL, and IxlO18GC / mL MOI. In some embodiments, a mutated or chimeric adeno-associated viruses of this disclosure are from about IxlO8GC / mL to about 3xl014GC / mL MOI, or are at most about IxlO1GC / mL, IxlO2GC / mL, IxlO3GC / mL, IxlO4GC / mL, IxlO5GC / mL, IxlO6GC / mL, IxlO7GC / mL, IxlO8GC / mL, IxlO9GC / mL,1 x io10GC / mL, I x lO11GC / mL, I x lO12GC / mL, I x lO13GC / mL, I x lO14GC / mL, I x lO15GC / mL, 1 x io16GC / mL, I x lO17GC / mL, and I x lO18GC / mL MOI.

[0116] In some embodiments, a non-viral vector or nucleic acid can be delivered without the use of a mutated or chimeric adeno-associated viral vector and can be measured according to the quantity of nucleic acid. Generally, any suitable amount of nucleic acid can be used with the compositions and methods of this disclosure. In some embodiments, nucleic acid can be at least about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g. In some embodiments, nucleic acid can be at most about 1 pg, 10 pg, 100 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 pg, 10 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng, 700 ng, 800 ng, 900 ng, 1 mg, 10 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 2 g, 3 g, 4 g, or 5 g.

[0117] Proteins, vectors, plasmids, compositions, systems, engineered proteins, and guide polynucleotides provided herein can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection, and protoplast fusion. The methods used to construct any embodiment of the compositions provided herein include genetic engineering, recombinant engineering, and synthetic techniques.

[0118] An engineered MHC molecule, a fusion protein comprising an engineered MHC molecule, or a conjugate provided herein can be delivered to a cell by electroporation. Electroporation using, for example, the NEON® Transfection System (Thermo Fisher Scientific) or the AMAXA® Nucleofector (AMAXA® Biosystems) can also be used for delivery of nucleic acids and proteins into a cell. For example, an engineered protein provided herein can be purified and complexed with a suitable guide polynucleotide for delivery into a cell. Electroporation parameters can be adjusted to optimize delivery efficiency and / or cell viability. Electroporation devices can have multiple electrical wave form pulse settings such as exponential decay, time constant and square wave. Every cell type has a unique optimal Field Strength (E) that is dependent on the pulse parameters applied (e.g., voltage, capacitance, and resistance). Application of optimal field strength causes electro-permeabilization through induction of transmembrane voltage, which allows nucleic acids to pass through the cell membrane. In some embodiments, the electroporation pulse voltage, the electroporation pulse width, number of pulses, cell density, and tip type can be adjusted to optimize transfection efficiency and / or cell viability.(5) Pharmaceutical Compositions, Dosing, and Administration

[0119] Provided herein is a pharmaceutical composition comprising the engineered MHC molecules provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient. Further provided herein is a pharmaceutical composition comprising a vector provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient. Further provided herein is a pharmaceutical composition comprising a vector provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient. Further provided herein is a pharmaceutical composition comprising an oncolytic virus provided herein; and a pharmaceutically acceptable diluent, carrier, or excipient.

[0120] In some embodiments, compositions provided herein (e.g., engineered proteins provided herein) are combined with pharmaceutically acceptable salts, excipients, and / or carriers to form a pharmaceutical composition. Pharmaceutical salts, excipients, and carriers may be chosen based on the route of administration, the location of the target issue, and the time course of delivery of the drug. A pharmaceutically acceptable carrier or excipient may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration.

[0121] In some embodiments, the pharmaceutical composition is in the form of a solid, semi-solid, liquid, or gas (aerosol). Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0122] In some embodiments, the pharmaceutical composition is in the form of a gel. In some embodiments, the gel is a fibrin gel, a fibrinogen gel, or a fibronectin gel formulated for delivery to a surgical site.

[0123] Compositions provided herein may be formulated in dosage unit form for ease of administration and uniformity of dosage. A dosage unit form is a physically discrete unit of acomposition provided herein appropriate for a subject to be treated. It will be understood, however, that the total usage of compositions provided herein will be decided by the attending physician within the scope of sound medical judgment. For any composition provided herein the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, such as mice, rabbits, dogs, pigs, or non-human primates. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50 / ED50. Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments. The data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for human use.

[0124] Provided herein are compositions and pharmaceutical compositions for administering to a subject in need thereof (e.g., as a treatment for cancer). In some embodiments, pharmaceutical compositions provided here are in a form which allows for compositions provided herein to be administered to a subject. In some embodiments, the pharmaceutical composition is formulated for intratumoral delivery. In some embodiments, administration of a pharmaceutical composition provided herein is local administration or systemic administration. In some embodiments, a pharmaceutical composition provided herein is formulated for administration / for use in administration via an intratumoral, intravascular (e.g., via the bladder), rectal, subcutaneous, topical, oral, intradermal, intramuscular, inhalation, intranasal, intravenous, intraperitoneal, intraocular, or intracranial route. In some embodiments, the administering is every 1, 2, 4, 6, 8, 12, 24, 36, or 48 hours. In some embodiments, the administering is daily, weekly, or monthly. In some embodiments, the administering is repeated at least about every 24 hours, every 48 hours, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, or every 56 days.

[0125] In some embodiments, the pharmaceutical composition described herein is in a form of a sustained release formulation. In some embodiments, the sustained release formulation comprises a carrier. In some embodiments, the carrier is a polymer matrix. In some embodiments, the polymer matrix comprises acetylated pullulan, alginate, chitosan, collagen, gelatin, glycerin, hyaluronic acid, poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA) polycaprolactone (PCL), polyethylene glycol (PEG), poly D, L-lactide-poly ethylene glycol(PELA), or a combination thereof. In some embodiments, the carrier is a liposome, an extracellular vesicle, a nanoparticle, or a lipid. In some embodiments, the carrier is a hydrogel.

[0126] Provided herein are devices, wherein the devices comprise a composition described herein. In some embodiments, the devices comprise an engineered MHC class I molecule described herein. In some embodiments, the devices comprise a pharmaceutical composition provided herein. In some embodiments, the devices comprise a vector provided herein. In some embodiments, the devices comprise a cell provided herein. In some embodiments, the device is an implantable device. For example, a device that is implanted following tumor resection in the site of the resected tumor in a subject (also referred to herein as a resection cavity). In some embodiments, the device is implanted in combination with cytoreductive surgery in a subject, administration of an anticancer agent to the subject, and / or administration of radiation to the subject.

[0127] A composition provided herein can be loaded by injection of a solution or suspension into a well inside the device followed by solvent removal by drying, evaporation, or lyophilization, or by placement of a composition provided herein in tablet or particulate form into the well. In some embodiments, the device comprises a biocompatible matrix. In some embodiments, the biocompatible matrix is a polymer, a silicone, a plastic, or a metal. In some embodiments, the biocompatible matrix comprises polysiloxane, silicone, or siloxane. In some embodiments, the device is implanted into a tissue of a subject via a catheter, cannula or biopsy needle. The device can be sized to permit placement using a catheter, cannula, or stylet. In some embodiments, the device has a guidewire to assist in placement and retrieval. The device may also include features that assist in maintaining spatial stability of tissue excised with the device, such as fins or stabilizers that can be expanded from the device prior to or at the time of removal. Optionally, the device has fiber optics, sensors and / or interactive features such as remote accessibility (such as Wi-Fi) to provide for in situ retrieval of information and modification of device release properties.

[0128] In some embodiments, the device, allows for the engineered protein or pharmaceutical composition provided herein to achieve a zero-order rate release profile into a tissue of a subject. Composition release pharmacokinetics are a function of the composition solubility, excipients, dimensions of the device or vessel within the device, and tissue into which the device is implanted (with greater rate of release into more highly vascularized tissue, than into less vascular tissue).

[0129] The devices provided herein may also include materials such as radiopaque materials or materials that can be imaged using ultrasound or MRI. They can be manufactured using techniques such as deep ion etching, nano imprint lithography, micromachining, laser etching, three- dimensional printing or stereolithography.(6) Cells Expressing an Engineered MHC Molecule

[0130] The cells provided herein are genetically modified to express a transgene. Provided herein are cells expressing an engineered MHC molecule provided herein. In some embodiments, the cell is a genetically modified cell. In some embodiments, the cell is contacted in vitro or ex vivo with a polynucleotide encoding for an engineered MHC molecule provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with a vector encoding for an engineered MHC molecule provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with an oncolytic virus encoding for an engineered MHC molecule provided herein. In some embodiments, the genetically modified cell is a chimeric antigen receptor (CAR) T cell, an engineered T cell receptor (TCR) T cell, or a tumor-infiltrating lymphocyte.

[0131] An engineered cell provided herein can be from any source, including but not limited to a mammal or a human. In some embodiments, the cell is a lymphocyte, a leukocyte, a myeloid cell, a T cell, a natural killer cell, a macrophage, a dendritic cell, a stem cell, an induced pluripotent stem-cell derived myeloid cell, an induced pluripotent stem-cell derived leukocyte, an induced pluripotent stem-cell derived lymphocyte. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a human induced pluripotent stem cell. In some embodiments, the stem cell is a human adult stem cell. In some embodiments, the stem cell is differentiated in-vitro or ex vivo to generate a cell comprising a least one differentiated cell marker. In some embodiments, the genetically modified cell is an in-vitro differentiated cell.

