US20230321206A1 - Mhc class ii t-cell modulatory multimeric polypeptides for treating type 1 diabetes mellitus (t1d) and methods of use thereof - Google Patents

Mhc class ii t-cell modulatory multimeric polypeptides for treating type 1 diabetes mellitus (t1d) and methods of use thereof Download PDF

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US20230321206A1
US20230321206A1 US18/107,244 US202318107244A US2023321206A1 US 20230321206 A1 US20230321206 A1 US 20230321206A1 US 202318107244 A US202318107244 A US 202318107244A US 2023321206 A1 US2023321206 A1 US 2023321206A1
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polypeptide
amino acid
acid sequence
cases
mhc class
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Ronald D. Seidel, III
Rodolfo J. Chaparro
John F. Ross
Chee Meng Low
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Cue Biopharma Inc
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Assigned to CUE BIOPHARMA, INC. reassignment CUE BIOPHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHAPARRO, RODOLFO J., LOW, CHEE MENG, ROSS, JOHN F., SEIDEL, RONALD D., III
Priority to US18/218,943 priority patent/US12029782B2/en
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
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    • 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
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01015—Glutamate decarboxylase (4.1.1.15)
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • A61K2039/645—Dendrimers; Multiple antigen peptides
    • C—CHEMISTRY; METALLURGY
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction

Definitions

  • APCs serve to capture and break the proteins from foreign organisms, or abnormal proteins (e.g., from genetic mutation in cancer cells), into smaller fragments suitable as signals for scrutiny by the larger immune system, including T cells.
  • APCs break down proteins into small peptide fragments, which are then paired with proteins of the major histocompatibility complex (“MHC”) and displayed on the cell surface.
  • MHC major histocompatibility complex
  • the peptide fragments can be pathogen-derived, tumor-derived, or derived from natural host proteins (self-proteins).
  • APCs can recognize other foreign components, such as bacterial toxins, viral proteins, viral DNA, viral RNA, etc., whose presence denotes an escalated threat level. The APCs relay this information to T cells through additional costimulatory signals in order to generate a more effective response.
  • T cells recognize peptide-major histocompatibility complex (“pMHC”) complexes through a specialized cell surface receptor, the T cell receptor (“TCR”).
  • TCR T cell receptor
  • the TCR is unique to each T cell; as a consequence, each T cell is highly specific for a particular pMHC target.
  • pMHC peptide-major histocompatibility complex
  • TCR T cell receptor
  • any given T cell, specific for a particular T cell peptide is initially a very small fraction of the total T cell population.
  • Such activated T cell responses are capable of attacking and clearing viral infections, bacterial infections, and other cellular threats including tumors, as illustrated below.
  • the broad, non-specific activation of overly active T cell responses against self or shared antigens can give rise to T cells inappropriately attacking and destroying healthy tissues or cells.
  • HLA class II gene loci include HLA-DM (HLA-DMA and HLA-DMB that encode HLA-DM ⁇ chain and HLA-DM f chain, respectively), HLA-DO (HLA-DOA and HLA-DOB that encode HLA-DO ⁇ chain and HLA-DO ⁇ chain, respectively), HLA-DP (HLA-DPA and HLA-DPB that encode HLA-DP ⁇ chain and HLA-DP ⁇ chain, respectively), HLA-DQ (HLA-DQA and HLA-DQB that encode HLA-DQ ⁇ chain and HLA-DQ ⁇ chain, respectively), and HLA-DR (HLA-DRA and HLA-DRB that encode HLA-DR ⁇ chain and HLA-DR ⁇ chain, respectively).
  • HLA-DM HLA-DMA and HLA-DMB that encode HLA-DM ⁇ chain and HLA-DM f chain, respectively
  • HLA-DO HLA-DOA and HLA-DOB that encode HLA-DO ⁇
  • the present disclosure provides T-cell modulatory multimeric polypeptides (TMMPs) comprising a type 1 diabetes (T1D)-associated peptide epitope, MHC class II polypeptides, and one or more immunomodulatory polypeptides.
  • T1D type 1 diabetes
  • a TMMP of the present disclosure is useful for modulating activity of a T cell.
  • the present disclosure provides compositions and methods for modulating the activity of T cells, as well as compositions and methods for treating persons who have T1D.
  • FIG. 1 A- 1 E present schematic depictions of TMMPs of the present disclosure, in which the immunomodulatory polypeptide is in Position 1.
  • FIG. 2 A- 2 D present schematic depictions of TMMPs of the present disclosure, in which the immunomodulatory polypeptide is in Position 2.
  • FIG. 3 A- 3 D present schematic depictions of TMMPs of the present disclosure, in which the immunomodulatory polypeptide is in Position 3.
  • FIG. 4 A- 4 D present schematic depictions of TMMPs of the present disclosure, in which the immunomodulatory polypeptide is in Position 5.
  • FIG. 5 is a schematic depiction of various possible positions of an immunomodulatory polypeptide in a TMMP of the present disclosure.
  • FIG. 6 provides amino acid sequence of an HLA Class II DRA ⁇ chain (SEQ ID NO:130).
  • FIG. 7 A- 7 D provide amino acid sequences of HLA Class II DRB1 ⁇ chains ( FIG. 7 A ), HLA Class II DRB3 ⁇ chains ( FIG. 7 B ), an HLA Class II DRB4 ⁇ chain ( FIG. 7 C ), and an HLA Class II DRB5 ⁇ chain ( FIG. 7 D ).
  • FIG. 8 provides amino acid sequences of HLA Class II DPA1 ⁇ chains.
  • FIG. 9 provides amino acid sequences of HLA Class II DPB1 ⁇ chains.
  • FIG. 10 provides amino acid sequences of HLA Class II DQA1 ⁇ chains.
  • FIG. 11 A- 11 B provide amino acid sequences of HLA Class II DQB1 ⁇ chains.
  • FIG. 12 A- 12 G provide amino acid sequences of immunoglobulin Fc polypeptides.
  • FIG. 13 A- 13 N provide amino acid sequences of wild-type ( FIG. 13 A ) and variant ( FIG. 13 B- 3 N ) DRA*0101 ⁇ chains.
  • FIG. 13 O provides an amino acid sequence of a PD-L1 polypeptide.
  • FIG. 14 A- 14 J provide amino acid sequences of wild-type ( FIG. 14 A ) and variant ( FIG. 14 B- 4 J ) DRB1*0401 ⁇ chains.
  • FIGS. 15 A- 15 Z and 15 AA- 15 SS provide amino acid sequences of first and second polypeptide chains of TMMPs and APPs of the present disclosure.
  • FIG. 16 A- 16 C are schematic depictions of disulfide-linked antigen-presenting polypeptides (APPs) of the present disclosure.
  • FIG. 17 A- 17 C depict various disulfide-linked heterodimers and their characterization. These disulfide-linked heterodimers do not include immunomodulatory polypeptides.
  • FIG. 18 A- 18 C depict various disulfide-linked heterodimers and their characterization. These disulfide-linked heterodimers include immunomodulatory polypeptides.
  • FIG. 19 A- 19 D depict production and monomer formation of exemplary TMMPs.
  • FIG. 20 A- 20 D depict production and monomer formation of exemplary TMMPs.
  • Linkers (G4S)6 (SEQ ID NO:77), (G4S)3 (SEQ ID NO:74), (G4S)4 (SEQ ID NO:75), GGSAAAGG (SEQ ID NO:83).
  • FIG. 21 A- 21 E depict the results of stability analysis of TMMP 3005-2639.
  • FIG. 22 A- 22 B depict the results of stability analysis of disulfide-stabilized TMMPs with the immunomodulatory polypeptide (MOD) at Position 3.
  • FIG. 23 depict the results of stability analysis of disulfide-stabilized TMMPs with the MOD at Position 1.
  • FIG. 24 presents a table (Table 6) that provides the expression level (in mg/mL) of various TMMPs.
  • Linkers GGCGS (SEQ ID NO:234) and GCGGS (SEQ ID NO:235)
  • FIG. 25 depicts functional response of Proinsulin-specific CD4 + T cells following administration with TMMP 3005-2639 or TMMP 3893-2938 as compared to control molecules.
  • FIG. 26 depicts antigen-specific, PD-L1-dependent suppression of PI-specific CD4 + T cells following administration of TMMP.
  • FIG. 27 depicts suppression of functional response of GAD65-specific CD4 + T cells by overnight treatment with TMMP 3005-2580 as compared to control molecules.
  • FIG. 28 depicts suppression of functional response of Proinsulin-specific CD4 + T cells stimulated for 5 days by treatment with TMMP 3005-2639 or TMMP 3893-2938 as compared to control molecules.
  • FIG. 29 depicts serum pharmacokinetics (PK) analysis of a single 5 mg/kg intravenous dose of TMMP 3893-2938.
  • FIG. 30 depicts the study design for in vivo analysis of TMMP 3893-2938.
  • FIG. 31 depicts frequencies of Proins- and HA-reactive T cells following administration of proinsulin (Proins) or hemagglutinin (HA) peptides.
  • FIG. 32 depicts frequencies of Proins- and HA-reactive T cells following administration of the TMMPs indicated.
  • FIG. 33 depicts the percent relative change in frequency of proinsulin (PI)- and HA-reactive T cells following treatment with TMMPs, relative to pre-treatment.
  • FIG. 34 depicts the effect of treatment with TMMPs on cytokine production.
  • FIG. 35 depicts data showing that cytokine-producing cells are CD4 + , CD44 hi , and PD-1 + .
  • polynucleotide and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.
  • peptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • a “polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity.
  • modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to polymerase chain reaction (PCR) amplification or other recombinant DNA methods.
  • References herein to a specific residue or residue number in a known polypeptide, e.g., position 72 or 75 of human DRA MHC class II polypeptide, are understood to refer to the amino acid at that position in the wild-type polypeptide (i.e. I72 or K75).
  • a reference herein to substitution of a specific amino acid as a specific position is understood to refer to a substitution of a cysteine for the amino acid at position 72 in the wild-type polypeptide, i.e., isoleucine. If, e.g., the wild-type polypeptide is altered to change the amino acid at position 72 from isoleucine to an alternate amino acid, then the reference to I72C will be understood to refer to the substitution of a cysteine for the alternate amino acid.
  • the reference to I72C will be understood to refer to the substitution of a cysteine for the alternate amino acid at the altered position number.
  • a reference to a “non-naturally occurring Cys residue” in a polypeptide means that the polypeptide comprises a Cys residue in a location where there is no Cys in the corresponding wild-type polypeptide. This can be accomplished through routine protein engineering in which a cysteine is substituted for the amino acid that occurs in the wild-type sequence, e.g., at position 72 or 75 of the DRA*0101 polypeptide (see FIG. 13 G and FIG. 13 H ) instead of the isoleucine (I) or lysine (K) residues that are present in the wild-type DRA*0101 polypeptide (see FIG. 13 A ).
  • a polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways.
  • sequences can be aligned using various convenient methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10. Unless otherwise stated, “sequence identity” as referred to herein is determined by BLAST (Basic Local Alignment Search Tool), as described in Altschul et al. (1990) J. Mol. Biol. 215:403.
  • BLAST Basic Local Alignment Search Tool
  • in vivo refers to any process or procedure occurring inside of the body, e.g., of a T1D patient.
  • in vitro refers to any process or procedure occurring outside of the body.
  • a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine.
  • Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine.
  • binding refers to a non-covalent interaction between two molecules.
  • Non-covalent binding refers to a direct association between two molecules, due to, for example, electrostatic, hydrophobic, ionic, and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • Covalent binding” or “covalent bond,” as used herein, refers to the formation of one or more covalent chemical bonds between two different molecules.
  • T cell includes all types of immune cells expressing CD3, including T-helper cells (CD4 + cells), cytotoxic T-cells (CD8 + cells), T-regulatory cells (Treg), and NK-T cells.
  • immunomodulatory polypeptide includes a polypeptide on an antigen presenting cell (APC) (e.g., a dendritic cell, a B cell, and the like), or a portion of the polypeptide on an APC, that specifically binds a cognate co-immunomodulatory polypeptide on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with a major histocompatibility complex (MHC) polypeptide loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • APC antigen presenting cell
  • MHC major histocompatibility complex
  • An immunomodulatory polypeptide can include, but is not limited to, a cytokine (e.g., IL-2; TGF ⁇ ), CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, and HVEM.
  • a cytokine e.g., IL-2; TGF ⁇
  • an “immunomodulatory polypeptide” specifically binds a cognate co-immunomodulatory polypeptide on a T cell (e.g., a target T cell).
  • PBS Phosphate buffer saline
  • sodium chloride 500 mM
  • sodium phosphate dibasic 10 mM
  • potassium phosphate monobasic 2 mM
  • potassium chloride 2.7 mM
  • the pH of the PBS is 7.5 ⁇ 0.15.
  • the buffer can be prepared in 18.2 megaohms DNase, and RNase free water and filtered through 0.22 micron filter.
