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WO2025213057A1 - Molécules du complexe majeur d'histocompatibilité modifiées et leurs utilisations - Google Patents

Molécules du complexe majeur d'histocompatibilité modifiées et leurs utilisations

Info

Publication number
WO2025213057A1
WO2025213057A1 PCT/US2025/023211 US2025023211W WO2025213057A1 WO 2025213057 A1 WO2025213057 A1 WO 2025213057A1 US 2025023211 W US2025023211 W US 2025023211W WO 2025213057 A1 WO2025213057 A1 WO 2025213057A1
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WO
WIPO (PCT)
Prior art keywords
seq
mhc class
amino acid
composition
engineered
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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PCT/US2025/023211
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English (en)
Inventor
Patrick HOLEC
Nishant Singh
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Fletcher Biosciences Inc
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Fletcher Biosciences Inc
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Application filed by Fletcher Biosciences Inc filed Critical Fletcher Biosciences Inc
Publication of WO2025213057A1 publication Critical patent/WO2025213057A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • MHC Major Histocompatibility Complex
  • TCR T-cell receptors
  • 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.
  • compositions 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 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.
  • 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
  • kits for treating a disease in a subject comprising: administering to the subject a composition provided herein, thereby treating the disease in the subject.
  • the disease is an autoimmune disease or a cancer.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • compositions 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, 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.
  • B2m microglobulin
  • kits for treating a disease in a subject comprising: administering to the subject a composition provided herein, thereby treating the disease in the subject.
  • the disease is an autoimmune disease or a cancer.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • 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.
  • MHC major histocompatibility complex
  • kits for treating a disease in a subject comprising: administering to the subject a composition provided herein, thereby treating the disease in the subject.
  • the disease is an autoimmune disease or a cancer.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • 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.
  • the MHC class I complex comprises an MHC class I heavy chain and a P2-microglobulin (B2m) protein
  • B2m P2-microglobulin
  • 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.
  • compositions further comprise an MHC class I ligand peptide.
  • methods of treating a disease in a subject comprising: administering to the subject a composition provided herein, thereby treating the disease in the subject.
  • the disease is an autoimmune disease or a cancer.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • compositions wherein the compositions further comprise an MHC class I ligand peptide.
  • methods of treating a disease in a subject comprising: administering to the subject a composition provided herein, thereby treating the disease in the subject.
  • methods, wherein the disease is an autoimmune disease or a cancer.
  • 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.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • 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
  • compositions wherein the compositions further comprise an MHC class I ligand peptide.
  • 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.
  • the disease is an autoimmune disease or a cancer.
  • 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.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • 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.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • methods of reducing inflammation in a subject comprise: administering to the subject the population of cells provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • 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.
  • methods of delivering a therapeutic nucleic acid to a cell comprising: contacting the cell with the composition provided herein, thereby delivering the therapeutic nucleic acid to the cell via internalization of the composition.
  • methods of treating a disease in a subject comprising: administering to the subject the composition provided herein, thereby treating the disease in the subject.
  • the disease is an autoimmune disease or a cancer.
  • methods of reducing inflammation in a subject comprising: administering to the subject a composition provided herein, thereby reducing inflammation in a subject.
  • methods of generating an immune response at a site of a tumor in a subject comprising: 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.
  • autoimmune disorder comprising administering to a subject in need thereof the compositions provided herein or the pharmaceutical compositions provided herein.
  • 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.
  • kits for generating an immune response at a site of a tumor in a subject 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.
  • 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.
  • P2m p2-microglobulin
  • 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.
  • 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.
  • 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.
  • 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.
  • Y- axis HLA-C*06:02 library percentage frequency.
  • A02.Q115E SEQ ID NO: 96
  • KD equilibrium dissociation constant
  • 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).
  • 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 (%).
  • FIGURE 14A-14F show graphs of thermal stability analysis (melting temperature, Tm) of engineered soluble pMHC variants bearing stabilization mutations.
  • MHC major histocompatibility complex
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • bind refers 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.
  • nucleotide 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.
  • 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)).
  • treating 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • antibody refers to the resistance of a complex of two or more agents to dissociate after dilution.
  • antibody and antigen preferentially binds
  • 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.
  • 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).
  • 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).
  • 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).
  • homologous when modified with an adverb such as “highly,” may refer to sequence similarity and does not necessarily relate to a common evolutionary origin.
  • 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 match over 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.
  • engineered MHC class I molecules with enhanced stability and/or potency and compositions comprising engineered MHC class I molecules or MHC class I complexes.
  • 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
  • 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.
  • CDVGS DWRFL RGYHQ YAYDG KDYIA LKEDL RSWTA ADMAA QTTKH KWEAA
  • 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 50
  • CDVGS DWRFL RGYHQ YAYDG KDYIA LKEDL RSWTA ADMAA QTTKH KWEAA
  • 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.