[0132] Provided herein are methods of targeting a leukocyte to kill a cancer cell. Further provided herein are methods of generating a cytotoxic leukocyte, the methods comprising: contacting a leukocyte ex vivo with a polynucleotide provided herein or a vector provided herein. In some embodiments, the method comprises contacting a cell with a polynucleotide encoding an engineered protein provided herein, a vector of provided herein, or an oncolytic virus provided herein. In some embodiments, the contacting is in vitro or in vivo. In some embodiments, the leukocyte is a T cell, a neutrophil, a natural killer cell, or a macrophage. In some embodiments, the leukocyte is an allogenic leukocyte or autologous leukocyte.

[0133] Cells provided herein can be administered to a subject in need thereof, e.g., a subject with cancer or an autoimmune disease. Therapeutic applications are discussed further below.(7) Therapeutic Applications

[0134] Provided herein are methods of treating a disease or a condition in a subject, wherein the methods comprise: administering to the subject a composition provided herein (e.g., the engineered protein, pharmaceutical composition, vector, cell, or device), thereby treating the disease or the condition in the subject. In some embodiments, the subject has an inflammatory disease or disorder. In some embodiments, the subject is suspected of having, has, or is at risk of developinga chronic inflammatory disease or has chronic inflammation. In some embodiments, the subject is suspected of having, has, or is at risk of developing an autoimmune disorder. In some embodiments, the subject is suspected of having, has, or is at risk of developing a hyperproliferative disease or condition. In some embodiments, the hyperproliferative disease is cancer.

[0135] The methods provided herein also include methods of modulating an immune response in a subject having a disease. In some embodiments, the methods comprise reducing inflammation in a subject. Provided herein are methods of treating a subject that has been diagnosed with, is suspected of having, or has an autoimmune disorder. An autoimmune disorder includes, but is not limited to Type-1 diabetes, rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barre syndrome, chronic thyroiditis, multiple sclerosis, multiple myositis, ankylosing spondylitis, fibrositis, or polyarteritis nodosa.

[0136] In some embodiments, the methods comprise generating an immune response at a site of a tumor in a subject to reduce tumor size. Provided herein are methods of treating a subject that has been diagnosed with, is suspected of having, or has cancer, the methods comprising administering a composition provided herein. Further provided herein are methods of reducing cancer cell proliferation and tumor growth. Further provided herein are methods of inducing an immune response to a cancer cell in a subject. Further provided herein are methods of inducing immune cell recruitment to a tumor. In some embodiments, compositions provided herein are used for reduction of a tumor size. In some embodiments, compositions provided herein are used for reduction of a tumor volume. In some embodiments, compositions provided herein are used for reduction of a cancer recurrence. In some embodiments, compositions provided herein are used for reduction of tumor metastasis. In some embodiments, the subject has been administered an anti-cancer agent prior to administration of the composition provided herein. In some embodiments, the subject has resistance to chemotherapeutic treatment of the cancer.

[0137] Provided herein are methods of treating a subject in need thereof by administering to the subject therapeutically effective dose of a composition or a pharmaceutical composition described herein. In some embodiments, the therapeutically effective dose is a dose sufficient to induce an inflammatory response, to promote tumor reduction, or both. In some embodiments, a therapeutically effective amount is an amount sufficient to reduce, ameliorate, or prevent at least one symptom of a disease or condition.Exemplary Embodiments:

[0138] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1 or any MHC class I heavy chain reference amino acid sequence provided herein; and a P2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1. Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117; and a p2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 80. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 80. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 80. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 81. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200,211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 81. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 82. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 82. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 83. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 83. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 84. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 84. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 85. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 85. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 86. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 86. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 87. Further provided herein are compositions, wherein the at least one amino acid substitution in theengineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 87. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 88. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 88. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 89. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 89. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 90. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 90. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 91. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 91. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 92. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 92. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 93. Furtherprovided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 93. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 94. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 94. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 95. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 95. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197-200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 117. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises 2, 3, 4, or 5 substitutions compared to an MHC class I heavy chain encoded by an HLA-A allele, an HLA-B allele, an HLA-C allele, or an HLA-E allele. Further provided herein are compositions, wherein the HLA-A allele is HLA-A*02:01 allele. Further provided herein are compositions, wherein the HLA-A allele is HLA-A*24:02 allele. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by 10% up to about 100% as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by at least about 10%, 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%, or at least about 100% as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class Icomplex to CD8 is increased by about 1.1-fold to about 10-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 1.1-fold, about 1.2- fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7- fold, about 8-fold, about 9-fold, or about 10-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold to about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60- fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, or about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 100 pM. Further provided herein are compositions, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 1 pM. Further provided herein are compositions, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 100 nM. Further provided herein are compositions, wherein the P2-microglobulin (B2m) protein comprises at least one amino acid substitution. Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is an amino acid substitution at a position selected from the group consisting of: 31, 32, 57, 58, 63, 64, 71, 72, 83, 84, and a combination thereof relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is at amino acid position S57 relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is a S57G substitution, a S57T substitution, a S57R substitution, or a S57V substitution relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is an amino acid substitution at amino acid position K58 relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is a K58F substitution, a K58T substitution, a K58G substitution, or a K58M substitution relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is a S57Vsubstitution, a K58Q substitution, a N83R substitution, an H84T substitution, or a combination thereof relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is an S27V substitution relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain comprises an amino acid substitution at an amino acid position selected from: 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, 265, and any combination thereof relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position corresponding to Hl 97 relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an H197E substitution or an H197R substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an A199L substitution, a T200A substitution, a L215V substitution, a T216M substitution, or a combination thereof relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a T216M substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a L215V substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V231G amino acid substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V247I substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V248E substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a Q255D substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the engineered MHC class I heavy chain further comprises an amino acid substitution at position 115 relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the amino acid substitution in Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises a QI 15E substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, orSEQ ID NO: 117 Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 96% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 97% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 98% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 99% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is 100% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the composition further comprises an MHC class I ligand peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide. Further provided herein are compositions, wherein the MHC class I antigen peptide is an MHC class I self-peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues. Further provided herein are compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein. Further provided herein are compositions, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide. Furtherprovided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, or SEQ ID NO: 118. Further provided herein are compositions, wherein the engineered MHC class I heavy chain, the P2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order: (i) the MHC class I ligand peptide; (ii) a first flexible linker; (iii) the B2m protein;(iv) a second flexible linker; and (v) the engineered MHC class I heavy chain. Further provided herein are compositions, wherein the engineered MHC class I heavy chain is present on a surface of a cell. Further provided herein are compositions, wherein the engineered MHC class I complex is in soluble form.

[0139] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain; and an engineered P2-microglobulin (B2m) protein, wherein the engineered B2m protein comprises at least one amino acid substitution relative to a corresponding B2m protein comprising an amino acid sequence of SEQ ID NO: 2, wherein the at least one amino acid substitution in the B2m protein provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered B2m protein is at an amino acid position selected from: 31, 32, 57, 58, 63, 64, 71, 72, 83, or 84 relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by 10% up to about 100% as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by at least about 10%, 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%, or at least about 100% as compared to the otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the binding affinity of the engineered MHC complex to CD8 is increased by about 1.1-fold to about 10-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 1.1-fold, about 1.2-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold as compared to the otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold to about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising an MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 80. Further provided herein are compositions, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60- fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, or about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2. Further provided herein are compositions, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 100 pM. Further provided herein are compositions, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 1 pM. Further provided herein are compositions, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 100 nM. Further provided herein are compositions, wherein the P2-microglobulin (B2m) protein comprises at least one amino acid substitution. Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is at amino acid position S57 relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is a S57G substitution, a S57T substitution, a S57R substitution, or a S57V substitution relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is an amino acid substitution at amino acid position K58 relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is a K58F substitution, a K58T substitution, a K58G substitution, or a K58M substitution relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is a S57V substitution, a K58Q substitution, a N83R substitution, an H84T substitution, or a combination thereof relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the at least one amino acid substitution in the B2m protein is an S27V substitution relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the engineered B2m protein comprises: anamino acid sequence that is at least 95% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148 Further provided herein are compositions, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 96% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148. Further provided herein are compositions, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 97% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148. Further provided herein are compositions, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 98% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148 Further provided herein are compositions, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 99% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148. Further provided herein are compositions, wherein the engineered B2m protein comprises: an amino acid sequence that is 100% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 148 Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain comprises an amino acid substitution at an amino acid position selected from: 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, 265, and any combination thereof relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position corresponding to H197 relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an H197E substitution or an H197R substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an A199L substitution, a T200A substitution, a L215V substitution, a T216M substitution, or a combination thereof relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a T216M substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a L215V substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V231G amino acid substitution relative to SEQ IDNO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V247I substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V248E substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a Q255D substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the engineered MHC class I heavy chain further comprises an amino acid substitution at position 115 relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the amino acid substitution in Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises a Q115E substitution relative to SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 96% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 97% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 98% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 99% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is 100% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, SEQ ID NO: 57 to SEQ ID NO: 79, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the composition further comprises an MHC class I ligand peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide. Further provided herein are compositions, wherein the MHC class I antigen peptide is an MHC class I self-peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues. Further provided herein are compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein. Further provided herein are compositions, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, or SEQ ID NO: 118. Further provided herein are compositions, wherein the engineered MHC class I heavy chain, the P2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order: (i) the MHC class I ligand peptide; (ii) a first flexible linker; (iii) the B2m protein;(iv) a second flexible linker; and (v) the engineered MHC class I heavy chain. Further provided herein are compositions, wherein the engineered MHC class I heavy chain is present on a surface of a cell. Further provided herein are compositions, wherein the engineered MHC class I complex is in soluble form.