  • Recombinant means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems.
  • DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system.
  • recombinant expression vector or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and at least one insert.
  • Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the insert(s), or for the construction of other recombinant nucleotide sequences.
  • the insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.
  • 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 (K D ).
  • antibody refers to the resistance of a complex of two or more agents to dissociation after dilution.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • treatment generally mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease or symptom in a mammal, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to acquiring the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease or symptom, i.e., arresting its development; and/or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • the subject therapy will desirably be administered during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • mammals include, e.g., humans, non-human primates, rodents (e.g., rats; mice), lagomorphs (e.g., rabbits), ungulates (e.g., cows, sheep, pigs, horses, goats, and the like), etc.
  • rodents e.g., rats; mice
  • lagomorphs e.g., rabbits
  • ungulates e.g., cows, sheep, pigs, horses, goats, and the like
  • an Ig Fc that “substantially does not induce cell lysis” means an Ig Fc that induces no cell lysis at all or that largely but not wholly induces no cell lysis.
  • the term “about” used in connection with an amount indicates that the amount can vary by 10%.
  • “about 100” means an amount of from 90-110.
  • the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range
  • “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range.
  • from about 100 to about 1000 means that the range extends from 90 to 1100.
  • the present disclosure provides T-cell modulatory multimeric polypeptides (TMMPs) comprising a type 1 diabetes (T1D)-associated peptide, MHC class II polypeptides, and one or more immunomodulatory polypeptides.
  • T1D type 1 diabetes
  • MHC class II polypeptides MHC class II polypeptides
  • immunomodulatory polypeptides A TMMP of the present disclosure is useful for modulating activity of a T cell.
  • the present disclosure provides compositions and methods for modulating the activity of T cells, as well as compositions and methods for treating individuals who have T1D.
  • TMMPs that comprise a disulfide-linked heterodimer comprising: a) a first polypeptide comprising: i) a peptide that, when present in a TMMP of this disclosure, presents a T1D-associated epitope capable of being bound by a T-cell receptor (TCR) on the surface of a T cell (hereinafter a “T1D peptide”); and ii) a first major histocompatibility complex (MHC) class II polypeptide; and b) a second polypeptide comprising a second MHC class II polypeptide.
  • TCR T-cell receptor
  • MHC major histocompatibility complex
  • One or both polypeptides of the heterodimer comprise one or more immunomodulatory polypeptides.
  • the first and the second polypeptides of the heterodimer are covalently linked to one another via at least one disulfide bond.
  • One of the polypeptides of the heterodimer optionally comprises an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold.
  • Ig immunoglobulin
  • a TMMP of the present disclosure can comprise two disulfide-linked heterodimers. When both heterodimers include Ig Fc polypeptides, disulfide bonds will spontaneously form between the respective Ig Fc polypeptides to covalently link the two heterodimers to one another (depicted schematically in FIG. 1 E ).
  • the first polypeptide and the second polypeptide of a heterodimer of a TMMP of the present disclosure are covalently linked to one another via at least one disulfide bond.
  • the at least one disulfide bond is present between: i) a Cys present in the first MHC class II polypeptide and a Cys present in the second MHC class II polypeptide; or ii) a Cys present in a peptide linker in the first polypeptide and a Cys present in an MHC class II polypeptide present in the second polypeptide; or iii) a Cys present in a peptide linker in the second polypeptide and a Cys present in an MHC class II polypeptide present in the first polypeptide.
  • the at least one disulfide bond formed between the first polypeptide and the second polypeptide of a heterodimer of a TMMP of the present disclosure provides for increased stability and/or expression of the TMMP, compared to a control TMMP lacking the at least one disulfide bond.
  • a TMMP of the present disclosure is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, more stable than a control TMMP lacking the at least one disulfide bond.
  • Stability can be determined by measuring the amount of intact TMMP monomer remaining after incubation of the TMMP for a specific period of time at a specific temperature (e.g., for 1 hour at 37° C.; 1 day at 37° C.; 5 days at 37° C.; 1 hour at 42° C.; 1 day at 42° C.; 5 days at 42° C.; and the like).
  • a disulfide-bonded TMMP of the present disclosure will in some cases exhibit a stability after 3 days at 37° C.
  • TMMP concentrations of 9.5 mg/mL, 1 mg/mL, or 0.1 mg/mL in the PBS buffer solution containing 500 mM NaCl (described above), as measured by the percent monomer remaining, that is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%.
  • a disulfide-bonded TMMP of the present disclosure will in some cases exhibit a stability after 5 days at 37° C. at TMMP concentrations of 9.5 mg/mL, 1 mg/mL, or 0.1 mg/mL in the PBS buffer solution containing 500 mM NaCl (described above), as measured by the percent monomer remaining, that is greater than 50%, greater than 60%, greater than 70%, or greater than 80%.
  • a disulfide-bonded TMMP of the present disclosure will in some cases exhibit a stability after 3 days at 42° C. at TMMP concentrations of 9.5 mg/mL, 1 mg/mL, or 0.1 mg/mL in the PBS buffer solution containing 500 mM NaCl (described above), as measured by the percent monomer remaining, that is greater than 30%, greater than 40%, greater than 50%, or greater than 60%.
  • TMMP concentrations 9.5 mg/mL, 1 mg/mL, or 0.1 mg/mL in the PBS buffer solution containing 500 mM NaCl (described above), as measured by the percent monomer remaining, that is greater than 30%, greater than 40%, greater than 50%, or greater than 60%.
  • a disulfide-bonded TMMP of the present disclosure will in some cases exhibit a stability after 5 days at 42° C.
  • TMMP concentrations of 9.5 mg/mL, 1 mg/mL, or 0.1 mg/mL in the PBS buffer solution containing 500 mM NaCl (described above), as measured by the percent monomer remaining, that is greater than 30%, greater than 40%, greater than 50%, or greater than 60%.
  • Whether a protein is present in solution as a monomer can be determined using size exclusion chromatography (SEC).
  • SEC size exclusion chromatography
  • Analytical SEC can be performed using a Superdex 200 Increase (3.2 ⁇ 300 mm) column (e.g., from GE Healthcare).
  • the running buffer can be PBS containing 500 mM NaCl (described above) and the flow rate can be 0.15 ml/minute.
  • a TMMP of the present disclosure is expressed (produced) in a Chinese hamster ovary (CHO) cell in vitro at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, higher than the expression level of a control TMMP lacking the at least one disulfide bond when expressed under the same conditions and in the same CHO cells.
  • CHO Chinese hamster ovary
  • Expression levels can be determined by: i) producing the TMMP in a mammalian cell in vitro in a CHO cell in vitro; and ii) determining the amount of TMMP produced by the mammalian cell.
  • a TMMP comprises an Ig Fc polypeptide
  • the TMMP can be isolated from the mammalian cells and/or from culture medium in which the mammalian cells are cultured, where isolation of the TMMP can be carried out by affinity chromatography, e.g., on a Protein A column, a Protein G column, or the like.
  • An example of a suitable mammalian cell is a CHO cell; e.g., an Expi-CHO-STM cell (e.g., ThermoFisher Scientific, Catalog #A29127).
  • a protein consisting of the desired TMMP is referred to herein as a “monomer.”
  • “monomer” can refer to a single heterodimer that is the desired TMMP when the heterodimer is not covalently bonded to another heterodimer, e.g., when the heterodimer does not include an optional Ig Fc polypeptide.
  • TMMP protein comprising 2 heterodimers, each comprising 2 polypeptide chains (for a total of 4 polypeptide chains), where 2 of the four polypeptide chains comprise Ig Fc polypeptides, wherein the Ig Fc polypeptides spontaneously form disulfide bonds that covalently link the heterodimers to each other, also is referred to herein as a “monomer.”
  • the term “monomer” does not include aggregates of monomers.
  • SEC size exclusion chromatography
  • Analytical SEC can be performed using a Superdex 200 Increase (3.2 ⁇ 300 mm) column (e.g., from GE Healthcare).
  • the running buffer can be PBS containing 500 mM NaCl (described above) and the flow rate can be 0.15 ml/minute.
  • a TMMP of the present disclosure exhibits at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, greater stability in the PBS buffer solution containing 500 mM NaCl (described above) in vitro over a specified period of time and at a specified temperature (e.g., in a solution at a temperature of 37° C. to 42° C.
  • a specified temperature e.g., in a solution at a temperature of 37° C. to 42° C.
  • TMMP can be present in the PBS buffer solution in a concentration of from 0.1 mg/mL to 10 mg/mL, and the buffer solution can be kept at 37° C. or 42° C.
  • stability is determined by measuring the percent of monomer (as defined above) remaining in the solution after a specified time in the solution at a specified temperature. Loss of monomer often is characterized by the formation of aggregates, or alternatively by the breakdown of the monomer into one or more separate polypeptide chains or fragments thereof.
  • “at least 10% greater stability” means that at least 10% more TMMP monomer is present in a test sample than in a control sample comprising the same polypeptide chains but lacking the at least one disulfide bond between the first polypeptide and the second polypeptide of the heterodimer.
  • the percent more monomer in the test sample is calculated based on the amount of monomer in the control sample.
  • a TMMP of the present disclosure exhibits at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, greater stability in a buffered solution such as the PBS containing 500 mM NaCl (described above) in vitro than the TMMP 3003-2579 ( FIG. 15 F and FIG. 15 E ) or than the TMMP 3003-2639 ( FIG. 15 F and FIG.
  • a TMMP of the present disclosure exhibits at least at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, greater stability in the PBS solution containing 500 mM NaCl (described above) in vitro than the TMMP 3005-2639 ( FIG. 15 D and FIG. 15 C ) when kept for 1 hour, 5 days, or 10 days at 37° C. or at 42° C.
  • the presence of the at least one disulfide bond between the first polypeptide and the second polypeptide of the heterodimer can lead to greater expression of the TMMP as compared to a TMMP lacking the at least one disulfide bond.
  • a TMMP of the present disclosure is expressed (produced) at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, higher than the expression level of the TMMP 3003-2579 ( FIG. 15 F and FIG. 15 E ) or of the TMMP 3003-2639 ( FIG. 15 F and FIG. 15 C ).
  • a TMMP of the present disclosure is expressed (produced) at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, higher than the expression level of the TMMP 3005-2639 ( FIG. 15 D and FIG. 15 C ).
  • stability can be expressed as the “% monomer” (as defined above) remaining after incubation of a TMMP of the present disclosure in the PBS buffer described above at a certain temperature for a certain amount of time.
  • the % monomers remaining after incubation of a TMMP of the present disclosure in the PBS buffer for a given time at a given temperature e.g., 37° C. for 1 hour; 37° C. for 1 day; 37° C. for 5 days; 37° C. for 10 days; 37° C. for 14 days, 37° C. for 28 days; 42° C. for 1 hour; 42° C. for 1 day; 42° C. for 5 days; 42° C.
  • a TMMP of the present disclosure can comprise two heterodimers.
  • the two heterodimers each include an Ig Fc polypeptide.
  • the TMMP comprises two heterodimers that are disulfide linked to one another via Cys residues present in the Ig Fc polypeptides. See, e.g., FIG. 1 E for a schematic depiction of an example of such a TMMP.
  • a TMMP of the present disclosure comprises one or more immunomodulatory polypeptides.
  • a TMMP of the present disclosure comprises a single immunomodulatory polypeptide.
  • the single immunomodulatory polypeptide is on the first polypeptide.
  • the single immunomodulatory polypeptide is on the second polypeptide.
  • the single immunomodulatory polypeptide is on the second polypeptide, and the second polypeptide comprises an MHC class II alpha polypeptide.
  • a TMMP of the present disclosure comprises two or more immunomodulatory polypeptides (e.g., 2, 3, 4, or 5 immunomodulatory polypeptides).
  • a TMMP of the present disclosure comprises a disulfide-linked heterodimer comprising a first polypeptide and a second polypeptide.
  • the two or more immunomodulatory polypeptides are present in the first polypeptide chain only.
  • the two or more immunomodulatory polypeptides are present in the second polypeptide chain only.
  • a TMPP of the present disclosure comprises a first polypeptide and a second polypeptide
  • at least one of the two or more immunomodulatory polypeptides are present in the first polypeptide chain
  • at least one of the two or more immunomodulatory polypeptides are present in the second polypeptide chain.
  • the immunomodulatory polypeptides are on the second polypeptide
  • the second polypeptide comprises an MHC class II alpha polypeptide.
  • a TMPP of the present disclosure comprises 2 or 3 copies of an immunomodulatory polypeptide, in some cases, the 2 or 3 copies are in tandem.
  • a TMPP of the present disclosure comprises 2 or 3 copies of an immunomodulatory polypeptide, in some cases, the 2 or 3 copies are separated from one another by a linker.
  • a TMPP of the present disclosure comprises two immunomodulatory polypeptides
  • the two immunomodulatory polypeptides have the same amino acid sequence, i.e., the TMPP comprises two copies of an immunomodulatory polypeptide.