  • the F pocket is composed of residues 77, 80, 81, 84, 95, 116, 123, 143, 146, and 147.
  • 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.
  • 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.
  • 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*02:03, 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.
  • 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.
  • an MHC composition provided herein can comprises any number or any combination of the amino acid substitutions provided in Table 1 below.
  • 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
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • an engineered MHC class I molecule comprises an amino acid substitution A139C, A139I, or A139V, relative to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 86, or SEQ ID NO: 91.
  • 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
  • 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.
  • 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.
  • Protein stability can be assessed in many ways.
  • the increased stability is assessed by increased thermostability.
  • 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.
  • the increased stability is assessed by monitoring the protein aggregation rate.
  • 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.
  • the increased thermal stability is determined by increased melting temperature (Tm) as measured by a spectroscopic method.
  • the spectroscopic method is circular dichroism or differential scanning calorimetry.
  • Tm is defined as the temperature at the inflection point of the fluorescence ratio measured at 350 nm over 330 nm.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • CTLs cytotoxic T cells
  • 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.
  • the binding of CD8 to MHC class I molecules recruits the Src family kinase Lck to the TCR signaling complex. This amplifies the signal required for T cell activation, leading to a robust immune response.
  • 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.
  • compositions comprising: at least one engineered MHC class I molecule complex with enhanced binding to CD8.
  • the engineered MHC class I complex comprises an MHC class I heavy chain; and a P2-microglobulin (B2m) protein.
  • an engineered MHC class I complex described herein comprises at least one amino acid substitution.
  • 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.
  • the at least one amino acid substitution is within a CD8 binding interface.
  • the at least one amino acid substitution is within an engineered MHC class I heavy chain.
  • the at least one amino acid substitution is within an engineered B2m protein.
  • 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.
  • the at least one amino acid substitution is within a CD8 binding interface.
  • the at least one amino acid substitution is within an engineered MHC class I heavy chain.
  • the at least one amino acid substitution is within an engineered B2m protein.
  • an engineered MHC class I complex provided herein comprises an engineered B2m protein.
  • the engineered B2m protein comprises at least one amino acid substitution relative to a B2m protein reference sequence.
  • the engineered B2m protein comprises at least one amino acid substitution relative to SEQ ID NO: 2.
  • 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 sequence identity to the B2m protein comprising an amino acid sequence of SEQ ID NO: 2.
  • sequence identity to a reference sequence for B2m can be determined by sequence alignment algorithm such as BLAST.
  • 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.
  • 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.
  • 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.
  • 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.
  • an engineered MHC class I molecule complex comprises an amino acid substitution in a B2m protein.
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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: 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.
  • 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.
  • 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. In some embodiments, an engineered MHC class I molecule comprises an amino acid substitution E198Y relative to the sequence of SEQ ID NO: 1.
  • 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.
  • 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.
  • 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.
  • an engineered MHC class I heavy chain provided herein can comprise two or more amino acid substitutions provided herein.
  • 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
  • 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
  • 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
  • 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,
  • 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.
  • 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
  • an engineered B2m protein provided herein comprises an amino acid substitution at position 57 relative to SEQ ID NO: 2
  • an engineered B2m protein provided herein comprises an amino acid substitution at position 57 and 58 relative to SEQ ID NO: 2.
  • 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%.
  • 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.
  • 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, about 95-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.
  • 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.
  • 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.
  • 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.
  • 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 CD8 is 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.
  • compositions 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.
  • compositions 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.
  • 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.
  • 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.
  • the antigen peptide sequence can comprise 9 contiguous amino acids.
  • a peptide sequence can be that of a protein fragment, wherein the protein is a pathogen protein or a cellular protein.
  • the pathogen protein or the cellular protein is a protein expressed by a cancer cell.
  • the pathogen protein or the cellular protein is a protein expressed by a virally infected cell.
  • 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.
  • the peptide-MHC complexes are engineered as single chain trimers (SCTs).
  • 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.
  • 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.
  • the first flexible linker has an amino acid sequence comprising GGGGSGGGGSGGGGS (SEQ ID NO: 53).
  • the second linker has an amino acid sequence comprising GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 54).
  • the linker has an amino acid sequence comprising GCGGSGGGGSGGGGS (SEQ ID NO: 119)
  • the linker has an amino acid sequence comprising GSGGGGSGGGGS (SEQ ID NO: 120).
  • the linker has an amino acid sequence comprising GSGGGGSGGGGS (SEQ ID NO: 121).
  • 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.
  • 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.
  • engineered MHC molecules conjugated to a functional moiety are engineered MHC molecules conjugated to a functional moiety.
  • the function moiety is a small molecule, a protein, a nucleic acid, an siRNA, an antibody, an antibody fragment, or an oligonucleotide.