[0140] Provided herein are compositions, wherein the compositions comprise: an engineered major histocompatibility complex (MHC) class I heavy chain comprising: at least one amino acid substitution in an F pocket in an antigen-binding groove, wherein the at least one amino acid substitution is at an amino acid residue selected from any one of positions 77 to 87, 95, or 135 to 145 relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117, and wherein the at least one amino acid substitution provides for increased stability of the engineered MHC class I heavy chain as compared to the stability of an otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises 1, 2, 3, 4, or 5 amino acid substitutions compared to an engineered MHC class I heavy chain encoded by an HLA-A allele. Further-n- provided herein are compositions, wherein the HLA-A allele is a HLA-A*02:01 allele. Further provided herein are compositions, wherein the HLA-A allele is an HLA-A*24:02 allele, an HLA- B*27:05 allele, or an HLA-C*06:02 allele. Further provided herein are compositions, wherein the increased stability is characterized by an increased thermal stability of the engineered MHC class I heavy chain as measured by a spectroscopic method. Further provided herein are compositions, wherein the spectroscopic method is differential scanning fluorimetry or circular dichroism. Further provided herein are compositions, wherein the spectroscopic method determines a melting temperature of the engineered MHC class I heavy chain or the otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1, wherein the melting temperature of the engineered MHC class I heavy chain is increased by about 2 degrees Celsius, 3 degrees Celsius, 4 degrees Celsius, 5 degrees Celsius, 6 degree Celsius, 7 degrees Celsius, 8 degrees Celsius, 9 degrees Celsius, 10 degrees Celsius, 11 degrees Celsius, 12 degrees Celsius, 13 degrees Celsius, 14 degrees Celsius, or 15 degrees Celsius, as compared to that of the otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1. Further provided herein are compositions, wherein the spectroscopic method determines a melting temperature of the at least one engineered MHC class I heavy chain or the otherwise equivalent molecule, wherein the melting temperature of the at least one engineered MHC class I heavy chain is increased by at least 5 degrees Celsius as compared to that of the otherwise MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1. Further provided herein are compositions, wherein the spectroscopic method determines a melting temperature of the engineered MHC class I heavy chain, wherein the melting temperature of the engineered MHC class I heavy chain is increased by at least 9 degrees Celsius as compared to that of the otherwise equivalent MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased yield of the engineered MHC class I heavy chain in an expression system as compared to an otherwise equivalent expression system comprising the MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1. Further provided herein are compositions, wherein the increased yield of the engineered MHC class I heavy chain in the expression system is 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 11 fold, about 12 fold, about 13 fold, about 14 fold, or about 15 fold greater than the otherwise equivalent expression system comprising the MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1. Further provided herein are compositions, wherein the at least one amino acid substitution provides for increased potency of the engineered MHC class I heavy chain as compared to the otherwise equivalent MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1. Further provided herein are compositions,wherein the increased potency of the engineered MHC class I heavy chain is measured by a binding affinity of the engineered MHC class I heavy chain to at least one antigen as compared to a binding affinity of the otherwise equivalent MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1 to the at least one antigen. Further provided herein are compositions, wherein the at least one amino acid substitution is at an amino acid residue position selected from: 78, 80, 81, 83, 84, 85, 138, or 139 of the engineered MHC class I heavy chain relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117, optionally wherein the engineered MHC class I heavy chain is encoded by an HLA-A *02:01 allele. Further provided herein are compositions, wherein the engineered MHC class I heavy chain is an HLA-A molecule, and wherein the at least one amino acid substitution is at amino acid residue position of G83, Y84, Y85, M138, or A139, relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution is G83K, Y84M, M138G, A139V, or any combination thereof relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution is G83K relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117. Further provided herein are compositions, wherein the at least one amino acid substitution is A84M relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 1171 Further provided herein are compositions, wherein the at least one amino acid substitution is M138G relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution is A139V relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the at least one amino acid substitution is G83K, A84M, M138P, A139V relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80 to SEQ ID NO: 95, or SEQ ID NO: 117 Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises an amino acid sequence that is at least about 95%, at least about 97%, or at least about 99% identical to any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprises an amino acid sequence comprising any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the composition further comprises a P2-microglobulin (B2m) protein. Further provided herein are compositions, wherein the composition further comprises an MHC class I ligand peptide. Further provided herein arecompositions, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide. Further provided herein are compositions, wherein the MHC class I antigen peptide is an MHC class I self-peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues. Further provided herein are compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein. Further provided herein are compositions, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, SEQ ID NO: 118, or SEQ ID NO: 150 Further provided herein are compositions, wherein the engineered MHC class I heavy chain, the P2- microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order: (i) the MHC class I ligand peptide; (ii) a first flexible linker; (iii) the B2m protein;(iv) a second flexible linker; and (v) the engineered MHC class I heavy chain. Further provided herein are compositions, wherein the engineered MHC class I heavy chain is present on a surface of a cell. Further provided herein are compositions, wherein the engineered MHC class I complex is in soluble form.

[0141] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I heavy chain comprising an amino acid sequence that is at least about 85% identical to any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116 Further provided herein are compositions, wherein the composition further comprises a B2m protein comprising at least one amino acid substitution relative to SEQ ID NO: 2 Further provided herein are compositions, wherein the composition further comprises a B2m protein comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 124 to SEQ ID NO: 148. Further provided herein are compositions, wherein the composition further comprises an MHC class I ligand peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide. Further provided herein are compositions, wherein the MHC class I antigen peptide is an MHC class I self- peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues. Further provided herein are compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein. Further provided herein are compositions, wherein the MHC class I ligand peptideis a virus peptide or tumor peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, SEQ ID NO: 118, or SEQ ID NO: 150. Further provided herein are compositions, wherein the engineered MHC class I heavy chain, the P2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order: (i) the MHC class I ligand peptide; (ii) a first flexible linker; (iii) the B2m protein;(iv) a second flexible linker; and (v) the engineered MHC class I heavy chain. Further provided herein are compositions, wherein the engineered MHC class I heavy chain is present on a surface of a cell. Further provided herein are compositions, wherein the engineered MHC class I complex is in soluble form.

[0142] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I heavy chain; and a B2m protein comprising an amino acid sequence that is at least about 85% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 124 to SEQ ID NO: 148. Further provided herein are compositions, wherein the engineered MHC class I heavy chain comprising an amino acid sequence that is at least about 85% identical to any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116. Further provided herein are compositions, wherein the composition further comprises an MHC class I ligand peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide. Further provided herein are compositions, wherein the MHC class I antigen peptide is an MHC class I self-peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues. Further provided herein are compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein. Further provided herein are compositions, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, SEQ ID NO: 118, or SEQ ID NO: 150 Further provided herein are compositions, wherein the engineered MHC class I heavy chain, the P2- microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order: (i) the MHC class I ligand peptide; (ii) a first flexible linker; (iii) the B2m protein;(iv) a secondflexible linker; and (v) the engineered MHC class I heavy chain. Further provided herein are compositions, wherein the engineered MHC class I heavy chain is present on a surface of a cell. Further provided herein are compositions, wherein the engineered MHC class I complex is in soluble form.

[0143] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 86; and a P2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 86. Further provided herein are compositions, wherein the composition further comprises a B2m protein comprising at least one amino acid substitution relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the composition further comprises a B2m protein comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 124 to SEQ ID NO: 148 Further provided herein are compositions, wherein the composition further comprises an MHC class I ligand peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide. Further provided herein are compositions, wherein the MHC class I antigen peptide is an MHC class I self-peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues. Further provided herein are compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein. Further provided herein are compositions, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, SEQ ID NO: 118, or SEQ ID NO: 150. Further provided herein are compositions, wherein the engineered MHC class I heavy chain, the p2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order: (i) the MHC class I ligand peptide; (ii) a first flexible linker; (iii) the B2m protein;(iv) a second flexible linker; and (v) the engineered MHC class I heavy chain. Further provided herein are compositions, wherein the engineered MHC class I heavy chain ispresent on a surface of a cell. Further provided herein are compositions, wherein the engineered MHC class I complex is in soluble form.