  • the two immunomodulatory polypeptides do not have the same amino acid sequence; e.g., one of the two immunomodulatory polypeptides comprises a first amino acid sequence and the second of the two immunomodulatory polypeptides comprises a second amino acid sequence, where the first and the second amino acid sequences are not identical.
  • the first and the second amino acid sequences differ from one another in amino acid sequence by from 1 amino acid to 10 amino acids, from 10 amino acids to 25 amino acids, or more than 25 amino acids. In some cases, the first and the second amino acid sequences share less than 98%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, or less than 70%, amino acid sequence identity with one another.
  • a TMMP of the present disclosure modulates activity of a T cell.
  • a TMMP of the present disclosure reduces activity of an autoreactive T cell and/or an autoreactive B cell, e.g., a T cell reactive with a T1D-associated epitope or a B cell reactive with a T1D-associated epitope (e.g., a B cell that produces antibodies that bind to a T1D-associated epitope).
  • a TMMP of the present disclosure increases the number and/or activity of a regulatory T cell (Treg), resulting in reduced activity of an autoreactive T cell and/or an autoreactive B cell.
  • a TMMP of the present disclosure is useful for treating T1D in an individual.
  • Immunomodulatory polypeptides that are suitable for inclusion in a TMPP of the present disclosure include, but are not limited to, IL-2, transforming growth factor-beta (TGF ⁇ ), JAG1, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, and HVEM.
  • TGF ⁇ transforming growth factor-beta
  • a TMMP of the present disclosure comprises one or more immunomodulatory polypeptides selected from the group consisting of an IL-2 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, a TGF- ⁇ polypeptide, and combinations thereof.
  • a TMMP of the present disclosure comprises one or more immunomodulatory polypeptides, where at least one of the one or more immunomodulatory polypeptides is a PD-L1 polypeptide.
  • a TMMP of the present disclosure comprises one or more immunomodulatory polypeptides, where at least one of the one or more immunomodulatory polypeptides is an IL-2 polypeptide.
  • a TMMP of the present disclosure comprises one or more immunomodulatory polypeptides, where at least one of the one or more immunomodulatory polypeptides is a TGF- ⁇ polypeptide. In some cases, a TMMP of the present disclosure comprises one or more immunomodulatory polypeptides, where at least one of the one or more immunomodulatory polypeptides is a FasL polypeptide.
  • an immunomodulatory polypeptide suitable for inclusion in a TMPP of the present disclosure comprises a wild-type amino acid sequence.
  • an immunomodulatory polypeptide suitable for inclusion in a TMPP of the present disclosure is a variant, e.g., comprising from 1 to 10 amino acid substitutions relative to a wild-type or naturally-occurring immunomodulatory polypeptide.
  • an immunomodulatory polypeptide suitable for inclusion in a TMPP of the present disclosure is a variant that exhibits reduced affinity to its cognate co-immunomodulatory polypeptide (e.g., a co-immunomodulatory polypeptide present on the surface of a T cell), compared to the affinity of the wild-type or naturally-occurring immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide.
  • a TMMP of the present disclosure comprises two disulfide-linked heterodimeric polypeptides comprising the following components: a) a T1D peptide; b) an MHC class II ⁇ polypeptide; c) an MHC class II ⁇ polypeptide; and d) one or more immunomodulatory polypeptides; and may also one or more peptide linkers; and may also include Ig Fc polypeptide(s).
  • the T1D peptide can be on same polypeptide with either the MHC class II ⁇ polypeptide or MHC class II ⁇ polypeptide, the T1D peptide typically will be on the same polypeptide chain as the MHC class II ⁇ polypeptide.
  • FIGS. 1 A- 1 E These components can be present in a TMMP of the present disclosure in various arrangements. Examples of such arrangements are depicted schematically in FIGS. 1 A- 1 E , FIGS. 2 A- 2 D , FIGS. 3 A- 3 D , and FIGS. 4 A- 4 D .
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) an MHC class II ⁇ polypeptide, optionally joined by a linker; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; and ii) an MHC class II ⁇ polypeptide, optionally joined by a linker, where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond.
  • a TMPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) an MHC class II ⁇ polypeptide, optionally joined by a linker; and b) a second polypeptide comprising: i) one or more immunomodulatory polypeptides (where two or more immunomodulatory polypeptides are present, they optionally may be joined by one or more linkers); ii) an MHC class II ⁇ polypeptide, optionally joined by a linker to the immunomodulatory polypeptide(s); and iii) an Ig Fc polypeptide, optionally joined by a linker to the MHC class II ⁇ polypeptide; where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond.
  • a TMPP of the present disclosure comprises a heterodimer comprising two disulfide-linked polypeptides, wherein the heterodimer comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) a T1D peptide; and ii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) an immunomodulatory polypeptide; ii) an MHC class II ⁇ polypeptide; and iii) an Ig Fc polypeptide.
  • a TMPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) a T1D peptide; and ii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including one or more linkers that do not comprise a Cys): i) an MHC class II ⁇ polypeptide; ii) one or more immunomodulatory polypeptides; and iii) an Ig Fc polypeptide. (See FIG.
  • a TMPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) a T1D peptide; and ii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) an MHC class II ⁇ polypeptide; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides. (See FIG. 5 , MOD position 3.).
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) one or more immunomodulatory polypeptides; ii) a T1D peptide; and iii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) an MHC class II ⁇ polypeptide; and ii) an Ig Fc polypeptide. (See FIG. 5 , MOD position 4.).
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) a T1D peptide; ii) an MHC class II ⁇ polypeptide; and iii) one or more immunomodulatory polypeptides; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including linkers): i) an MHC class II ⁇ polypeptide; and ii) an Ig Fc polypeptide.
  • the immunomodulatory polypeptide(s) can be on the same polypeptide chain as the MHC class II ⁇ polypeptides.
  • potential locations in the heterodimer for disulfide bonds are where residues in the first and second polypeptides of the heterodimer are separated by a distance of 5 angstroms or less. Such locations represent potential locations where Cys residues, if not naturally present, can be substituted for the residues that exist in the polypeptides.
  • a first polypeptide and a second polypeptide thus can be linked via a disulfide bond between two Cys residues that are generally no more than about 5 angstroms apart from one another in the heterodimer. In some cases, one or both of the Cys residues are non-naturally-occurring.
  • amino acid in the alpha chain and an amino acid in the beta chain of MHC class II polypeptides that are no more than 5 angstroms from one another in an MHC class II heterodimer represent amino acids that, when substituted with a Cys, can form a disulfide bond in a TMMP of the present disclosure.
  • a disulfide bond can be formed i) between a Cys residue in a linker and a Cys residue in an MHC class II polypeptide, or ii) between a Cys residue in a linker in one polypeptide of the heterodimer and a Cys residue in a linker in the other polypeptide of the heterodimer, where the two Cys residues are no more than about 5 angstroms apart from each other in the heterodimer.
  • not all pairs of residues separated by about 5 angstroms or less will be suitable for formation of a disulfide bond or provide a disulfide bond that stabilizes the resulting heterodimer.
  • the TMMP comprises a peptide linker between the T1D peptide and the MHC class II polypeptide.
  • the linker can comprise a Cys that can be used to form a disulfide bond between the two polypeptides in the heterodimer.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including one or more linkers that do not comprise a Cys): i) one or more immunomodulatory polypeptides; ii) an MHC class II ⁇ chain; and (optionally) iii) an Ig Fc polypeptide.
  • the peptide linker comprising a Cys can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof, and in some cases the immunomodulatory polypeptide is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the MHC class II ⁇ chain comprises an amino acid substitution of an amino acid (other than a Cys) with a Cys, such that the Cys in the MHC class II ⁇ chain forms a disulfide bond with the Cys in the first polypeptide linker.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including one or more linkers that do not comprise a Cys): i) an MHC class II ⁇ chain; ii) one or more immunomodulatory polypeptides; and iii) an Ig Fc polypeptide. (See FIG.
  • the peptide linker comprising a Cys can comprise, e.g., an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • Other peptide linkers comprising a Cys may be used.
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof, and in some cases, the immunomodulatory polypeptide is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the MHC class II ⁇ chain comprises an amino acid substitution of an amino acid (other than a Cys) with a Cys, such that the Cys in the MHC class II ⁇ chain forms a disulfide bond with the Cys in the linker.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus (optionally including one or more linkers that do not comprise a Cys): i) an MHC class II ⁇ chain; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides. (See FIG.
  • the peptide linker comprising a Cys can comprise, e.g., an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • Other peptide linkers comprising a Cys may be used.
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof. In any of the above embodiments, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide or variant thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the MHC class II ⁇ chain comprises an amino acid substitution of an amino acid (other than a Cys) with a Cys, such that the Cys in the MHC class II ⁇ chain forms a disulfide bond with the Cys in the linker.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a T1D peptide; ii) a peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class II ⁇ chain; and (optionally) ii) an Ig Fc polypeptide, optionally joined by a linker that does not comprise a Cys. (See FIG.
  • the peptide linker comprising a Cys can comprise, e.g., an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • Other peptide linkers comprising a Cys may be used.
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof, and in some cases the immunomodulatory polypeptide is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the MHC class II ⁇ chain comprises an amino acid substitution of an amino acid (other than a Cys) with a Cys, such that the Cys in the MHC class II ⁇ chain forms a disulfide bond with the Cys in the linker.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a peptide linker comprising a Cys; iii) an MHC class II ⁇ polypeptide; and iv) one or more immunomodulatory polypeptides; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class II ⁇ chain; and (optionally) ii) an Ig Fc polypeptide, optionally joined by a linker that does not comprise a Cys. (See FIG.
  • the peptide linker comprising a Cys can comprise, e.g., an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • Other peptide linkers comprising a Cys may be used.
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof, and in some cases the immunomodulatory polypeptide is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the MHC class II ⁇ chain comprises an amino acid substitution of an amino acid (other than a Cys) with a Cys, such that the Cys in the MHC class II ⁇ chain forms a disulfide bond with the Cys in the linker.
  • the TMMP comprises a first Cys-containing peptide linker between the T1D peptide and the MHC class II polypeptide in the first polypeptide, and a second Cys-containing peptide linker between two of the components in the second polypeptide.
  • the Cys residues in the first and second Cys-containing linkers can be used to form a disulfide bond between the two polypeptides in the heterodimer.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a first peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide (e.g., a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a first peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide (e.g., a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at
  • a second polypeptide comprising, in order from N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) an MHC class II ⁇ chain (e.g., a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 13 A ); and (optionally) iii) an Ig Fc polypeptide. (See FIG.
  • the second polypeptide comprises a second peptide linker that comprises a Cys, where the second peptide linker is positioned either between the immunomodulatory polypeptides and the MHC class II ⁇ chain or between the MHC class II ⁇ chain and the Ig Fc, if present.
  • the second polypeptide optionally may include one or more linkers that do not comprise a Cys.
  • the peptide linkers comprising a Cys can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof, and in some cases the immunomodulatory polypeptide is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a first peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide (e.g., a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a first peptide linker comprising a Cys; and iii) an MHC class II ⁇ polypeptide (e.g., a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at
  • a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC class II ⁇ chain (e.g., a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 13 A ); ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides. (See FIG.
  • the second polypeptide comprises a second peptide linker that comprises a Cys, where the second peptide linker is positioned either between the MHC class II ⁇ chain and the Ig Fc or between the IG Fc and the one or more immunomodulatory polypeptides.
  • the second polypeptide optionally may include one or more linkers that do not comprise a Cys.
  • the peptide linkers comprising a Cys can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof, in some cases the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof, and in some cases the immunomodulatory polypeptide is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the peptide linkers comprising a Cys can comprise, e.g., an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • Other peptide linkers comprising a Cys may be used.
  • the immunomodulatory polypeptide is a PD-L1 polypeptide or variant thereof. In any of the above embodiments, in some cases the immunomodulatory polypeptide is a TGF- ⁇ polypeptide or variant thereof. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is an IL-2 polypeptide or variant thereof. In any of the above embodiments, in some cases, the immunomodulatory polypeptide is a FasL polypeptide or variant thereof. In some cases, the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • a disulfide-linked TMMP can be constructed in which the one or more immunomodulatory polypeptides are at Position 2, 4 or 5 as those positions are described above and depicted schematically in FIG. 5 .
  • the first polypeptide of a heterodimer of a TMMP of the present disclosure comprises a first MHC class II polypeptide comprising an amino acid substitution that results in a Cys (a “first Cys”); and the second polypeptide comprises a second MHC class II polypeptide comprising an amino acid substitution that results in a Cys (a “second Cys”); where the heterodimer comprises a disulfide bond formed between the first Cys and the second Cys.