  • 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.
  • 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.
  • compositions comprising MHC-cell conjugates.
  • an engineered MHC class I molecule or an engineered MHC class I complex described 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.
  • MHC-drug conjugates 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.
  • modified viruses are replication defective retroviruses, adenoviruses and adeno-associated viruses, lentiviruses; herpes viruses, poxviruses.
  • 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.
  • regulatory sequences include the following: a transcriptional promoter and enhancer, RNA polymerase binding sequence, or a ribosomal binding sequence (including a translation initiation signal).
  • 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.
  • vectors that comprise nucleic acids coding for at least one engineered MHC molecule in the present disclosure.
  • the polypeptides comprising the engineered MHC molecules or complexes provided herein may also be produced using cell free protein expression systems.
  • 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.
  • 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.
  • 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.
  • the engineered MHC molecules, complexes, or fusion proteins described herein couple to bovine serum albumin (BSA) or keyhole limpet hemocyanin.
  • BSA bovine serum albumin
  • compositions comprising an engineered MHC molecule, a fusion protein or a conjugate comprising an engineered MHC and a delivery vehicle.
  • 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.
  • 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.
  • 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.
  • compositions as described herein can be admixed with a delivery vehicle that permits delivery of the system to the target nucleic acid sequence.
  • 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 include an 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.
  • 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.
  • SUVs small unilamellar vesicles
  • 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.
  • 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®).
  • 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.
  • 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.
  • the methods comprise generating an immune response at a site of a tumor in a subject to reduce tumor size.
  • 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.
  • 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.
  • methods of inducing immune cell recruitment to a tumor are used for reduction of a tumor size.
  • compositions provided herein are used for reduction of a tumor volume.
  • 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.
  • compositions 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 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the HLA-A allele is HLA-A*02:01 allele.
  • compositions, wherein the HLA-A allele is HLA-A*24:02 allele.
  • 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
  • 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
  • compositions, wherein the at least one amino acid substitution in the B2m protein is an S27V substitution relative to SEQ ID NO: 2.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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, 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • the engineered MHC class I heavy chain is present on a surface of a cell.
  • compositions, wherein the engineered MHC class I complex is in soluble form.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions, wherein the engineered MHC class I complex binds to CD8 is with a Kd of less than 100 pM.
  • 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.
  • 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
  • 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
  • compositions, wherein the at least one amino acid substitution in the B2m protein is an S27V substitution relative to SEQ ID NO: 2.
  • compositions 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: 148
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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
  • 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 are further provided herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the composition further comprises an MHC class I ligand peptide.
  • the MHC class I ligand peptide comprises an MHC class I antigen peptide.
  • compositions, wherein the MHC class I antigen peptide is an MHC class I self-peptide.
  • 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.
  • the HLA-A allele is a HLA-A*02:01 allele.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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
  • 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
  • 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
  • compositions wherein the engineered MHC class I heavy chain
  • compositions wherein the composition further comprises an MHC class I ligand peptide.
  • the MHC class I ligand peptide comprises an MHC class I antigen peptide.
  • the MHC class I antigen peptide is an MHC class I self-peptide.
  • compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues.
  • compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein.
  • 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.
  • 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
  • compositions wherein the composition further comprises an MHC class I ligand peptide.
  • the MHC class I ligand peptide comprises an MHC class I antigen peptide.
  • the MHC class I antigen peptide is an MHC class I self- peptide.
  • the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues.
  • compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein.
  • 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.
  • 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.
  • compositions wherein the engineered MHC class I heavy chain is present on a surface of a cell.
  • compositions, wherein the engineered MHC class I complex is in soluble form.
  • compositions wherein the composition further comprises an MHC class I ligand peptide.
  • the MHC class I ligand peptide comprises an MHC class I antigen peptide.
  • the MHC class I antigen peptide is an MHC class I self-peptide.
  • compositions, wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues.
  • compositions, wherein the MHC class I ligand peptide is a fragment of a cellular protein.
  • 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.
  • compositions 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.
  • 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
  • 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.
  • 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.
  • 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.
  • compositions wherein the engineered MHC class I heavy chain is present on a surface of a cell.
  • compositions, wherein the engineered MHC class I complex is in soluble form.
  • compositions 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions wherein the engineered MHC class I heavy chain is present on a surface of a cell.
  • compositions wherein the engineered MHC class I complex is in soluble form.
  • 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.
  • the target cell comprises an immune cell or a cancer cell.
  • 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.
  • HCAb heavy-chain only antibody
  • ScFab single chain antigen-binding fragment
  • Fc fragment antigen-binding
  • Fc fragment antigen-binding
  • Fc fragment antigen-binding
  • a single chain variable fragment e.g. scFv
  • 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.