[0144] Provided herein are compositions, wherein the compositions comprise: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 91; and a P2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 91. Further provided herein are compositions, wherein the composition further comprises a B2m protein comprising at least one amino acid substitution relative to SEQ ID NO: 2. Further provided herein are compositions, wherein the composition further comprises a B2m protein comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 124 to SEQ ID NO: 148 Further provided herein are compositions, wherein the composition further comprises an MHC class I ligand peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide. Further provided herein are compositions, wherein the MHC class I antigen peptide is an MHC class I self-peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues. Further provided herein are compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein. Further provided herein are compositions, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide. Further provided herein are compositions, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, SEQ ID NO: 118, or SEQ ID NO: 150. Further provided herein are compositions, wherein the engineered MHC class I heavy chain, the p2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order: (i) the MHC class I ligand peptide; (ii) a first flexible linker; (iii) the B2m protein;(iv) a second flexible linker; and (v) the engineered MHC class I heavy chain. Further provided herein are compositions, wherein the engineered MHC class I heavy chain is present on a surface of a cell. Further provided herein are compositions, wherein the engineered MHC class I complex is in soluble form.

[0145] Provided herein are compositions, wherein the compositions comprise: any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein; and an antibody or an antibody fragment that specifically binds to a target cell. Further provided herein are compositions, wherein the target cell comprises an immune cell or a cancer cell. Further provided herein are compositions, wherein the antibody or the antibody fragment comprises a single domain antibody, a heavy-chain only antibody (HCAb), a single chain antigen-binding fragment (ScFab), a fragment antigen-binding (e.g., Fab, Fab', Fab'- SH, F(ab')2) domain, a fragment crystallizable (Fc) domain, a single chain variable fragment (e.g. scFv), single-chain antibody molecules, a minibody, an antibody, diabodies, or linear antibodies.

[0146] Provided herein are compositions, wherein the compositions comprise: a nucleic acid encoding any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein. Provided herein are compositions, wherein the compositions comprise: a vector encoding any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein. Further provided herein are compositions, wherein the compositions further comprise a nucleic acid encoding for a MHC class I ligand peptide.

[0147] Provided herein are compositions, wherein the compositions comprise: a vector comprising a nucleic acid encoding any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein. Further provided herein are compositions, wherein the compositions further comprise a nucleic acid encoding for a MHC class I ligand peptide.

[0148] Provided herein are compositions comprising a population of cells, wherein the population of cells comprise: any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition, or any nucleic acid provided herein. Further provided herein are compositions, wherein the population of cells further comprise: a MHC class I ligand peptide or a nucleic acid encoding the MHC class I ligand peptide. Further provided herein are methods of treating a disease in a subject, wherein the methods comprise: administering to the subject the population of cells provided herein, thereby treating the disease in the subject. Further provided herein are methods, wherein the disease is an autoimmune disease or a cancer. Further provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject the population of cells provided herein, thereby reducing inflammation in a subject. Further provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject the population of cells provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size.

[0149] Provided herein are compositions, wherein the compositions comprise: any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition, or any nucleic acid provided herein; and a nucleic acid. Further provided herein are compositions, wherein the composition is linked to the nucleic acid. Further provided herein are compositions, wherein the composition is conjugated to the nucleic acid. Further provided herein are compositions, wherein the nucleic acid comprises a therapeutic nucleic acid. Further provided herein are compositions, wherein the therapeutic nucleic acid comprises an RNA, a DNA, an siRNA, a guide nucleic acid, a gene editing system, or an oligonucleotide. Further provided herein are compositions, wherein the compositions further comprise a MHC class I ligand peptide or a nucleic acid encoding for the MHC class I ligand peptide. Further provided herein are methods of delivering a therapeutic nucleic acid to a cell, wherein the methods comprise: contacting the cell with a composition provided herein and a therapeutic nucleic acid, thereby delivering the therapeutic nucleic acid to the cell via internalization of the composition.

[0150] Provided herein are pharmaceutical compositions, wherein the pharmaceutical compositions comprise: (a) any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, or any composition provided herein; and (b) a pharmaceutically acceptable excipient, diluent, or carrier.

[0151] Provided herein are methods of treating an autoimmune disorder in a subject, wherein the methods comprise: administering to the subject any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition provided herein, or pharmaceutical composition provided herein, thereby treating the autoimmune disorder in the subject. Further provided herein are methods, wherein the autoimmune disorder is Type 1 diabetes, Celiac disease, rheumatoid arthritis, multiple sclerosis, axial spondylarthritis, birdshot uveitis, psoriasis, ankylosing spondylitis, lupus erythematosus, psoriatic arthritis, scleroderma, inflammatory bowel disease, Sjogren syndrome, or Addison disease. Further provided herein are methods, wherein the administering is local administration or systemic administration. Further provided herein are methods, wherein the administering is subcutaneous administration or intravenous administration. Further provided herein are methods, wherein the administering is every 8 hours, every 12 hours, every 24 hours, every 48 hours, every 72 hours, every 96 hours, or every 120 hours. Further provided herein are methods, wherein the administering is every 6 days, every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, or every 56 days.

[0152] Provided herein are methods of treating cancer in a subject, wherein the methods comprise: administering to the subject any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition provided herein, or pharmaceuticalcomposition provided herein, thereby treating the cancer in the subject. Further provided herein are methods, wherein the administering is local administration or systemic administration. Further provided herein are methods, wherein the administering is intratumoral administration, intravenous administration, intravascular administration, rectal administration, subcutaneous administration, topical administration, oral administration, intradermal administration, intramuscular administration, via inhalation, intranasal administration, intraperitoneal administration, intraocular administration, or intracranial administration. Further provided herein are methods, wherein the subject has a solid tumor. Further provided herein are methods, wherein the solid tumor is a carcinoma, a melanoma, or a sarcoma. Further provided herein are methods, wherein the subject has a blood cancer. Further provided herein are methods, wherein the blood cancer is a lymphoma or a leukemia. Further provided herein are methods, wherein the administering is every 8 hours, every 12 hours, every 24 hours, every 48 hours, every 72 hours, every 96 hours, or every 120 hours. Further provided herein are methods, wherein the administering is every 6 days, every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, or every 56 days.

[0153] Provided herein are methods of reducing inflammation in a subject, wherein the methods comprise: administering to the subject any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition provided herein, or pharmaceutical composition provided herein, thereby reducing inflammation in a subject. Further provided herein are methods, wherein the inflammation is caused by an autoimmune condition. Further provided herein are methods, wherein the autoimmune condition is Type 1 diabetes, Celiac disease, rheumatoid arthritis, multiple sclerosis, axial spondylarthritis, birdshot uveitis, psoriasis, ankylosing spondylitis, lupus erythematosus, psoriatic arthritis, scleroderma, inflammatory bowel disease, Sjogren syndrome, or Addison disease. Further provided herein are methods, wherein the administering is local administration or systemic administration. Further provided herein are methods, wherein the administering is subcutaneous administration or intravenous administration. Further provided herein are methods, wherein the administering is every 8 hours, every 12 hours, every 24 hours, every 48 hours, every 72 hours, every 96 hours, or every 120 hours. Further provided herein are methods, wherein the administering is every 6 days, every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, or every 56 days.

[0154] Provided herein are methods of generating an immune response at a site of a tumor in a subject, wherein the methods comprise: administering to the subject any engineered MHC class I heavy chain, any engineered B2m protein, any MHC class I molecule complex, any composition provided herein, or pharmaceutical composition provided herein, thereby generating an immune response at the site of the tumor in the subject and reducing tumor size. Further provided herein are methods, wherein the administering is local administration or systemic administration. Furtherprovided herein are methods, wherein the administering is intratumoral administration, intravenous administration, intravascular administration, rectal administration, subcutaneous administration, topical administration, oral administration, intradermal administration, intramuscular administration, via inhalation, intranasal administration, intraperitoneal administration, intraocular administration, or intracranial administration. Further provided herein are methods, wherein the subject has a solid tumor. Further provided herein are methods, wherein the solid tumor is a carcinoma, a melanoma, or a sarcoma. Further provided herein are methods, wherein the administering is every 8 hours, every 12 hours, every 24 hours, every 48 hours, every 72 hours, every 96 hours, or every 120 hours. Further provided herein are methods, wherein the administering is every 6 days, every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, or every 56 days.EXAMPLESEXAMPLE 1: MHC CLASS I MOLECULE CONSTRUCTION, EXPRESSION AND PURIFICATION

[0155] A pMHC SCT was constructed with antigen peptide at the N termini peptide (NLV), followed by a gly-ser linker, followed by the B2m, gly-ser linker and an MHC class I (HLA-A02) heavy chain (SEQ ID NO: 52). The pMHC SCT was expressed either in Expi293F or ExpiCHO cell lines, and the protein was purified from the supernatant using Nickel based affinity chromatography and a size exclusion chromatography. Site-directed mutagenesis was performed to generate mutations in either the B2m or the MHC class I heavy chain polypeptide.EXAMPLE 2: AMINO ACID SUBSTITUTIONS IN THE F POCKET

[0156] To identify amino acid substitutions that enhance the stability of MHC class I molecules, high-throughput assays were performed to examine 6 amino acid positions in the F pocket of MHC class I molecule: G83, A(Y)84, Y85, M138, A139, and A140. Every combination of mutations at these six residues are explored in a screening pipeline. Briefly, recombinant MHC class I molecules were expressed and displayed on the surface of cells. Each cell possesses a different genetically encoded variant of the MHC consisting of modifications to the 6 amino acid positions. Millions of cells with different variants were evaluated as one heterogeneous population for binding to either expression markers such as Myc tag, FLAG tag, or HA tag. Multiple rounds of selection for cells possessing the highest expressing MHC variants generates a population of cells bearing genetic encodings of MHC variants with mutations that correlate to superior expression, which correlates with stability. These cells can be evaluated for genetic content at scale via next-generation sequencing, where the fold-change from starting population to selected population is expressed in “enrichment”.