  • the first polypeptide comprises an MHC class II ⁇ polypeptide comprising an amino acid substitution that results in a Cys (a “first Cys”); and the second polypeptide comprises an MHC class II ⁇ polypeptide comprising an amino acid substitution that results in a Cys (a “second Cys”); where the heterodimer comprises a disulfide bond formed between the first Cys and the second Cys.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “first Cys”), the components of the first polypeptide optionally being joined by a linker that does not comprise a Cys; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a second MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “second Cys”); and iii) optionally an Ig Fc polypeptide, the components of the second polypeptide optionally being joined by one or more linkers that do not comprise a Cys; where
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is a TGF- ⁇ polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is an IL-2 polypeptide or variant thereof, and in some cases the one or more immunomodulatory polypeptides is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the first MHC class II polypeptide is an MHC class II ⁇ chain polypeptide and the second MHC class II polypeptide is an MHC class II ⁇ chain polypeptide.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “first Cys”), the components of the first polypeptide optionally being joined by a linker that does not comprise a Cys; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “second Cys”); ii) one or more immunomodulatory polypeptides; and iii) optionally an Ig Fc polypeptide, the components of the second polypeptide optionally being joined by one or more linkers that do not comprise a Cys;
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is a TGF- ⁇ polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is an IL-2 polypeptide or variant thereof, and in some cases the one or more immunomodulatory polypeptides is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the first MHC class II polypeptide is an MHC class II ⁇ chain polypeptide and the second MHC class II polypeptide is an MHC class II ⁇ chain polypeptide.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “first Cys”), the components of the first polypeptide optionally being joined by a linker that does not comprise a Cys; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “second Cys”); ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides, the components of the second polypeptide optionally being joined by a linker that does not comprise a Cys; where the first
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is a TGF- ⁇ polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is an IL-2 polypeptide or variant thereof, and in some cases the one or more immunomodulatory polypeptides is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the first MHC class II polypeptide is an MHC class II ⁇ chain polypeptide and the second MHC class II polypeptide is an MHC class II ⁇ chain polypeptide.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a T1D peptide; and iii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “first Cys”), the components of the first polypeptide optionally being joined by one or more linkers that do not comprise a Cys; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “second Cys”); and ii) optionally an Ig Fc polypeptide, where when the optional Ig Fc is present, the components of the second polypeptide are optionally joined by a
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is a TGF- ⁇ polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is an IL-2 polypeptide or variant thereof, and in some cases the one or more immunomodulatory polypeptides is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the first MHC class II polypeptide is an MHC class II ⁇ chain polypeptide and the second MHC class II polypeptide is an MHC class II ⁇ chain polypeptide.
  • a TMMP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “first Cys”); and iii) one or more immunomodulatory polypeptides, the components of the first polypeptide optionally being joined by one or more linkers that do not comprise a Cys; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “second Cys”); and ii) optionally an Ig Fc polypeptide, where when the optional Ig Fc is present, the components of the second polypeptide are optionally joined by a
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is a TGF- ⁇ polypeptide or variant thereof, in some cases the one or more immunomodulatory polypeptides is an IL-2 polypeptide or variant thereof, and in some cases the one or more immunomodulatory polypeptides is a FasL polypeptide or variant thereof.
  • the T1D peptide is a proinsulin peptide. In some cases, the T1D peptide is a GAD peptide.
  • the first MHC class II polypeptide is an MHC class II ⁇ chain polypeptide and the second MHC class II polypeptide is an MHC class II ⁇ chain polypeptide.
  • a TMMP of the present disclosure comprises MHC class II polypeptides.
  • Naturally occurring MHC class II polypeptides comprise an ⁇ chain and a ⁇ chain.
  • MHC class II polypeptides include human leukocyte antigen (HLA) ⁇ - and ⁇ -chains.
  • MHC class II polypeptides include MHC class II DP ⁇ and ⁇ polypeptides, DM ⁇ and ⁇ polypeptides, DOA ⁇ and ⁇ polypeptides, DOB ⁇ and ⁇ polypeptides, DQ ⁇ and ⁇ polypeptides, and DR ⁇ and ⁇ polypeptides.
  • a “MHC class II polypeptide” can comprise a MHC class II ⁇ chain polypeptide, a MHC class II ⁇ chain polypeptide, or only a portion of a MHC class II ⁇ or ⁇ chain polypeptide.
  • a “MHC class II polypeptide” can be a polypeptide that includes: i) only the ⁇ 1 domain of a MHC class II ⁇ chain polypeptide; ii) only the ⁇ 2 domain of a MHC class II ⁇ chain; iii) only the ⁇ 1 domain and an ⁇ 2 domain of a MHC class II ⁇ chain; iv) only the ⁇ 1 domain of a MHC class II ⁇ chain; v) only the ⁇ 2 domain of a MHC class II ⁇ chain; vi) only the ⁇ 1 domain and the ⁇ 2 domain of a MHC class II chain; vii) the ⁇ 1 domain of a MHC class II ⁇ chain, the ⁇ 1 domain of a MHC class II chain, and the ⁇ 2 domain of a MHC class II; and the like.
  • MHC class II polypeptides include allelic forms.
  • the HLA locus is highly polymorphic in nature.
  • MHC class II polypeptide includes allelic forms of any known MHC class II polypeptide.
  • a TMMP of the present disclosure comprises an MHC class II ⁇ chain (e.g., a DPA1, DRA1, or DQA1 ⁇ chain), without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring MHC class II ⁇ chain.
  • MHC class II ⁇ chain e.g., a DPA1, DRA1, or DQA1 ⁇ chain
  • intracellular portions e.g., cytoplasmic tails
  • a TMMP of the present disclosure comprises only the ⁇ 1 and ⁇ 2 portions of an MHC class II ⁇ chain (e.g., only the ⁇ 1 and ⁇ 2 portions of a DPA1, DRA1, or DQA1 ⁇ chain); and does not include the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring MHC class II ⁇ chain.
  • a TMMP of the present disclosure comprises an MHC class II ⁇ chain (e.g., a DPB1, DRB1, or DQB1 ⁇ chain), without the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring MHC class II ⁇ chain.
  • MHC class II ⁇ chain e.g., a DPB1, DRB1, or DQB1 ⁇ chain
  • intracellular portions e.g., cytoplasmic tails
  • a TMMP of the present disclosure comprises only the ⁇ 1 and ⁇ 2 portions of an MHC class II ⁇ chain (e.g., only the ⁇ 1 and ⁇ 2 portions of a DPB1, DRB1, or DQB1 ⁇ chain); and does not include the leader, transmembrane, and intracellular portions (e.g., cytoplasmic tails) that may be present in a naturally-occurring MHC class II ⁇ chain.
  • MHC class II alpha chains comprise an ⁇ 1 domain and an ⁇ 2 domain.
  • the ⁇ 1 domain and the ⁇ 2 domain present in an antigen-presenting cell are from the same MHC class II ⁇ chain polypeptide.
  • the ⁇ 1 domain and the ⁇ 2 domain present in an antigen-presenting cell are from two different MHC class II ⁇ chain polypeptides.
  • MHC class II alpha chains suitable for inclusion in a TMMP of the present disclosure lack a signal peptide.
  • An MHC class II alpha chain suitable for inclusion in a multimeric polypeptide of the present disclosure can have a length of from about 60 amino acids to about 190 amino acids; for example, an MHC class II alpha chain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 60 amino acids to about 80 amino acids, from about 80 amino acids to about 100 amino acids, from about 100 amino acids to about 120 amino acids, from about 120 amino acids to about 140 amino acids, from about 140 amino acids to about 160 amino acids, from about 160 amino acids to about 180 amino acids, or from about 180 amino acids to about 200 amino acids.
  • An MHC class II ⁇ 1 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 95 amino acids; for example, an MHC class II ⁇ 1 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 60 amino acids, from about 60 amino acids to about 70 amino acids, from about 70 amino acids to about 80 amino acids, from about 80 amino acids to about 90 amino acids, or from about 90 amino acids to about 95 amino acids.
  • An MHC class II ⁇ 2 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 95 amino acids; for example, an MHC class II ⁇ 2 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 60 amino acids, from about 60 amino acids to about 70 amino acids, from about 70 amino acids to about 80 amino acids, from about 80 amino acids to about 90 amino acids, or from about 90 amino acids to about 95 amino acids.
  • an MHC class II ⁇ chain polypeptide present in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type MHC class II ⁇ chain polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • an amino acid substitution can occur between amino acid 55 and 110 of the MHC class II ⁇ chain polypeptide.
  • an amino acid substitution that replaces an amino acid other than a Cys with a Cys can be present between amino acids 55 and 60, between amino acids 60 and 65, between amino acids 65 and 70, between amino acids 70 and 75, between amino acids 75 and 80, between amino acids 80 and 85, between amino acids 85 and 90, between amino acids 90 and 95, between amino acids 95 and 100, between amino acids 100 and 105, or between amino acids 105 and 110.
  • a suitable MHC class II ⁇ chain polypeptide is a DRA polypeptide, more specifically a DRA1 polypeptide.
  • a DRA polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 26-203 of the DRA amino acid sequence depicted in FIG. 6 .
  • the DRA polypeptide has a length of about 178 amino acids (e.g., 175, 176, 177, 178, 179, or 180 amino acids).
  • a “DRA polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITNV PPEVTVLTNSPVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLPSTEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO:6, amino acids 26-203 of DRA*01:02, see FIG.
  • a DRA polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRA polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a TMMP of the present disclosure comprises a variant DRA polypeptide that comprises a non-naturally occurring Cys residue.
  • a TMMP of the present disclosure comprises a variant DRA polypeptide that comprises an amino acid substitution selected from E3C, E4C, F12C, G28C, D29C, I72C, K75C, T80C, P81C, I82C, T93C, N94C, and S95C.
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLECMTK RSNYTPITNV PPEVTVLTNSPVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLPSTEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO:7), where amino acid 172 is substituted with a Cys (shown in bold text).
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTC RSNYTPITNV PPEVTVLTNSPVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLPSTEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO:8), where amino acid K75 is substituted with a Cys (shown in bold text).
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAV DKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTT GVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPET (SEQ ID NO:9).
  • an amino acid between amino acids 55 and 110 is substituted with a Cys.
  • an amino acid between amino acids 70 and 85 is substituted with a Cys.
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEHVIIQAEFYLNPDQSGEFMFDFDGCEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAV DKANLEIMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTT GVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPET (SEQ ID NO:10), where D29 is substituted with a Cys (shown in bold text).
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAV DKANLECMTKRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTT GVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPET (SEQ ID NO:11), where 172 is substituted with a Cys (shown in bold text).
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAV DKANLEIMTCRSNYTPITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTT GVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPET (SEQ ID NO:12), where K75 is substituted with a Cys (shown in bold text).
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAV DKANLEIMTKRSNYTCITNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTT GVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPET (SEQ ID NO:13), where P81 is substituted with a Cys (shown in bold text).
  • a suitable DRA polypeptide comprises the following amino acid sequence: IKEEHVIIQAEFYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAV DKANLEIMTKRSNYTPCTNVPPEVTVLTNSPVELREPNVLICFIDKFTPPVVNVTWLRNGKPVTT GVSETVFLPREDHLFRKFHYLPFLPSTEDVYDCRVEHWGLDEPLLKHWEFDAPSPLPET (SEQ ID NO:14), where 182 is substituted with a Cys (shown in bold text).
  • a suitable DRA ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITN (SEQ ID NO:15); and can have a length of about 84 amino acids (e.g., 80, 81, 82, 83, 84, 85, or 86 amino acids).
  • a suitable DRA ⁇ 1 domain can comprise the following amino acid sequence: VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITN (SEQ ID NO:15), or a naturally-occurring allelic variant.
  • a suitable DRA ⁇ 1 domain can comprise the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGCEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPITN (SEQ ID NO:16), where amino acid D29C is substituted with a Cys (shown in bold text).
  • a suitable DRA ⁇ 1 domain can comprise the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLECMTK RSNYTPITN (SEQ ID NO:17), where amino acid 172 is substituted with a Cys (shown in bold text).
  • a suitable DRA ⁇ 1 domain can comprise the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTC RSNYTPITN (SEQ ID NO:18), where amino acid K75 is substituted with a Cys (shown in bold text).
  • a suitable DRA ⁇ 1 domain can comprise the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTCITN (SEQ ID NO:19), where amino acid P81 is substituted with a Cys (shown in bold text).
  • a suitable DRA ⁇ 1 domain can comprise the following amino acid sequence: IKEEH VIIQAEFYLN PDQSGEFMFD FDGDEIFHVD MAKKETVWRL EEFGRFASFE AQGALANIAV DKANLEIMTK RSNYTPCTN (SEQ ID NO:20), where amino acid 182 is substituted with a Cys (shown in bold text).
  • a suitable DRA ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: V PPEVTVLTNSPVELREPNVL ICFIDKFTPP VVNVTWLRNG KPVTTGVSET VFLPREDHLF RKFHYLPFLPSTEDVYDCRV EHWGLDEPLL KHW (SEQ ID NO:21); and can have a length of about 94 amino acids (e.g., 90, 91, 92, 93, 94, 95, 96, 97, or 98 amino acids).