  • 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.
  • compositions further comprise a nucleic acid encoding for a MHC class I ligand peptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • the administering is local administration or systemic administration.
  • the administering is subcutaneous administration or intravenous administration.
  • 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.
  • methods of treating cancer in a subject 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 cancer in the subject.
  • the administering is local administration or systemic administration.
  • 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.
  • methods wherein the subject has a solid tumor.
  • the solid tumor is a carcinoma, a melanoma, or a sarcoma.
  • methods, wherein the subject has a blood cancer Further provided herein are methods, wherein the blood cancer is a lymphoma or a leukemia.
  • 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.
  • 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.
  • methods of reducing inflammation in a subject 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.
  • 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.
  • EXAMPLE 1 MHC CLASS I MOLECULE CONSTRUCTION, EXPRESSION AND PURIFICATION
  • 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).
  • NLV N termini peptide
  • B2m B2m
  • gly-ser linker MHC class I (HLA-A02) heavy chain
  • 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.
  • 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”.
  • 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.
  • 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.
  • 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.
  • 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.
  • EXAMPLE 5 CO-RECEPTOR BINDING ASSAY VIA SPR (AFFINITY VALIDATION).
  • SPR assays were conducted using a BIACORETM 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.
  • 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.
  • 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).
  • 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.
  • SPR Surface plasmon resonance 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))
  • A02.Q115E SEQ ID NO: 96
  • KD equilibrium dissociation constant
  • 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).
  • 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.
  • 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.
  • PBS phosphate-buffered saline
  • 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.
  • 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.
  • HEK Human embryonic kidney
  • 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 10 A 6 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 10 A 6 cells/mL in 50 mL Expi293 Expression Medium.
  • Transfection was performed using the ExpiFectamineTM 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-MEMTM Reduced Serum Medium (Thermo Fisher Scientific). Separately, 160 pL of ExpiFectamineTM 293 reagent was diluted in 3 mL of Opti-MEMTM 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, ExpiFectamineTM 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne des compositions de molécules du CMH (complexe majeur d'histocompatibilité) modifiées pour le traitement d'une maladie ou d'un trouble. Les compositions selon la présente invention comprennent une chaîne lourde de CMH modifiée ; une protéine bêta 2-microglobuline (B2m) modifiée ; et/ou un complexe de molécules de classe I du CMH qui ont été modifiées pour présenter une affinité de liaison accrue aux co-récepteurs cellulaires, tels que CD8, et présentent également une internalisation accrue dans des cellules immunitaires par rapport à une chaîne lourde du CMH de classe I autrement comparable, une protéine B2m, ou un complexe de molécules de classe I du CMH. L'invention concerne également diverses constructions protéiques et des compositions pharmaceutiques des molécules du CMH modifiées. L'invention concerne en outre des méthodes d'utilisation des constructions protéiques et des compositions pharmaceutiques des molécules du CMH modifiées.
PCT/US2025/023211 2024-04-04 2025-04-04 Molécules du complexe majeur d'histocompatibilité modifiées et leurs utilisations Pending WO2025213057A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070031442A1 (en) * 2003-02-17 2007-02-08 Andrew Sewell Method and compositions for boosting immune response
US20210230542A1 (en) * 2018-06-06 2021-07-29 Stemcell Technologies Canada Inc. Kits, compositions and methods for cell enrichment
US20230054274A1 (en) * 2019-07-02 2023-02-23 Immunocore Limited Peptide-mhc complexes
US20230265157A1 (en) * 2020-06-24 2023-08-24 Repertoire Immune Medicines, Inc. Mhc multimer expression constructs and uses thereof
WO2024059740A1 (fr) * 2022-09-14 2024-03-21 Synthego Corporation Polynucléotides génétiquement modifiés et cellules exprimant des protéines mhc modifiées et leurs utilisations
US20240092899A1 (en) * 2021-02-07 2024-03-21 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Bispecific antibody

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070031442A1 (en) * 2003-02-17 2007-02-08 Andrew Sewell Method and compositions for boosting immune response
US20210230542A1 (en) * 2018-06-06 2021-07-29 Stemcell Technologies Canada Inc. Kits, compositions and methods for cell enrichment
US20230054274A1 (en) * 2019-07-02 2023-02-23 Immunocore Limited Peptide-mhc complexes
US20230265157A1 (en) * 2020-06-24 2023-08-24 Repertoire Immune Medicines, Inc. Mhc multimer expression constructs and uses thereof
US20240092899A1 (en) * 2021-02-07 2024-03-21 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Bispecific antibody
WO2024059740A1 (fr) * 2022-09-14 2024-03-21 Synthego Corporation Polynucléotides génétiquement modifiés et cellules exprimant des protéines mhc modifiées et leurs utilisations

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