[0157] 36 variant MHC class I molecules identified compose of mutations that were enriched in the screening pipeline were found to be soluble in expression system and a subset were evaluated for thermal stability as measured by its melting temperature (Tm). Briefly, the 36 MHC variants in solutions were heated to the final melting temperatures. The Tm of each sample was determined by performing a thermal ramp with a 1 degree Celsius / minute at a heating rate from 20 degrees Celsius to 95 degrees Celsius. Tm was defined as the temperature at the inflection point of the fluorescence ratio measured at 350nm over 330nm. The results for five mutant MHC class I molecules as compared to wild-type are summarized in Table 5. As shown in Table 5, the Tm of M138G, A139V, and G83K / A84M / M138P / A139V quadruple mutants increased by about 9 degrees Celsius, 4 degrees Celsius, and 7 degrees Celsius, respectively, as compared to that of the wild-type MHC Class I molecule.Table 5. Melting Temperatures of MHC Class I Molecule Variant.EXAMPLE 3: AMINO ACID SUBSTITUTIONS IN THE CO-RECEPTOR BINDING INTERFACE

[0158] To develop engineered MHC Class I molecules with enhanced binding to CD8, high- throughput coreceptor screening assays were performed to identify amino acid substitutions with enhanced binding to CD8 in B2m domains and alpha3 domains. Recombinant protein screening was performed to determine the enrichment regions of the mutations and frequency of mutations in B2m domains and alpha3 domains. FIG. 4A shows the enrichment map from the screening results with the positive hits for improved CD8 binding marked. The amino acid substitutions showing the % of final frequency and enrichment are shown in Table 6 and Table 7. Increased final frequency and / or enrichment indicate enhanced binding to CD8.Table 6. Top B2m Mutations.Table 7. Top alpha3 Mutations.EXAMPLE 4: IDENTIFICATION OF CD8 VARIANTS THROUGH HIGH- THROUGHPUT SCREENING.Recombinant DNA encoding HLA alleles of class I MHC molecules were generated through mutagenesis of a targeted region spanning residues structurally identified areas of the pMHC that are binding sites for CD8. Plasmids were transformed and evaluated for MHC surface expression. Separately, CD8 was recombinantly expressed using mammalian cell expression systems and tetramerized via biotin conjugation and association with strepavadin. The CD8 molecules were used in a multi-round enrichment assay to identify MHC variants across the library with enhanced CD8 binding. Table 8 provides the human CD8a, CD8b, and CD8a- high affinity sequences below.Table 8. human CD8 sequences.

[0159] Selection pressure was applied via iterative rounds of enrichment and flow-based sorting for co-receptor binding. Representative variants and enriched mutations were identified by sequencing and validated for binding affinity and functional activity. Enrichment analyses from mutagenesis screens identifying mutations in P2m and the u3 domain of the MHC sequences that significantly contribute to improved affinity towards the CD8 co-receptor are shown in FIG. 4A, FIG. 4B, and FIG. 4C as indicated. Data shown in FIG. 4B and FIG. 4C represent enrichment scores from two sequential rounds (round 3 vs. round 4) of selection.

[0160] Frequency of amino acids are shown before selection and after four rounds of selection in HLA-A*02:01 (FIG. 5). Amino acid mutations that contribute to selection criteria that includes binding to CD8. The amino acid mutations were marked by read frequency that deviated from wild-type.EXAMPLE 5: CO-RECEPTOR BINDING ASSAY VIA SPR (AFFINITY VALIDATION).

[0161] SPR assays were conducted using a BIACORE™ T200 instrument (GE Healthcare). Soluble CD8a / p heterodimer was immobilized onto a streptavidin-coated sensor chip (SA Chip, GE Healthcare). pMHC monomer, prepared in HBSEP+ buffer, was flowed over the chip surface as the analyte at concentrations ranging from 0 uM to 200 uM. The flow rate was maintained at 10 pL / min, with a contact time of 200 seconds followed by a dissociation phase of 300 seconds. The experiments were carried out at 25°C. Data were analyzed using the BiaEval software employing a 1 : 1 steady-state binding model to calculate the dissociation constant (Kd).

[0162] CD8 affinity enhancing mutations identified screens were aligned to a representative set of class I MHC allele sequences. Wild-type residues for the beta-2-microglobulin for each HLA allele are represented by single-letter amino acid symbols with conserved positions across all alleles indicated by a dot (FIG. 5). The amino acid mutations identified at each position are shown as a bar graph, showing these mutations are in highly conserved regions of a pMHC (FIG. 6). The reference sequence for each HLA allele are provided in Table 9 below.Table 9. HLA Allele Sequences.

[0163] Wild-type residues for each HLA allele are represented by single-letter amino acid symbols with conserved positions across all alleles indicated by a dot (FIG. 5). The amino acid mutations identified at each position are shown as a bar graph, showing these mutations are in highly conserved regions of a pMHC (FIG. 7).

[0164] Follow-up screens were performed on HLA-A*02:01 (A02) as well as two additional HLA alleles, HLA-B*27:05 (B27) and HLA-C*06:02 (C06) (FIG. 8). This approach targeted specificpositions that function in the specific HLA allele. Higher frequency corresponded to mutations that contribute to CD8 affinity.

[0165] Amino acid frequency in the A02 screen correlated against either the B27 or C06 amino acid frequencies (FIG. 9A, FIG. 9B). Mutations that contribute to CD8 affinity in original A02 screen correlated strongly with mutations identified in alternative HLA allele demonstrating broad function of these mutations when incorporated into class I MHC alleles.

[0166] Surface plasmon resonance (SPR) analysis demonstrated enhanced CD8 binding affinity of soluble NLV-A02 monomer variants that incorporate different combinations of mutations identified in screen (A02.var3 (SEQ ID NO: 108), A02.var5 (SEQ ID NO: 109), A02.var6 (SEQ ID NO: 97), A02 var7 (SEQ ID NO: 110), A02 var8 (SEQ ID NO: 111)) This was compared against A02.Q115E (SEQ ID NO: 96) that contributes to overall CD8 affinity in MHC class I, as measured by equilibrium dissociation constant (KD) (FIG. 10). The list of variants are provided in Tables 10-11 below.Table 10. Recombinant monomer MHC class I heavy chain variant sequences in FIG. 10.Table 11. Recombinant MHC Class I monomer and homodimer sequences in FIGs. 11-12.EXAMPLE 6: CO-RECEPTOR BINDING ASSAY VIA BLI

[0167] The affinity of the engineered MHCs for CD8 was evaluated by a co-receptor binding assay. Biotinylated human CD8a / B was diluted into running buffer (IX PBS) at a final concentration of 3 ug / mL in 96-well black tilt plates (Gator Bio #130282), loaded onto streptavidin probes (Gator Bio #160002) and incubated with varying concentrations of pMHC monomer (from 50 uM to 68 uM) or pMHC-Fc dimers (from 4 uM to 6.25 uM). Approximate run parameters (and associated solutions) were: baseline 120 seconds (running buffer), loading 120 seconds (biotinylated CD8 diluted in running buffer), baseline 2-120 seconds (running buffer), association 600 seconds (pMHC construct diluted in running buffer), dissociation 600 seconds (running buffer). Data was analyzed using the GatorOne™ analysis software (Gator Bio) to evaluate global curve fits and GraphPad Prism was used for plotting. Monomer pMHC curves were processed with a 1 : 1 fit while dimeric pMHC curves were processed with a 2: 1 fit to account for avid interactions.

[0168] Biolayer interferometry (BLI) analysis demonstrated that soluble A02 monomer variants (var6 and varl2) and B27 fusion variants (var6 and varl2) had increased CD8 binding affinity relative to wild-type monomers. The affinity for both variants greatly exceeded the natural affinity between MHC and CD8 (estimated at 200 pM). The mutations identified in the BLI screen led to engineered MHC molecules with improved engagement of CD8.EXAMPLE 7: INTERNALIZATION ASSAY IN T CELLS.

[0169] Cells were seeded in a 96-well plate at a density of 100,000 cells per well in 100 pL of culture medium. Separately, protein was labeled with AF647 using an NHS ester labeling kit (Thermo Fisher Scientific) to track internalization. Cells were treated with 20 nM of the AF647- labeled protein, a concentration sufficient to coat the cells, with the labeled protein added at various time points to monitor internalization dynamics. Cells were divided into two populations in V- bottom 96-well plates. One population served as a control and was treated with PBS alone, leaving labeled protein present on both the cell surface and interior. The other population was treated with Proteinase K (NEB, catalog #P8107S, 800 units / mL; stock concentration 20 mg / mL) diluted to a final concentration of 0.5 mg / mL in phosphate-buffered saline (PBS) to selectively degrade surface-bound, non-intemalized protein, leaving only internalized protein for analysis. For Proteinase K treatment, cells were pelleted at 400 xg for 3 minutes, washed twice with PBS, and resuspended in 50 pL of either PBS (control) or PBS containing Proteinase K. Following incubation at 37°C for 20 minutes, the reaction was quenched with 100 pL of IX FACS buffer, and cells were washed thoroughly, including a final wash with a 1 :3000 dilution of DAPI in FACS buffer to stain nuclei. Samples were analyzed using an Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were processed and analyzed using FlowJo software.