  • a suitable MHC class II ⁇ chain polypeptide is a DPA1 polypeptide.
  • a DPA1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 29-209 of the DPA1 amino acid sequence depicted in FIG. 8 .
  • the DPA1 polypeptide has a length of about 181 amino acids (e.g., 178, 179, 180, 181, 182, 183, or 184 amino acids).
  • a DPA polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DPA polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a “DPA1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DPA1 polypeptide comprises the following amino acid sequence: AG AIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANIAILNNN LNTLIQRSNH TQATNDPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO:22; amino acids 29-209 of DPA1*01:03:01:01, see FIG.
  • a DPA polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DPA polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a suitable DPA1 ⁇ 1 domain may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: AIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANIAILNNN LNTLIQRSNH TQATN (SEQ ID NO:23); and can have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids).
  • a suitable DPA1 ⁇ 1 domain can comprise the following amino acid sequence: AIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANIAILNNN LNTLIQRSNH TQATN (SEQ ID NO:23), or a naturally-occurring allelic variant.
  • a suitable DPA1 ⁇ 2 domain may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO:24); and can have a length of about 97 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).
  • a suitable DPA1 ⁇ 2 domain can comprise the following amino acid sequence: DPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO:24), or a naturally-occurring allelic variant thereof.
  • DPA1 polypeptides comprise the sequence: MRPEDRMFHIRAVILRALSLAFLLSLRGAGAIKADHVSTYAAFVQTHRPTGEFMFEFDEDEQFY VDLDKKETVWHLEEFGRAFSFEAQGGLANIAILNNNLNTLIQRSNHTQAANDPPEVTVFPKEPV ELGQPNTLICHIDRFFPPVLNVTWLCNGEPVTEGVAESLFLPRTDYSFHKFHYLTFVPSAEDVYD CRVEHWGLDQPLLKHWEAQEPIQMPETTETVLCALGLVLGLVGIIVGTVLIIKSLRSGHDPRAQ GPL (SEQ ID NO:25; amino acids 29-209 of DPA1*02:01:01:01, see FIG. 8 ), or variant thereof having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity.
  • a suitable DPA1 ⁇ 1 domain may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acids 29-115 of DPA1*02:01:01:01, SEQ ID NO:25; and can have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids.
  • a suitable DPA1 ⁇ 2 domain may comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 116 to 209 of DPA1*02:01:01:01, SEQ ID NO:25; and can have a length of about 97 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).
  • a suitable MHC class II ⁇ chain polypeptide is a DQA1 polypeptide.
  • a DQA1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 24-204 of any of the DQA1 amino acid sequences depicted in FIG. 10 .
  • the DQA1 polypeptide has a length of about 181 amino acids (e.g., 177, 178, 179, 180, 181, 182, or 183 amino acids).
  • a DQA1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQA1*01:01 ⁇ chain amino acid in FIG. 10 , ImMunoGeneTics (“IMGT”)/HLA Acc No:HLA00601.
  • IMGT ImMunoGeneTics
  • a DQA1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQA1*03:01: ⁇ chain amino acid in FIG.
  • a DQA1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQA1*04:01 ⁇ chain amino acid in FIG. 10 , IMGT/HLA Acc No:HLA00612.
  • a DQA1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQA1*05:01 ⁇ chain amino acid in FIG. 10 , IMGT/HLA Acc No:HLA00613.
  • a DQA polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DQA polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a “DQA1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DQA1 polypeptide comprises the following amino acid sequence: EDIVADH VASCGVNLYQ FYGPSGQYTH EFDGDEQFYV DLERKETAWR WPEFSKFGGF DPQGALRNMA VAKHNLNIMI KRYNSTAATN EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO:26), or an allelic variant thereof.
  • a DQA polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DQA polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a suitable DQA1 ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: EDIVADH VASCGVNLYQ FYGPSGQYTH EFDGDEQFYV DLERKETAWR WPEFSKFGGF DPQGALRNMA VAKHNLNIMI KRYNSTAATN (SEQ ID NO:27); and can have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids).
  • a suitable DQA1 ⁇ 1 domain can comprise the following amino acid sequence: EDIVADH VASCGVNLYQ FYGPSGQYTH EFDGDEQFYV DLERKETAWR WPEFSKFGGF DPQGALRNMA VAKHNLNIMI KRYNSTAATN (SEQ ID NO:28), or a naturally-occurring allelic variant.
  • a suitable DQA1 ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO:29); and can have a length of about 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).
  • a suitable DQA1 ⁇ 2 domain can comprise the following amino acid sequence: EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO:29), or a naturally-occurring allelic variant thereof.
  • MHC class II beta chains comprise a ⁇ 1 domain and a ⁇ 2 domain.
  • the ⁇ 1 domain and the ⁇ 2 domain present in an antigen-presenting cell are from the same MHC class II ⁇ chain polypeptide.
  • the ⁇ 1 domain and the ⁇ 2 domain present in an antigen-presenting cell are from two different MHC class II ⁇ chain polypeptides.
  • MHC class II ⁇ chain polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type MHC class II ⁇ chain polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • MHC class II beta chains suitable for inclusion in a TMMP of the present disclosure lack a signal peptide.
  • An MHC class II beta chain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 60 amino acids to about 210 amino acids; for example, an MHC class II beta chain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 60 amino acids to about 80 amino acids, from about 80 amino acids to about 100 amino acids, from about 100 amino acids to about 120 amino acids, from about 120 amino acids to about 140 amino acids, from about 140 amino acids to about 160 amino acids, from about 160 amino acids to about 180 amino acids, from about 180 amino acids to about 200 amino acids, or from about 200 amino acids to about 210 amino acids.
  • An MHC class II 31 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 105 amino acids; for example, an MHC class II ⁇ 1 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 60 amino acids, from about 60 amino acids to about 70 amino acids, from about 70 amino acids to about 80 amino acids, from about 80 amino acids to about 90 amino acids, from about 90 amino acids to about 95 amino acids, from about 95 amino acids to about 100 amino acids, or from about 100 amino acids to about 105 amino acids.
  • An MHC class II ⁇ 2 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 105 amino acids; for example, an MHC class II ⁇ 2 domain suitable for inclusion in a TMMP of the present disclosure can have a length of from about 30 amino acids to about 40 amino acids, from about 40 amino acids to about 50 amino acids, from about 50 amino acids to about 60 amino acids, from about 60 amino acids to about 70 amino acids, from about 70 amino acids to about 80 amino acids, from about 80 amino acids to about 90 amino acids, from about 90 amino acids to about 95 amino acids, from about 95 amino acids to about 100 amino acids, or from about 100 amino acids to about 105 amino acids.
  • the MHC class II ⁇ chain polypeptide is a variant DRB MHC class II polypeptide that comprises a non-naturally occurring Cys residue.
  • the MHC class II ⁇ chain polypeptide is a variant DRB1 MHC class II polypeptide that comprises an amino acid substitution selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C.
  • the MHC class II ⁇ chain polypeptide is a variant DRB1 polypeptide comprising an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the following DRB1 amino acid sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWR ARSESAQSKM (SEQ ID NO:30), and comprising an amino acid substitution selected from the group consisting of P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C
  • the MHC class II ⁇ chain polypeptide is a variant DRB3 polypeptide, a variant DRB4 polypeptide, or a variant DRB5 polypeptide comprising an amino acid substitution corresponding to any of the foregoing amino acid substitutions in a variant DRB1 polypeptide.
  • a variant DRB3 polypeptide a variant DRB4 polypeptide
  • a variant DRB5 polypeptide comprising an amino acid substitution corresponding to any of the foregoing amino acid substitutions in a variant DRB1 polypeptide.
  • an amino acid corresponding to P5 of DRB1 would be P5 of a mature DRB3 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSSLAALTVTLMVLSSRLAFA (SEQ ID NO:31)); ii) an amino acid corresponding to F7 of DRB1 would be F7 of a mature DRB3 polypeptide; iii) an amino acid corresponding to Q10 of DRB1 would be L10 of a mature DRB3 polypeptide; iv) an amino acid corresponding to N19 of DRB1 would be N19 of a mature DRB3 polypeptide; v) an amino acid corresponding to G20 of DRB1 would be G20 of a mature DRB3 polypeptide; vi) an amino acid corresponding to H33 of DRB1 would be N33 of a mature DRB3 polypeptide; vii) an amino acid corresponding to G151 of DRB1 would be G151 of a mature DRB3 polypeptide; viii
  • an amino acid corresponding to P5 of DRB1 would be P15 of a mature DRB4 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSCMAALTVTL (SEQ ID NO:32)); ii) an amino acid corresponding to F7 of DRB1 would be F17 of a mature DRB4 polypeptide; iii) an amino acid corresponding to Q10 of DRB1 would be Q20 of a mature DRB4 polypeptide; iv) an amino acid corresponding to N19 of DRB1 would be N29 of a mature DRB4 polypeptide; v) an amino acid corresponding to G20 of DRB1 would be G30 of a mature DRB4 polypeptide; vi) an amino acid corresponding to H33 of DRB1 would be N43 of a mature DRB4 polypeptide; vii) an amino acid corresponding to G151 of DRB1 would be G161 of a mature DRB4
  • an amino acid corresponding to P5 of DRB1 would be P15 of a mature DRB5 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSYMAKLTVTL (SEQ ID NO:33)); ii) an amino acid corresponding to F7 of DRB1 would be F17 of a mature DRB5 polypeptide; iii) an amino acid corresponding to Q10 of DRB1 would be Q20 of a mature DRB5 polypeptide; iv) an amino acid corresponding to N19 of DRB1 would be N29 of a mature DRB5 polypeptide; v) an amino acid corresponding to G20 of DRB1 would be G30 of a mature DRB5 polypeptide; vi) an amino acid corresponding to H33 of DRB1 would be N43 of a mature DRB5 polypeptide; vii) an amino acid corresponding to G151 of DRB1 would be G161 of a mature D
  • a suitable MHC class II ⁇ chain polypeptide is a DRB1 polypeptide.
  • a DRB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of any DRB1 amino acid sequence depicted in FIG. 7 , which displays the DRB1 precursor proteins in which amino acids 1-29 are the signal sequence, 30-124 form the ⁇ 1 region, 125-227 form the ⁇ 2 region, and 228-250 form the transmembrane region.
  • a DRB1 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRB1 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a DRB1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-3 (DRB1*03:01) beta chain amino acid sequence sp P01912.2 in FIG. 7 .
  • a DRB1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-4 (DRB1*04:01) beta chain amino acid sequence sp P13760.1 in FIG. 7 .
  • a DRB1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-8 (DRB1*08:01) beta chain amino acid sequence sp Q30134.2 in FIG. 7 .
  • a DRB1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-9 (DRB1*09:01) beta chain amino acid sequence sp Q9TQE0.1 in FIG. 7 .
  • a DRB1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-16 (DRB1*16:01) beta chain amino acid sequence sp Q29974.1 (also HLA00876) in FIG. 7 .
  • the DRB1 ⁇ chain polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).
  • a “DRB1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DRB1 polypeptide comprises the following amino acid sequence: DTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQ KDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSI EVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRA RSESAQSK (SEQ ID NO:34) (amino acids 31-227 of DRB1-4, see FIG. 7 ), or an allelic variant thereof.
  • a suitable DRB1 polypeptide can comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWR ARSESAQSKM (SEQ ID NO:30); and can have a length of from 195 to 205 amino acids, e.g., 199 amino acids.
  • a suitable DRB1 polypeptide can comprise the following amino acid sequence: GDTRCRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWR ARSESAQSKM (SEQ ID NO:35), where P5 is substituted with a Cys (shown in bold text).
  • a suitable DRB1 polypeptide can comprise the following amino acid sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNCDWTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWR ARSESAQSKM (SEQ ID NO:36), where G151 is substituted with a Cys (shown in bold text).
  • a suitable DRB1 polypeptide can comprise the following amino acid sequence: GDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLVCSVNGFYPA SIEVRWFRNGQEEKTGVVSTGLIQNGDCTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWR ARSESAQSKM (SEQ ID NO:37), where W153 is substituted with a Cys (shown in bold text).
  • a suitable DRB1 ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQ KDLLEQKRAAVDTYCRHNYGVGESFTVQRRV (SEQ ID NO:38); and can have a length of about 95 amino acids (e.g., 92, 93, 94, 95, 96, 97, or 98 amino acids).
  • a suitable DRB1 ⁇ 1 domain can comprise the following amino acid sequence: DTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQ KDLLEQKRAAVDTYCRHNYGVGESFTVQRRV (SEQ ID NO:38), or a naturally-occurring allelic variant.
  • a suitable DRB1 ⁇ 1 domain can comprise the following amino acid sequence: GDTRCRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNS QKDLLEQKRAAVDTYCRHNYGVGESFTVQRRV (SEQ ID NO:39), where P5 is substituted with a Cys (shown in bold text).
  • a suitable DRB1 ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTL VMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSK (SEQ ID NO:40); and can have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, 105, or 106 amino acids).