[0170] Flow cytometry-based internalization assays were performed for soluble pMHC variants (A02.Var6 (SEQ ID NO: 97), A02.Varl2 (SEQ ID NO: 98), B27.Var6 (SEQ ID NO: 123), B27. Varl2). The CD8+ T cells had enhanced intake of the pMHC variants over time, as compared to A02.Q115E (SEQ ID NO: 96) and A02 wild-type controls (FIG. 12A and FIG. 12B). The increased internalization of pMHC variants by CD8+ T cells was associated with elevated CD8 binding affinity, over time as compared to A02.Q115E (SEQ ID NO: 96) and A02 wild-type controls. Increased internalization correlated strongly with elevated CD8 binding affinity. The internalization rates are shown in Table 12 below.Table 12. Internalization of pMHC class I variants.EXAMPLE 8: CONSTRUCT EXPRESSION AND PURIFICATION.

[0171] Human embryonic kidney (HEK) Expi293F cells (Thermo Fisher Scientific) were cultured in Expi293 Expression Medium. On the day prior to transfection, cells were seeded at a density of approximately 2.5-3 x 10A6 cells / mL and incubated overnight at 37°C with 8% CO2 in an orbital shaker incubator set at 125 rpm. On the day of transfection, cells were diluted to a final density of 3 x 10A6 cells / mL in 50 mL Expi293 Expression Medium.

[0172] Transfection was performed using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific). Specifically, 50 pg of plasmid DNA encoding His-tagged peptide-major histocompatibility complex fused to Fc fragment (pMHC-Fc) was diluted in 3 mL of Opti-MEM™ Reduced Serum Medium (Thermo Fisher Scientific). Separately, 160 pL of ExpiFectamine™ 293 reagent was diluted in 3 mL of Opti-MEM™ and incubated at room temperature for 5 minutes. Subsequently, the diluted DNA and reagent were combined and incubated at room temperature for 15 minutes. The DNA-lipid complexes were then added slowly to the cell culture with gentle swirling, and the culture was incubated under the conditions described above. Approximately 18- 22 hours post-transfection, ExpiFectamine™ 293 Enhancer 1 (300 pL) and Enhancer 2 (3 mL) were added to the culture. The cells were further incubated for 5 days post-transfection. At the time of harvest, cells were removed by centrifugation at 15,000 x g for 30 minutes at 4°C, and the resulting supernatant was filtered through a 0.22 pm membrane.EXAMPLE 9: PURIFICATION OF RECOMBINANT pMHC.

[0173] Filtered supernatant was subjected to immobilized metal affinity chromatography using Ni- IMAC resin (Pierce). The resin was equilibrated with phosphate-buffered saline (PBS, pH 7.4), and protein binding was performed at 4°C overnight with gentle rotation. The resin wassubsequently washed three times with PBS containing 20 mM imidazole. His-tagged pMHC-Fc protein was eluted with PBS containing 500 mM imidazole, collecting 5 mL fractions.

[0174] Elution fractions containing the protein of interest were pooled and further purified by size exclusion chromatography (SEC) using a Superdex 200 column (GE Healthcare) equilibrated with PBS containing 0.5M NaCl, pH 7.4. The protein was eluted in PBS with 0.5M NaCl and peak fractions were identified based on absorbance at 280 nm. The purified recombinant His-tagged pMHC-Fc protein fractions were pooled, concentrated to the desired concentration, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for purity, and quantified by UV absorbance at 280 nm. Aliquots of purified protein were stored at -80°C in 10% glycerol until use.EXAMPLE 10: EVALUATION OF THERMAL STABILITY VIA THERMAL MELT.

[0175] Thermal unfolding assays were conducted using the NanoTemper Prometheus instrument. 10 pL of the purified pMHC monomer was loaded into high-sensitivity Prometheus capillaries. Samples were subjected to a temperature gradient increasing from 25°C to 95°C at a rate of l°C / min. Fluorescence at 330 and 350 nm was continuously recorded during the temperature ramp, and the melting temperature (Tm) was determined by plotting the first derivative of the 350 / 330 nm fluorescence ratio against temperature.

[0176] Thermal stability analysis (melting temperature, Tm) of the engineered soluble pMHC variants bearing stabilization mutations were performed. Screens were performed on HLA- A*02:01 (A02) to identify stabilizing mutations in the F pocket for two different peptides. This approach introduces mutations at key residues in the F pocket region. Amino acid frequencies for each selected library are shown in FIG. 13, with higher frequency corresponding to mutations that contribute to overall protein stability.

[0177] Stability enhancements were quantified by increased melting temperatures as compared to wild-type pMHC, with significant improvements observed in variants such as A2.G83K, A2.M138G, and multi -mutant constructs. These results demonstrated that the variants have improved protein stability conferred by specific amino acid substitutions in the peptide-binding region (F pocket) of the MHC (FIG. 14A - FIG. 14F). Table 13 provides the MHC variant sequences used in FIGS. 14A-14F.Table 13. MHC Class I monomer variants in FIG. 14A-14F.

[0178] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments 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. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A composition comprising: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1; and a P2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1.

2. The composition of claim 1, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO:

13. The composition of claim 1, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position selected from: 189-192, 197- 200, 211, 215, 216, 224, 231, 245-248, 255, and 265 relative to SEQ ID NO: 1.

4. The composition of claim 1, wherein the engineered MHC class I heavy chain comprises 2, 3, 4, or 5 substitutions compared to an MHC class I heavy chain encoded by an HLA-A allele.

5. The composition of claim 4, wherein the HLA-A allele is HLA-A*02:01 allele.

6. The composition of claim 4, wherein the HLA-A allele is an HLA-A*24:02 allele, an HLA-B*27:05 allele, or an HLA-C*06:02 allele.

7. The composition of claim 1, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by 10% up to about 100% as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1.

8. The composition of claim 1, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by at least about 10%, 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%, atleast about 90%, or at least about 100% as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1.

9. The composition of claim 1, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 1.1-fold to about 10-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1.

10. The composition of claim 1, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 1.1-fold, about 1.2-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1.

11. The composition of claim 1, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold to about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1.

12. The composition of claim 1, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200- fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800- fold, about 900-fold, or about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 1.

13. The composition of claim 1, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 100 pM.

14. The composition of claim 1, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 1 pM.

15. The composition of claim 1, wherein the P2-microglobulin (B2m) protein comprises at least one amino acid substitution.

16. The composition of claim 15, wherein the at least one amino acid substitution in the B2m protein is an amino acid substitution at a position selected from the group consisting of: 31, 32, 57, 58, 63, 64, 71, 72, 83, 84, and a combination thereof relative to SEQ ID NO: 2.

17. The composition of claim 16, wherein the at least one amino acid substitution in the B2m protein is at amino acid position S57 relative to SEQ ID NO: 2.

18. The composition of claim 17, wherein the at least one amino acid substitution in the B2m protein is a S57G substitution, a S57T substitution, a S57R substitution, or a S57V substitution relative to SEQ ID NO:

219. The composition of claim 16, wherein the at least one amino acid substitution in the B2m protein is an amino acid substitution at amino acid position K58 relative to SEQ ID NO: 2.

20. The composition of claim 19, wherein the at least one amino acid substitution in the B2m protein is a K58F substitution, a K58T substitution, a K58G substitution, or a K58M substitution relative to SEQ ID NO:

221. The composition of claim 16, wherein the at least one amino acid substitution in the B2m protein is a S57V substitution, a K58Q substitution, a N83R substitution, an H84T substitution, or a combination thereof relative to SEQ ID NO:

222. The composition of claim 16, wherein the at least one amino acid substitution in the B2m protein is an S27V substitution relative to SEQ ID NO: 2.

23. The composition of claim 1, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain comprises an amino acid substitution at an amino acid position selected from: 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, 265, and any combination thereof relative to SEQ ID NO: 1.

24. The composition of claim 23, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position corresponding to Hl 97 relative to SEQ ID NO:

125. The composition of claim 23, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an H197E substitution or an H197R substitution relative to SEQ ID NO:

126. The composition of claim 23, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an A199L substitution, a T200A substitution, a L215V substitution, a T216M substitution, or a combination thereof relative to SEQ ID NO:

127. The composition of claim 23, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a T216M substitution relative to SEQ ID NO: 1.

28. The composition of claim 23, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a L215V substitution relative to SEQ ID NO: 1.

29. The composition of claim 23, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V247I substitution relative to SEQ ID NO: 1.

30. The composition of claim 23, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V248E substitution relative to SEQ ID NO: 1.

31. The composition of claim 1, wherein the engineered MHC class I heavy chain further comprises an amino acid substitution at position 115 relative to SEQ ID NO: 1.

32. The composition of claim 31, wherein the amino acid substitution in the engineered MHC class I heavy chain comprises a QI 15E substitution relative to SEQ ID NO: 1.

33. The composition of claim 1, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, or SEQ ID NO: 57 to SEQ ID NO: 7934. The composition of claim 1, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 96% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, or SEQ ID NO: 57 to SEQ ID NO: 7935. The composition of claim 1, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 97% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, or SEQ ID NO: 57 to SEQ ID NO: 7936. The composition of claim 1, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 98% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, or SEQ ID NO: 57 to SEQ ID NO: 7937. The composition of claim 1, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is at least 99% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, or SEQ ID NO: 57 to SEQ ID NO: 7938. The composition of claim 1, wherein the engineered MHC class I heavy chain comprises: an amino acid sequence that is 100% identical to any one of SEQ ID NO: 7 to SEQ ID NO: 10, or SEQ ID NO: 57 to SEQ ID NO: 7939. The composition of claim 1, wherein the composition further comprises an MHC class I ligand peptide.

40. The composition of claim 39, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide.

41. The composition of claim 40, wherein the MHC class I antigen peptide is an MHC class I self-peptide.

42. The composition of claim 39, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues.

43. The composition of claim 39, wherein the MHC class I ligand peptide is a fragment of a cellular protein.

44. The composition of claim 39, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide.

45. The composition of claim 39, wherein the MHC class I ligand peptide is an HLA-A restricted peptide.

46. The composition of claim 39, wherein the MHC class I ligand peptide is an HLA- A*0201 restricted peptide.

47. The composition of claim 39, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51 or SEQ ID NO: 11848. The composition of claim 39, wherein the engineered MHC class I heavy chain, the P2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order:(i) the MHC class I ligand peptide;(ii) a first flexible linker;(iii) the B2m protein;(iv) a second flexible linker; and(v) the engineered MHC class I heavy chain.

49. The composition of claim 1, wherein the engineered MHC class I heavy chain is present on a surface of a cell.

50. The composition of claim 1, wherein the engineered MHC class I complex is in soluble form.

51. A composition comprising: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain; and an engineered p2-microglobulin (B2m) protein, wherein the engineered B2m protein comprises at least one amino acid substitution relative to a corresponding B2m protein comprising an amino acid sequence of SEQ ID NO: 2, wherein the at least one amino acid substitution in the B2m protein provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2.

52. The composition of claim 51, wherein the at least one amino acid substitution provides for increased internalization of the engineered MHC class I complex in a cell as compared to an otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2.

53. The composition of claim 51, wherein the at least one amino acid substitution in the engineered B2m protein is at an amino acid position selected from: 31, 32, 57, 58, 63, 64, 71, 72, 83, or 84 relative to SEQ ID NO: 2.

54. The composition of claim 51, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by 10% up to about 100% as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 2.

55. The composition of claim 51, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by at least about 10%, 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%, or at least about 100% as compared to the otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2.

56. The composition of claim 51, wherein the binding affinity of the engineered MHC complex to CD8 is increased by about 1.1-fold to about 10-fold as compared to the otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 2.

57. The composition of claim 51, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 1.1-fold, about 1.2-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold as compared to the otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2.

58. The composition of claim 51, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold to about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising an MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 80.

59. The composition of claim 51, wherein the binding affinity of the engineered MHC class I complex to CD8 is increased by about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200- fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800- fold, about 900-fold, or about 1000-fold as compared to the otherwise equivalent MHC class I complex comprising a B2m protein comprising an amino acid sequence of SEQ ID NO: 2.

60. The composition of claim 51, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 100 pM.

61. The composition of claim 51, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 1 pM.

62. The composition of claim 51, wherein the P2-microglobulin (B2m) protein comprises at least one amino acid substitution.

63. The composition of claim 51, wherein the at least one amino acid substitution in the B2m protein is at amino acid position S57 relative to SEQ ID NO: 2.

64. The composition of claim 63, wherein the at least one amino acid substitution in the B2m protein is a S57G substitution, a S57T substitution, a S57R substitution, or a S57V substitution relative to SEQ ID NO:

265. The composition of claim 51, wherein the at least one amino acid substitution in the B2m protein is an amino acid substitution at amino acid position K58 relative to SEQ ID NO: 2.

66. The composition of claim 65, wherein the at least one amino acid substitution in the B2m protein is a K58F substitution, a K58T substitution, a K58G substitution, or a K58M substitution relative to SEQ ID NO:

267. The composition of claim 51, wherein the at least one amino acid substitution in the B2m protein is a S57V substitution, a K58Q substitution, a N83R substitution, an H84T substitution, or a combination thereof relative to SEQ ID NO:

268. The composition of claim 51, wherein the at least one amino acid substitution in the B2m protein is an S27V substitution relative to SEQ ID NO: 2.

69. The composition of claim 51, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11770. The composition of claim 69, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain comprises at an amino acid substitution at a position selected from the group consisting of: 189, 191, 192, 197-200, 211, 215, 216, 224, 231, 245, 247, 248, 255, 265, and any combination thereof relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11771. The composition of claim 70, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is at an amino acid position corresponding to Hl 97 relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117.

72. The composition of claim 71, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an H197E substitution or an H197R substitution relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117.

73. The composition of claim 69, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is an A199L substitution, a T200A substitution, a L215V substitution, a T216M substitution, or a combination thereof relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11774. The composition of claim 69, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a T216M substitution relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 117-I l l-75. The composition of claim 69, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a L215V substitution relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11776. The composition of claim 69, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V247I substitution relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11777. The composition of claim 69, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain is a V248E substitution relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11778. The composition of claim 51, wherein the engineered MHC class I heavy chain further comprises an amino acid substitution at position 115 relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11779. The composition of claim 78, wherein the amino acid substitution in the engineered MHC class I heavy chain comprises a Q115E substitution relative to an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 80-95, or SEQ ID NO: 11780. The composition of claim 51, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 95% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 14881. The composition of claim 51, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 96% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 14882. The composition of claim 51, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 97% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 14883. The composition of claim 51, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 98% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 14884. The composition of claim 51, wherein the engineered B2m protein comprises: an amino acid sequence that is at least 99% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 14885. The composition of claim 51, wherein the engineered B2m protein comprises: an amino acid sequence that is 100% identical to any one of SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 124 to SEQ ID NO: 14886. The composition of claim 51, wherein the composition further comprises an MHC class I ligand peptide.

87. The composition of claim 86, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide.

88. The composition of claim 87, wherein the MHC class I antigen peptide is an MHC class I self-peptide.

89. The composition of claim 86, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues.

90. The composition of claim 86, wherein the MHC class I ligand peptide is a fragment of a cellular protein.

91. The composition of claim 86, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide.

92. The composition of claim 86, wherein the MHC class I ligand peptide is an HLA-A restricted peptide.

93. The composition of claim 86, wherein the MHC class I ligand peptide is an HLA- A*0201 restricted peptide.

94. The composition of claim 86, wherein the MHC class I ligand peptide comprises any one of SEQ ID NO: 46 to SEQ ID NO: 51, or SEQ ID NO: 11895. The composition of claim 86, wherein the engineered MHC class I heavy chain, the engineered p2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chain trimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order:(i) the MHC class I ligand peptide;(ii) a first flexible linker;(iii) the engineered p2-microglobulin protein;(iv) a second flexible linker; and(v) the engineered MHC class I heavy chain.

96. The composition of claim 51, wherein the engineered MHC class I heavy chain is present on a surface of a cell.

97. The composition of claim 51, wherein the engineered MHC class I complex is a soluble form of the MHC class I complex.

98. A composition comprising: an engineered major histocompatibility complex (MHC) class I heavy chain comprising: at least one amino acid substitution in an F pocket in an antigen-binding groove, wherein the at least one amino acid substitution is at an amino acid residueselected from any one of positions 77 to 87, 95, or 135 to 145 relative to SEQ ID NO: 1, and wherein the at least one amino acid substitution provides for increased stability of the engineered MHC class I heavy chain as compared to the stability of an otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1.

99. The composition of claim 98, wherein the engineered MHC class I heavy chain comprises 1, 2, 3, 4, or 5 amino acid substitutions compared to an engineered MHC class I heavy chain encoded by an HLA-A allele, an HLA-B allele, an HLA-C allele, or an HLA-E allele.

100. The composition of claim 99, wherein the HLA-A allele is a HLA-A*02:01 allele.

101. The composition of claim 99, wherein the engineered MHC class I heavy chain is encoded by an HLA-A*24:02 allele, an HLA-B*27:05 allele, or an HLA-C*06:02 allele.

102. The composition of claim 98, wherein the increased stability is characterized by an increased thermal stability of the engineered MHC class I heavy chain as measured by a spectroscopic method.

103. The composition of claim 102, wherein the spectroscopic method is differential scanning fluorimetry or circular dichroism.

104. The composition of claim 102, wherein the spectroscopic method determines a melting temperature of the engineered MHC class I heavy chain or the otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1, wherein the melting temperature of the engineered MHC class I heavy chain is increased by about 2 degrees Celsius, 3 degrees Celsius, 4 degrees Celsius, 5 degrees Celsius, 6 degree Celsius, 7 degrees Celsius, 8 degrees Celsius, 9 degrees Celsius, 10 degrees Celsius, 11 degrees Celsius, 12 degrees Celsius, 13 degrees Celsius, 14 degrees Celsius, or 15 degrees Celsius, as compared to that of the otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1.