  • a suitable DRB1 ⁇ 2 domain can comprise the following amino acid sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTL VMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSK (SEQ ID NO:40), or a naturally-occurring allelic variant thereof.
  • a suitable DRB1 ⁇ 2 domain can comprise the following amino acid sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPASIEVRWFRNGQEEKTGVVSTGLIQNCDWTFQTL VMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSKM (SEQ ID NO:41), where G151 is substituted with a Cys (shown in bold text).
  • a suitable DRB1 ⁇ 2 domain can comprise the following amino acid sequence: YPEVTVYPAKTQPLQHHNLLVCSVNGFYPASIEVRWFRNGQEEKTGVVSTGLIQNGDCTFQTLV MLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSKM (SEQ ID NO:42), where W153 is substituted with a Cys (shown in bold text).
  • a suitable MHC class II ⁇ chain polypeptide is a DRB3 polypeptide.
  • a DRB3 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of any DRB3 amino acid sequence depicted in FIG. 7 B , which displays the DRB3 precursor proteins in which amino acids 1-29 are the signal sequence (underlined), 30-124 form the ⁇ 1 region (shown bolded), 125-227 for the ⁇ 2 region, and 228-250 the transmembrane region.
  • a DRB3 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-3 (DRB3*01:01) beta chain amino acid sequence GenBank NP_072049.1 in FIG. 7 B .
  • a DRB3 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-3 beta chain amino acid sequence in GenBank accession EAX03632.1 in FIG. 7 B .
  • a DRB3 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-3 (DRB3*02:01) beta chain amino acid sequence GenBank CAA23781.1 in FIG. 7 B .
  • a DRB3 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB1-3 (DRB3*03:01) beta chain amino acid sequence GenBank AAN15205.1 in FIG. 7 B .
  • a DRB3 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRB3 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a “DRB3 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DRB3 polypeptide comprises the following amino acid sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRVHPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO:43), or an allelic variant thereof.
  • a DRB3 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRB3 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • the MHC class II ⁇ chain polypeptide is a variant DRB3 MHC class II polypeptide that comprises a non-naturally occurring Cys residue; e.g., where the variant DRB3 MHC class II polypeptide comprises an amino acid substitution selected from the group consisting of P5C, F7C, L10C, N19C, G20C, N33C, G151C, D152C, and W153C (of a mature DRB3 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSSLAALTVTLMVLSSRLAFA (SEQ ID NO:31) depicted in FIG. 7 B ).
  • a suitable DRB3 ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRV (SEQ ID NO:44); and can have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids).
  • a suitable DRB3 ⁇ 1 domain can comprise the following amino acid sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRV (SEQ ID NO:44), or a naturally-occurring allelic variant.
  • a suitable DRB3 ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: HPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO:45); and can have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, or 105 amino acids).
  • a suitable DRB3 ⁇ 2 domain can comprise the following amino acid sequence: HPQVTV YPAKTQPLQH HNLLVCSVSG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SALTVEWRAR SESAQSK (SEQ ID NO:45), or a naturally-occurring allelic variant thereof.
  • a suitable MHC class II ⁇ chain polypeptide is a DRB4 polypeptide.
  • a DRB4 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB4 amino acid sequence depicted in FIG. 7 C .
  • the DRB4 polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).
  • a DRB4 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a “DRB4 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DRB4 polypeptide comprises the following amino acid sequence: T VLSSPLALAG DTQPRFLEQA KCECHFLNGT ERVWNLIRYI YNQEEYARYN SDLGEYQAVT ELGRPDAEYW NSQKDLLERR RAEVDTYCRY NYGVVESFTV QRRVQPKVTV YPSKTQPLQH HNLLVCSVNG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSMM SPLTVQWSAR SESAQSK (SEQ ID NO:46), or an allelic variant thereof.
  • a DRB4 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRB4 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • the MHC class II ⁇ chain polypeptide is a variant DRB4 MHC class II polypeptide that comprises a non-naturally occurring Cys residue; e.g., where the variant DRB4 MHC class II polypeptide comprises an amino acid substitution selected from the group consisting of P15C, F17C, Q20C, N29C, G30C, N43C, G161C, D162C, and W163C (of a mature DRB4 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSCMAALTVTL (SEQ ID NO:32) depicted in FIG. 7 C ).
  • a suitable DRB4 ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: T VLSSPLALAG DTQPRFLEQA KCECHFLNGT ERVWNLIRYI YNQEEYARYN SDLGEYQAVT ELGRPDAEYW NSQKDLLERR RAEVDTYCRY NYGVVESFTV QRRV (SEQ ID NO:47); and can have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids).
  • a suitable DRB4 ⁇ 1 domain can comprise the following amino acid sequence: T VLSSPLALAG DTQPRFLEQA KCECHFLNGT ERVWNLIRYI YNQEEYARYN SDLGEYQAVT ELGRPDAEYW NSQKDLLERR RAEVDTYCRY NYGVVESFTV QRRV (SEQ ID NO:47), or a naturally-occurring allelic variant.
  • a suitable DRB4 ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QPKVTV YPSKTQPLQH HNLLVCSVNG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSMM SPLTVQWSAR SESAQSK (SEQ ID NO:48); and can have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, or 105 amino acids).
  • a suitable DRB4 ⁇ 2 domain can comprise the following amino acid sequence: QPKVTV YPSKTQPLQH HNLLVCSVNG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSMM SPLTVQWSAR SESAQSK (SEQ ID NO:48), or a naturally-occurring allelic variant thereof.
  • a suitable MHC class II ⁇ chain polypeptide is a DRB5 polypeptide.
  • a DRB5 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DRB5 amino acid sequence depicted in FIG. 7 D .
  • the DRB5 polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).
  • a DRB5 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRB5 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a “DRB5 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DRB5 polypeptide comprises the following amino acid sequence: M VLSSPLALAG DTRPRFLQQD KYECHFFNGT ERVRFLHRDI YNQEEDLRFD SDVGEYRAVT ELGRPDAEYW NSQKDFLEDR RAAVDTYCRH NYGVGESFTV QRRVEPKVTV YPARTQTLQH HNLLVCSVNG FYPGSIEVRW FRNSQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SPLTVEWRAQ SESAQS (SEQ ID NO:49), or an allelic variant thereof.
  • a DRB5 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DRB5 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • the MHC class II ⁇ chain polypeptide is a variant DRB5 MHC class II polypeptide that comprises a non-naturally occurring Cys residue; e.g., where the variant DRB5 MHC class II polypeptide comprises an amino acid substitution selected from the group consisting of P15C, F17C, Q20C, N29C, G30C, N43C, G161C, D162C, and W163C (of a mature DRB5 polypeptide (lacking the N-terminal signal peptide MVCLKLPGGSYMAKLTVTL (SEQ ID NO:33) depicted in FIG. 7 D ).
  • a suitable DRB5 ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: M VLSSPLALAG DTRPRFLQQD KYECHFFNGT ERVRFLHRDI YNQEEDLRFD SDVGEYRAVT ELGRPDAEYW NSQKDFLEDR RAAVDTYCRH NYGVGESFTV QRRV (SEQ ID NO:50); and can have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids).
  • a suitable DRB5 ⁇ 1 domain can comprise the following amino acid sequence: M VLSSPLALAG DTRPRFLQQD KYECHFFNGT ERVRFLHRDI YNQEEDLRFD SDVGEYRAVT ELGRPDAEYW NSQKDFLEDR RAAVDTYCRH NYGVGESFTV QRRV (SEQ ID NO:50), or a naturally-occurring allelic variant.
  • a suitable DRB5 ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: EPKVTV YPARTQTLQH HNLLVCSVNG FYPGSIEVRW FRNSQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SPLTVEWRAQ SESAQS (SEQ ID NO:51); and can have a length of about 103 amino acids (e.g., 100, 101, 102, 103, 104, or 105 amino acids).
  • a suitable DRB5 ⁇ 2 domain can comprise the following amino acid sequence: EPKVTV YPARTQTLQH HNLLVCSVNG FYPGSIEVRW FRNSQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSVT SPLTVEWRAQ SESAQS (SEQ ID NO:51), or a naturally-occurring allelic variant thereof.
  • a suitable MHC class II ⁇ chain polypeptide is a DPB1 polypeptide.
  • a DPB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-215 of any of the DPB1 amino acid sequences depicted in FIG. 9 .
  • the DPB1 polypeptide has a length of about 186 amino acids (e.g., 184, 185, 186, 187, or 188 amino acids).
  • a DRB1 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DPB1*02:02 ⁇ chain amino acid sequence in FIG. 9 , IMGT/HLA Acc No. HLA00519.
  • a DPB1 DRB3 ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DPB1*03:01 ⁇ chain amino acid sequence in FIG. 9 , IMGT/HLA Acc No.:HLA00520.
  • a “DPB1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DPB1 polypeptide comprises the following amino acid sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR RVQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL NGQEETAGVV STNLIRNGDW TFQILVMLEM TPQQGDVYTC QVEHTSLDSP VTVEW (SEQ ID NO:52), or an allelic variant thereof.
  • a DPB1 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DPB1 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a suitable DPB1 ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR R (SEQ ID NO:53); and can have a length of about 92 amino acids (e.g., 90, 91, 92, 93, or 94 amino acids).
  • a suitable DPB1 ⁇ 1 domain can comprise the following amino acid sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR R (SEQ ID NO:53), or a naturally-occurring allelic variant.
  • a suitable DPB1 ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: VQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL NGQEETAGVV STNLIRNGDW TFQILVMLEM TPQQGDVYTC QVEHTSLDSP VTVEW (SEQ ID NO:54); and can have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).
  • a suitable DPB1 ⁇ 2 domain can comprise the following amino acid sequence: VQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL NGQEETAGVV STNLIRNGDW TFQILVMLEM TPQQGDVYTC QVEHTSLDSP VTVEW (SEQ ID NO:54), or a naturally-occurring allelic variant thereof.
  • a suitable MHC class II ⁇ chain polypeptide is a DQB1 polypeptide.
  • a DQB1 polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 33-220 of any one of the DQB1 amino acid sequences depicted in FIG. 11 A- 11 B .
  • the DQB1 polypeptide has a length of about 188 amino acids (e.g., 186, 187, 188, 190, 191, or 192 amino acids).
  • a DQB1 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DQB1 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a “DQB1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DQB1 polypeptide comprises the following amino acid sequence: RDSPEDFV FQFKGMCYFT NGTERVRLVT RYIYNREEYA RFDSDVGVYR AVTPQGRPDA EYWNSQKEVL EGTRAELDTV CRHNYEVAFR GILQRRVEPT VTISPSRTEA LNHHNLLVCS VTDFYPGQIK VRWFRNDQEE TAGVVSTPLI RNGDWTFQIL VMLEMTPQRG DVYTCHVEHP SLQSPITVEW (SEQ ID NO:55), or an allelic variant thereof.
  • a DQB1 polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid substitution, relative to a wild-type DQB1 polypeptide, where the amino acid substitution replaces an amino acid (other than a Cys) with a Cys.
  • a suitable DQB1 ⁇ 1 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: RDSPEDFV FQFKGMCYFT NGTERVRLVT RYIYNREEYA RFDSDVGVYR AVTPQGRPDA EYWNSQKEVL EGTRAELDTV CRHNYEVAFR GILQRR (SEQ ID NO:56); and can have a length of about 94 amino acids (e.g., 92, 93, 94, 95, or 96 amino acids).
  • a suitable DQB1 ⁇ 1 domain can comprise the following amino acid sequence: RDSPEDFV FQFKGMCYFT NGTERVRLVT RYIYNREEYA RFDSDVGVYR AVTPQGRPDA EYWNSQKEVL EGTRAELDTV CRHNYEVAFR GILQRR (SEQ ID NO:56), or a naturally-occurring allelic variant.
  • a suitable DQB1 ⁇ 2 domain comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: VEPT VTISPSRTEA LNHHNLLVCS VTDFYPGQIK VRWFRNDQEE TAGVVSTPLI RNGDWTFQIL VMLEMTPQRG DVYTCHVEHP SLQSPITVEW (SEQ ID NO:57); and can have a length of about 94 amino acids (e.g., 92, 93, 94, 95, or 96 amino acids).
  • a suitable DQB1 ⁇ 2 domain can comprise the following amino acid sequence: VEPT VTISPSRTEA LNHHNLLVCS VTDFYPGQIK VRWFRNDQEE TAGVVSTPLI RNGDWTFQIL VMLEMTPQRG DVYTCHVEHP SLQSPITVEW (SEQ ID NO:57), or a naturally-occurring allelic variant thereof.
  • a DQB ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQB1*02:01 beta chain amino acid sequence in FIG. 11 A- 11 B , IMGT/HLA Acc No: HLA00622.
  • a DQB ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQB1*03:02 beta chain amino acid sequence in FIG. 11 A- 11 B , IMGT/HLA Acc No: HLA00627.