105. The composition of claim 102, wherein the spectroscopic method determines a melting temperature of the at least one engineered MHC class I heavy chain or the otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1, wherein the melting temperature of the at least one engineered MHC class I heavy chain is increased by at least 5 degrees Celsius as compared to that of the otherwise equivalent MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 1.

106. The composition of claim 102, wherein the spectroscopic method determines a melting temperature of the engineered MHC class I heavy chain, wherein the melting temperature of the engineered MHC class I heavy chain is increased by at least 9 degrees Celsius as comparedto that of the otherwise equivalent MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1107. The composition of claim 98, wherein the at least one amino acid substitution provides for increased yield of the engineered MHC class I heavy chain in an expression system as compared to an otherwise equivalent expression system comprising the MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1.

108. The composition of claim 107, wherein the increased yield of the engineered MHC class I heavy chain in the expression system is 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 11 fold, about 12 fold, about 13 fold, about 14 fold, or about 15 fold greater than the otherwise equivalent expression system comprising the MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1109. The composition of claim 98, wherein the at least one amino acid substitution provides for increased potency of the engineered MHC class I heavy chain as compared to the otherwise equivalent MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1110. The composition of claim 109, wherein the increased potency of the engineered MHC class I heavy chain is measured by a binding affinity of the engineered MHC class I heavy chain to at least one antigen as compared to a binding affinity of the otherwise equivalent MHC class I heavy chain comprising the amino acid sequence of SEQ ID NO: 1 to the at least one antigen.

111. The composition of claim 98, wherein the at least one amino acid substitution is at an amino acid residue position selected from: 78, 80, 81, 83, 84, 85, 138, or 139 of the engineered MHC class I heavy chain relative to SEQ ID NO: 1, optionally wherein the engineered MHC class I heavy chain is encoded by an HLA-A *02:01 allele.

112. The composition of claim 111, wherein the engineered MHC class I heavy chain is an HLA-A molecule, and wherein the at least one amino acid substitution is at amino acid residue position of G83, Y84, Y85, M138, or A139, relative to SEQ ID NO: 1.

113. The composition of claim 111, wherein the at least one amino acid substitution is G83K, Y84M, M138G, A139V, or any combination thereof relative to SEQ ID NO: 1.

114. The composition of claim 111, wherein the at least one amino acid substitution is G83K relative to SEQ ID NO: 1.

115. The composition of claim 111, wherein the at least one amino acid substitution is A84M relative to SEQ ID NO: 1.

116. The composition of claim 111, wherein the at least one amino acid substitution is M138G relative to SEQ ID NO: 1.

117. The composition of claim 111, wherein the at least one amino acid substitution is A139V relative to SEQ ID NO: 1.

118. The composition of claim 111, wherein the at least one amino acid substitution is G83K, A84M, M138P, A139V relative to SEQ ID NO: 1.

119. The composition of claim 98, wherein the engineered MHC class I heavy chain comprises an amino acid sequence that is at least about 95%, at least about 97%, or at least about 99% identical to any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116120. The composition of claim 98, wherein the engineered MHC class I heavy chain comprises an amino acid sequence comprising any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116121. The composition of claim 98, wherein the composition further comprises a P2- microglobulin (B2m) protein.

122. The composition of claim 121, wherein the composition further comprises an MHC class I ligand peptide.

123. The composition of claim 122, wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide.

124. The composition of claim 123, wherein the MHC class I antigen peptide is an MHC class I self-peptide.

125. The composition of claim 122, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues.

126. The composition of claim 122, wherein the MHC class I ligand peptide is a fragment of a cellular protein.

127. The composition of claim 122, wherein the MHC class I ligand peptide is a virus peptide or tumor peptide.

128. The composition of claim 122, wherein the MHC class I ligand peptide is an HLA-A restricted peptide.

129. The composition of claim 122, wherein the MHC class I ligand peptide is an HLA-A*0201 restricted peptide.

130. The composition of claim 122, wherein the MHC class I ligand peptide comprises an amino acid sequence of any one of SEQ ID NO: 46 to SEQ ID NO: 51, or SEQ ID NO: 118.

131. The composition of claim 122, wherein the engineered MHC class I heavy chain, a P2-microglobulin (B2m) protein, and the MHC class I ligand peptide are within a single chaintrimer molecule, wherein the single chain trimer molecule comprises, in amino-to-carboxy terminal order:(i) the MHC class I ligand peptide;(ii) a first flexible linker;(iii) the p2-microglobulin protein;(iv) a second flexible linker; and(v) the engineered MHC class I heavy chain.

132. The composition of claim 98, wherein the engineered MHC class I heavy chain is present on a surface of a cell.

133. The composition of claim 98, wherein the engineered MHC class I heavy chain is in soluble form.

134. A composition comprising: an engineered MHC class I heavy chain comprising an amino acid sequence that is at least about 85% identical to any one of SEQ ID NO: 11 to SEQ ID NO: 45, SEQ ID NO: 96 to SEQ ID NO: 98, or SEQ ID NO: 108 to SEQ ID NO: 116135. A composition comprising: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 86; and a P2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 86.

136. The composition of claim 135, wherein the at least one amino acid substitution comprises an amino acid substitution at position 84, 115, 189, 197, 198, 215, 216, 217, 224, 231, or 255 relative to the amino acid sequence of SEQ ID NO: 86.

137. The composition of claim 135, wherein the composition comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 122 to SEQ ID NO: 124.

138. The composition of claim 135, further comprising an MHC peptide ligand.

139. The composition of claim 138, wherein the MHC peptide ligand comprises an amino acid sequence of any one of SEQ ID NO: 45 to SEQ ID NO: 51, SEQ ID NO: 118, or SEQ ID NO: 150140. A composition comprising: an engineered MHC class I complex, wherein the engineered MHC class I complex comprises: an engineered MHC class I heavy chain, wherein the engineered MHC class I heavy chain comprises at least one amino acid substitution relative to a corresponding MHC class I heavy chain comprising an amino acid sequence of SEQ ID NO: 91; and a P2-microglobulin (B2m) protein, wherein the at least one amino acid substitution in the engineered MHC class I heavy chain provides for increased binding affinity of the engineered MHC class I complex to CD8 on a cell surface as compared to an otherwise equivalent MHC class I complex comprising an amino acid sequence of SEQ ID NO: 91.

141. A composition comprising: the composition of any one of claims 1 to 140; and an antibody or an antibody fragment that specifically binds to a target cell.

142. The composition of claim 141, wherein the target cell comprises an immune cell or a cancer cell.

143. A composition comprising a nucleic acid encoding any one of the compositions of claims 1 to 140.

144. A composition comprising: a population of cells comprising a composition of any one of claims 1 to 140.

145. A composition comprising: a composition of any one of claims 1 to 144; and a nucleic acid.

146. The composition of claim 145, wherein the composition is linked or conjugated to the nucleic acid.

147. A pharmaceutical composition comprising:(a) a composition of any one of claims 1 to 146; and(b) a pharmaceutically acceptable excipient, diluent, or carrier.

148. A method of treating an autoimmune disorder in a subject, the method comprising: administering to the subject the composition of any one of claims 1 to 146; or the pharmaceutical composition of claim 147, thereby treating the autoimmune disorder in the subject.

149. The method of claim 148, wherein the autoimmune disorder is Type 1 diabetes, Celiac disease, rheumatoid arthritis, multiple sclerosis, axial spondylarthritis, birdshot uveitis,psoriasis, ankylosing spondylitis, lupus erythematosus, psoriatic arthritis, scleroderma, inflammatory bowel disease, Sjogren syndrome, or Addison disease.

150. The method of claim 148, wherein the administering is local administration or systemic administration.

151. The method of claim 148, wherein the administering is subcutaneous administration or intravenous administration.

152. The method of claim 148, wherein the administering is every 8 hours, every 12 hours, every 24 hours, every 48 hours, every 72 hours, every 96 hours, or every 120 hours.

153. The method of claim 148, wherein the administering is every 6 days, every 7 days, every 14 days, every 21 days, every 28 days, every 35 days, every 42 days, or every 56 days.

154. A method of treating cancer in a subject, the method comprising: administering to the subject the composition of any one of claims 1 to 146; or the pharmaceutical composition of claim 147, thereby treating the cancer in the subject.

155. The method of claim 154, wherein the administering is local administration or systemic administration.

156. The method of claim 154, wherein the administering is intratumoral administration, intravenous administration, intravascular administration, rectal administration, subcutaneous administration, topical administration, oral administration, intradermal administration, intramuscular administration, via inhalation, intranasal administration, intraperitoneal administration, intraocular administration, or intracranial administration.

157. The method of claim 154, wherein the subject has a solid tumor.

158. The method of claim 157, wherein the solid tumor is a carcinoma, a melanoma, or a sarcoma.

159. The method of claim 158, wherein the subject has a blood cancer.

160. The method of claim 159, wherein the blood cancer is a lymphoma or a leukemia.

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