  • a DQB ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQB1*03:03 beta chain amino acid sequence in FIG. 11 A- 11 B , IMGT/HLA Acc No: HLA00629.
  • a DQB ⁇ chain polypeptide can have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-227 of the DQB1*05:01 beta chain amino acid sequence in FIG. 11 A- 11 B , IMGT/HLA Acc No: HLA00638.
  • T1D Type 1 Diabetes Mellitus
  • Alleles/isoforms showing increased association with T1D represent suitable sources of MHC II ⁇ 1, ⁇ 2, ⁇ 1, and ⁇ 2 polypeptide sequences for incorporation into TMMPs directed to the treatment of T1D.
  • T1D is associated with alleles belonging to the HLA-DR3 and HLA-DR4 haplotypes/serotypes, with the strongest risk associated with the HLA-DQ8, (e.g., HLA-DQB1*03:02) and alleles of the HLA-DQ2 serotype.
  • HLA-DQ8 e.g., HLA-DQB1*03:02
  • Some high and moderate risk haplotypes and their association with various DR serotypes are shown in Table 1, below, which is adapted from Kantirova and Buc, Physiol. Res. 56: 255-266 (2007).
  • the stereotypically defined DR3 and DR4 protein isoforms/haplotypes of the DRB1 gene are associated with increased risk that an individual expressing such alleles will develop T1D.
  • the DR3 serotype includes the alleles encoding the DRB1*03:01, *03:02, *03:03, and *03:04 proteins, with the HLA-DRB1*0301 allele often found associated with a predisposition to T1D.
  • the DR4 serotype includes the alleles encoding the DRB1*04:01, *04:02, *04:03, *04:04, *04:05, *04:06, *04:07, *04:08, *04:09, *04:10, *04:11, *04:12, and *04:13 proteins.
  • Certain HLA-DR4 alleles e.g., HLA-DRB1*0401, HLA-DRB1*0402, and HLA-DRB1*0405
  • predispose individuals to T1D predispose individuals to T1D
  • HLA-DRB1*04:03 allele/isoform may afford protection.
  • DRB1* 16:01 also show an increased frequency in diabetic children relative to healthy controls (Deja, et al., Mediators of Inflammation 2006:1-7 (2006)).
  • HLA-DQ8 serotype e.g., the HLA-DQB1*03:02 isoform
  • HLA DQ2 serotype e.g., HLA-DQB1*0201
  • HLA-DQ8 T1D susceptible serotypes include the HLA-DQ8.1 serotype (HLA-DQA1*03:01/DQB1* 03:02)
  • the HLA-DQ2 serotype (HLA-DQB1*02) associated with T1D include DQB1*02:01). Jones, et al., Nat.
  • the DQB1 locus alone has also been reported to be associated with T1D when position ⁇ 57 is a neutral residue such as Ala or Ser.
  • Both the DQ2 and DQ8 serotypes, which are associated with T1D lack an Asp at the 570 position, and instead have an Ala in its place (see e.g., Ala 89 in FIG. 19 B HLA-DQB1*02:01 and FIG. 19 C , HLA-DQB1*03:02 respectively) conferred T1D susceptibility.
  • DQB1*06:02 which has an Asp) at position 057 of DQB1 (position 89 in FIG. 11 A- 11 B ) was found to be associated with resistance to T1D.
  • HLA-DR4.1 HLA-DRA1*01:01/DRB1*04:01
  • HLA-DR4.5 HLA-DRA1*01:01/DRB1*04:05
  • HLA-DQ2.5 HLA-DQA1*05:01/DQB1*02:01
  • HLA-DQ8.1 HLA-DQA1*03:01/DQB1*03:02
  • the DR ⁇ 1*04:05-DQ ⁇ 1*04:01/DR ⁇ 1*08:02-DQ ⁇ 1*03:02 genotype has shown to be associated with acute-onset and slow progressive T1D. Fulminant diabetes has been associated with DR ⁇ 1*04:05-DQ ⁇ 1*04:01/DR ⁇ 1*04:05-DQ ⁇ 1*04:01 genotype, in a Japanese population study Kawabata, et al., Diabetologia 2009, 52:2513-21.
  • HLA-DR associations with T1D are not as strong as those of HLA-DQ, insulin-reactive T cells derived from lymph nodes draining the pancreas of patients with T1D appear to be HLA-DR4.1 restricted rather than HLA-DQ8 or HLA-DQ2 restricted (Kent et al., Nature 2005 435: 224-228).
  • the above-mentioned alleles associated with an increased risk of T1D represent suitable candidates from which the ⁇ 1, ⁇ 2, ⁇ 1, and/or ⁇ 2 polypeptide sequences present in a TMMP of the present disclosure may be taken.
  • the TMMP is DQ2.5-like with the ⁇ 1 and ⁇ 2 polypeptides from DQA1*0501, and the ⁇ 1 and ⁇ 2 polypeptides taken from DQB1*0201.
  • the TMMP is DQ8.1-like with the ⁇ 1 and ⁇ 2 polypeptides from DQA1*0301, and the ⁇ 1 and ⁇ 2 polypeptides taken from DQB1*0302.
  • a TMMP of the present disclosure can comprise an immunoglobulin or non-immunoglobulin scaffold.
  • a TMMP polypeptide of the present disclosure can comprise an Fc polypeptide, or can comprise another suitable scaffold polypeptide.
  • Suitable scaffold polypeptides include antibody-based scaffold polypeptides and non-antibody-based scaffolds.
  • Non-antibody-based scaffolds include, e.g., albumin, an XTEN (extended recombinant) polypeptide, transferrin, an Fc receptor polypeptide, an elastin-like polypeptide (see, e.g., Hassouneh et al. (2012) Methods Enzymol.
  • SELP silk-elastin-like polypeptide
  • Suitable XTEN polypeptides include, e.g., those disclosed in WO 2009/023270, WO 2010/091122, WO 2007/103515, US 2010/0189682, and US 2009/0092582; see also Schellenberger et al. (2009) Nat Biotechnol. 27:1186).
  • Suitable albumin polypeptides include, e.g., human serum albumin.
  • Suitable scaffold polypeptides will in some cases be half-life extending polypeptides.
  • a suitable scaffold polypeptide increases the in vivo half-life (e.g., the serum half-life) of the multimeric polypeptide, compared to a control multimeric polypeptide lacking the scaffold polypeptide.
  • a scaffold polypeptide increases the in vivo half-life (e.g., the serum half-life) of the multimeric polypeptide, compared to a control multimeric polypeptide lacking the scaffold polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold.
  • the in vivo half-life e.g., the serum half-life
  • an Fc polypeptide increases the in vivo half-life (e.g., the serum half-life) of the multimeric polypeptide, compared to a control multimeric polypeptide lacking the Fc polypeptide, by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or more than 100-fold.
  • the in vivo half-life e.g., the serum half-life
  • the first or the second polypeptide chain of a TMMP of the present disclosure comprises an Fc polypeptide.
  • the Fc polypeptide of a TMMP of the present disclosure can be a human IgG1 Fc, a human IgG2 Fc, a human IgG3 Fc, a human IgG4 Fc, etc.
  • the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence of an Fc region depicted in FIG. 12 A- 12 G .
  • the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgG1 Fc polypeptide depicted in FIG. 12 A .
  • the Fc region comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to the human IgG1 Fc polypeptide depicted in FIG. 12 A , and comprises a substitution of N77; e.g., the Fc polypeptide comprises an N77A substitution.
  • the Ig Fc polypeptide induces cell lysis through activation of complement-dependent cytotoxicity (CDC).
  • the Ig Fc polypeptide is a variant that substantially does not induce cell lysis through activation of CDC, e.g., an IgG1 Fc polypeptide comprising L234A and L235A substitutions (L14A and L15A substitution of the amino acid sequence depicted in FIG. 12 A ).
  • the TMMPs of this disclosure that employ an Ig Fc polypeptide are meant to engage a target T cell through the MHC-epitope complex and then modulate the activity of a T cell through the immunomodulatory polypeptides, and thus will employ a variant that substantially does not induce cell lysis through activation of CDC.
  • the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgG2 Fc polypeptide depicted in FIG. 12 A ; e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 99-325 of the human IgG2 Fc polypeptide depicted in FIG. 12 A .
  • the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgG3 Fc polypeptide depicted in FIG. 12 A ; e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 19-246 of the human IgG3 Fc polypeptide depicted in FIG. 12 A .
  • the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgM Fc polypeptide depicted in FIG. 12 B ; e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 1-276 to the human IgM Fc polypeptide depicted in FIG. 2 B .
  • the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgA Fc polypeptide depicted in FIG. 12 C ; e.g., the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 1-234 to the human IgA Fc polypeptide depicted in FIG. 12 C .
  • the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the human IgG4 Fc polypeptide depicted in FIG. 12 C .
  • the Fc polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to amino acids 100 to 327 of the human IgG4 Fc polypeptide depicted in FIG. 12 C .
  • the IgG4 Fc polypeptide comprises the following amino acid sequence: PPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:58).
  • the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 A (human IgG1 Fc). In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 A (human IgG1 Fc), except for a substitution of N297 (N77 of the amino acid sequence depicted in FIG. 12 A ) with an amino acid other than asparagine. In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 C (human IgG1 Fc comprising an N297A substitution, which is N77 of the amino acid sequence depicted in FIG. 12 A ).
  • the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 A (human IgG1 Fc), except for a substitution of L234 (L14 of the amino acid sequence depicted in FIG. 12 A ) with an amino acid other than leucine.
  • the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 A (human IgG1 Fc), except for a substitution of L235 (L15 of the amino acid sequence depicted in FIG. 2 A ) with an amino acid other than leucine.
  • the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 E . In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 F . In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 G (human IgG1 Fc comprising an L234A substitution and an L235A substitution, corresponding to positions 14 and 15 of the amino acid sequence depicted in FIG. 12 G ). In some cases, the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG.
  • the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 A (human IgG1 Fc), except for substitutions at L234 and L235 (L14 and L15 of the amino acid sequence depicted in FIG. 12 A ) with amino acids other than leucine.
  • the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG.
  • the Fc polypeptide present in a TMMP comprises the amino acid sequence depicted in FIG. 12 E (human IgG1 Fc comprising L234F, L235E, and P331S substitutions (corresponding to amino acid positions 14, 15, and 111 of the amino acid sequence depicted in FIG. 12 E ).
  • the Fc polypeptide present in a TMMP is an IgG1 Fc polypeptide that comprises L234A and L235A substitutions (substitutions of L14 and L15 of the amino acid sequence depicted in FIG. 12 A with Ala), as depicted in FIG. 12 G .
  • a TMMP of the present disclosure comprises an Ig Fc polypeptide comprising the following amino acid sequence: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSPG (SEQ ID NO:59), which is an IgG1 Fc polypeptide comprising L234A and L235A substitutions (L14A and L15A in this sequence; shown in bold text).
  • a TMMP of the present disclosure can include one or more linker peptides between components of the first and second polypeptides of the TMMP, e.g., between a T1D peptide and an MHC polypeptide; between an MHC polypeptide and an Ig Fc polypeptide; between a first MHC polypeptide and a second MHC polypeptide; between an immunomodulatory polypeptide and an MHC polypeptide; etc.
  • a TMMP of the present disclosure can include a Cys-containing peptide linker between the T1D peptide and an MHC class II polypeptide, e.g., between the T1D peptide and an MHC class II ⁇ chain polypeptide.
  • a Cys-containing peptide linker will be used in either the first or second polypeptide of a TMMP in order to intentionally facilitate formation of a disulfide bond between the linker and a desired site on the other polypeptide.
  • a Cys-containing linker is inserted in one polypeptide of a TMMP
  • the remaining linkers in the TMMP will not include a Cys in order to prevent formation of a disulfide bond at an unwanted site in the TMMP, with the exception that a Cys-containing linker could be used in each of the first and second polypeptides when it is desired to link the first and second polypeptides through a disulfide bond formed between the linkers.
  • a TMMP of the present disclosure thus can include: a) a Cys-containing peptide linker between the T1D peptide and an MHC class II polypeptide, e.g., between the T1D peptide and an MHC class II ⁇ chain polypeptide; and b) at least one additional peptide linker, where the at least one additional peptide linker does not include a Cys.
  • Suitable linkers can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid to 25 amino acids, from 3 amino acids to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids.
  • a suitable linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • a suitable linker can be from 25 to 35 amino acids in length.
  • a suitable linker can be 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids in length.
  • a suitable linker can be from 35 to 45 amino acids in length.
  • a suitable linker can be 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 amino acids in length.
  • a suitable linker can be from 45 to 50 amino acids in length.
  • a suitable linker can be 45, 46, 47, 48, 49, or 50 amino acids in length.
  • a peptide linker comprising a Cys can comprise an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • a TMMP of the present disclosure comprises a heterodimer comprising a first and a second polypeptide, where the first polypeptide comprises a T1D peptide and an MHC class II polypeptide (e.g., an MHC class II chain polypeptide), and comprises a linker between the T1D peptide and the MHC class II polypeptide (e.g., the MHC class II chain polypeptide), where the linker comprises an amino acid sequence selected from (CGGGS)(GGGGS)n (SEQ ID NO:1), (GCGGS)(GGGGS)n (SEQ ID NO:2), (GGCGS)(GGGGS)n (SEQ ID NO:3), (GGGCS)(GGGGS)n (SEQ ID NO:4), and (GGGGC)(GGGGS)n (SEQ ID NO:5), where n is an integer from 1 to 10 (e.g., where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).
  • Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGGS) n (SEQ ID NO:60) and (GGGS) n (SEQ ID NO:61), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components.
  • Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
  • Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO:62), GGSGG (SEQ ID NO:63), GSGSG (SEQ ID NO:64), GSGGG (SEQ ID NO:65), GGGSG (SEQ ID NO:66), GSSSG (SEQ ID NO:67), and the like.
  • Exemplary linkers can include, e.g., Gly(Ser 4 )n, (SEQ ID NO:68) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • a linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO:69), where n is 4.
  • a linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO:70), where n is 5.
  • Exemplary linkers can include, e.g., (GlyGlyGlyGlySer)n (SEQ ID NO:71) (also referred to as “G4S” linkers), where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:72), where n is 1. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:73), where n is 2. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:74), where n is 3. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:75), where n is 4. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:76), where n is 5. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:77), where n is 6.
  • a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:78), where n is 7. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:79), where n is 8. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:80), where n is 9. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:81), where n is 10. In some cases, a linker comprises the amino acid sequence AAAGG (SEQ ID NO:82). In some cases, a linker comprises the amino acid sequence GGSAAAGG (SEQ ID NO:83).
  • the AAAGG (SEQ ID NO:82) and GGSAAAGG (SEQ ID NO:83) linkers have been found to be useful for linking an MHC class II alpha chain polypeptide (e.g., a DRA class II polypeptide) to an Ig Fc polypeptide (e.g., a human IgG1 Fc polypeptide sequence depicted in FIG. 12 G comprising an L234A substitution and an L235A substitution, corresponding to positions 14 and 15 of the amino acid sequence depicted in FIG. 12 G ).
  • MHC class II alpha chain polypeptide e.g., a DRA class II polypeptide
  • Ig Fc polypeptide e.g., a human IgG1 Fc polypeptide sequence depicted in FIG. 12 G comprising an L234A substitution and an L235A substitution, corresponding to positions 14 and 15 of the amino acid sequence depicted in FIG. 12 G ).
  • a “T1D peptide” is a peptide that, when present in a TMMP of this disclosure, presents a T1D-associated epitope capable of being bound by a TCR on the surface of a T cell.
  • a T1D peptide can have a length of from about 4 amino acids to about 25 amino acids, e.g., the T1D peptide can have a length of from 4 amino acids (aa) to 10 aa, from 8 aa to 12 aa, from 10 aa to 15 aa, from 12 aa to 20 aa, from 15 aa to 20 aa, from 15 aa to 25 aa, or from 20 aa to 25 aa.
  • a T1D peptide present in a TMPP of the present disclosure can have a length of 4 amino acids (aa), 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, 20 aa, 21 aa, 22 aa, 23 aa, 24 aa, or 25 aa.
  • a T1D peptide present in a TMPP of the present disclosure has a length of from 5 amino acids to 10 amino acids, e.g., 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa.
  • Antigens associated with type 1 diabetes include, e.g., preproinsulin, proinsulin, insulin, insulin B chain, insulin A chain, 65 kDa isoform of glutamic acid decarboxylase (GAD65), 67 kDa isoform of glutamic acid decarboxylase (GAD67), tyrosine phosphatase (IA-2), heat-shock protein HSP65, islet-specific glucose6-phosphatase catalytic subunit related protein (IGRP), islet antigen 2 (IA2), and zinc transporter (ZnT8).
  • preproinsulin proinsulin
  • insulin insulin B chain
  • insulin A chain 65 kDa isoform of glutamic acid decarboxylase
  • GAD67 isoform of glutamic acid decarboxylase
  • HSP65 tyrosine phosphatase
  • HSP65 heat-shock protein
  • IGRP islet-specific glucose6-phosphatase catalytic subunit related protein
  • An antigen “associated with” a particular autoimmune disorder is an antigen that is a target of autoantibodies and/or autoreactive T cells present in individuals with that autoimmune disorder, where such autoantibodies and/or autoreactive T cells mediate a pathological state associated with the autoimmune disorder.
  • a suitable T1D peptide for inclusion in a TMMP of the present disclosure can be an epitope-presenting T1D peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned T1D-associated antigens.
  • a T1D peptide is proinsulin 73-90 (GAGSLQPLALEGSLQKR; SEQ ID NO:84).
  • a T1D peptide is the following insulin (InsA (1-15) peptide: GIVDQCCTSICSLYQ (SEQ ID NO:85).
  • a T1D peptide is the following insulin (InsA(1-15; D4E) peptide: GIVEQCCTSICSLYQ (SEQ ID NO:86).
  • a T1D peptide is the following GAD65 (555-567) peptide; NFFRMVISNPAAT (SEQ ID NO:87).
  • a T1D peptide is the following GAD65 (555-567; F557I) peptide; NFIRMVISNPAAT (SEQ ID NO:88).
  • a T1D peptide is the following islet antigen 2 (IA2) peptide: SFYLKNVQTQETRTLTQFHF (SEQ ID NO:89).
  • IA2 islet antigen 2
  • a T1D peptide is the following proinsulin peptide: SLQPLALEGSLQSRG (SEQ ID NO:90).
  • a T1D peptide is the following proinsulin peptide GSLQPLALEGSLQSRGIV (SEQ ID NO:91; proIns 75-92(K88S)).
  • a suitable T1D peptide comprises from 4 to 25 contiguous amino acids of an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 25-110 of the following human preproinsulin amino acid sequence (wherein amino acids 1-24 (underlined) is a signal peptide): MALWMRLLPL LALLALWGPD PAAA FVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN (SEQ ID NO:92); where the T1D peptide has a length of 4 amino acids (aa), 5 aa, 6 aa, 7, aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa,
  • the T1D peptide has the amino acid sequence: GAGSLQPLALEGSLQKRG (SEQ ID NO:93) (proIns 73-90). In some cases, the T1D peptide has the amino acid sequence: SLQPLALEGSLQKRG (SEQ ID NO:94) (proIns 76-90). In some cases, the T1D peptide has the amino acid sequence: SLQPLALEGSLQSRG (SEQ ID NO:90) (proIns 76-90; K88S). In some cases, the T1D peptide has the amino acid sequence: QPLALEGSLQKRG (SEQ ID NO:95). In some cases, the T1D peptide has the amino acid sequence: QPLALEGSLQSRG (SEQ ID NO:96).
  • MODs Immunomodulatory Polypeptides
  • immunomodulatory polypeptides that are suitable for inclusion in a TMPP of the present disclosure include, but are not limited to, IL-2, TGF ⁇ , CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, Fas ligand (FasL), inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, and HVEM.
  • IL-2 intercellular adhesion molecule
  • the immunomodulatory polypeptide is selected from an IL-2 polypeptide, a 4-1BBL polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, a TGF ⁇ polypeptide, and a PD-L2 polypeptide.
  • the immunomodulatory polypeptide can comprise a wild-type amino acid sequence, or can comprise one or more amino acid substitutions relative to a wild-type amino acid sequence.
  • the immunomodulatory polypeptide can comprise only the extracellular portion of a full-length immunomodulatory polypeptide.
  • the immunomodulatory polypeptide can in some cases exclude one or more of a signal peptide, a transmembrane domain, and an intracellular domain normally found in a naturally-occurring immunomodulatory polypeptide.
  • an immunomodulatory polypeptide suitable for inclusion in a TMPP of the present disclosure comprises all or a portion of (e.g., an extracellular portion of) the amino acid sequence of a naturally-occurring immunomodulatory polypeptide.
  • an immunomodulatory polypeptide suitable for inclusion in a TMPP of the present disclosure is a variant immunomodulatory polypeptide that comprises at least one amino acid substitution compared to the amino acid sequence of a naturally-occurring immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide exhibits a binding affinity for a co-immunomodulatory polypeptide that is lower than the affinity of a corresponding naturally-occurring immunomodulatory polypeptide (e.g., an immunomodulatory polypeptide not comprising the amino acid substitution(s) present in the variant) for the co-immunomodulatory polypeptide.
  • Suitable immunomodulatory domains that exhibit reduced affinity for a co-immunomodulatory domain can have from 1 amino acid (aa) to 20 aa differences from a wild-type immunomodulatory domain.
  • a variant immunomodulatory polypeptide present in a TMPP of the present disclosure differs in amino acid sequence by 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa, from a corresponding wild-type immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMPP of the present disclosure differs in amino acid sequence by 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, 19 aa, or 20 aa, from a corresponding wild-type immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMPP of the present disclosure includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide suitable for inclusion in a TMPP of the present disclosure exhibits reduced affinity for a cognate co-immunomodulatory polypeptide, compared to the affinity of a corresponding wild-type immunomodulatory polypeptide for the cognate co-immunomodulatory polypeptide.
  • Exemplary pairs of immunomodulatory polypeptide and cognate co-immunomodulatory polypeptide include, but are not limited to those set out in Table 2, below:
  • a variant immunomodulatory polypeptide present in a TMPP of the present disclosure has a binding affinity for a cognate co-immunomodulatory polypeptide that is from 100 nM to 100 M.
  • a variant immunomodulatory polypeptide present in a TMPP of the present disclosure has a binding affinity for a cognate co-immunomodulatory polypeptide that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM
  • Binding affinity between an immunomodulatory polypeptide and its cognate co-immunomodulatory polypeptide can be determined by bio-layer interferometry (BLI) using purified immunomodulatory polypeptide and purified cognate co-immunomodulatory polypeptide, following the procedure set forth in published PCT Application WO 2020/132138 A1.
  • At least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure comprises the amino acid sequence of a wild-type PD-L1 polypeptide.
  • at least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure is a variant PD-L1 polypeptide. Wild-type PD-L1 and variant PD-L1 polypeptides bind to PD1.
  • a wild-type human PD-L1 polypeptide can comprise the following amino acid sequence: MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKICLT LSPST (SEQ ID NO:97).
  • a wild-type PD-L1 polypeptide suitable for inclusion in a TMMP of the present disclosure can comprise the following amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO:98).
  • a wild-type PD-L1 polypeptide suitable for inclusion in a TMMP of the present disclosure can comprise the following amino acid sequence:
  • a wild-type PD-1 polypeptide can comprise the following amino acid sequence:
  • a variant PD-L1 polypeptide exhibits reduced binding affinity to PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence depicted above), compared to the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYR QRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVD PVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTF RRLDPEENHTAELVIPELPLAHPPNER (SEQ ID NO:99) or the amino acid sequence: FT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EED
  • a variant PD-L1 polypeptide of the present disclosure binds PD-1 (e.g., a PD-1 polypeptide comprising the PD-1 amino acid sequence depicted above) with a binding affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25% less, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of a PD-L1 polypeptide comprising the amino acid sequence: FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYR QRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADY
  • a variant PD-L1 polypeptide has a binding affinity to PD-1 that is from 1 nM to 1 mM. In some cases, a variant PD-L1 polypeptide of the present disclosure has a binding affinity to PD-1 that is from 100 nM to 100 ⁇ M.
  • a variant PD-L1 polypeptide has a binding affinity for PD1 (e.g., a PD1 polypeptide comprising the PD-1 amino acid sequence depicted above) that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900 nM to about 1 ⁇ M, to about 1 ⁇ M to about 5 ⁇ M, from about 5 M to about 10 ⁇ M, from about 10 ⁇ M to about 15 ⁇ M, from about 15
  • a variant PD-L1 polypeptide has a single amino acid substitution compared to one of the PD-L1 amino acid sequences depicted above. In some cases, a variant PD-L1 polypeptide has from 2 to 10 amino acid substitutions compared to one of the PD-L1 amino acid sequences depicted above.
  • At least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure comprises the amino acid sequence of a wild-type IL-2 polypeptide.
  • at least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure is a variant IL-2 polypeptide. Wild-type IL-2 and variant IL-2 polypeptides bind to IL-2 receptor (IL-2R) (e.g., bind to one or more polypeptides of an IL-2R).
  • IL-2R IL-2 receptor
  • a wild-type IL-2 amino acid sequence can be as follows: APTSSSTKKT QLQL EH LLL D LQMILNGINN YKNPKLTRML T F KF Y MPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFC Q SIIS TLT (SEQ ID NO:101).
  • Wild-type IL-2 binds to an IL2 receptor (IL2R) on the surface of a cell.
  • An IL2 receptor is in some cases a heterotrimeric polypeptide comprising an alpha chain (IL-2R ⁇ ; also referred to as CD25), a beta chain (IL-2R ⁇ ; also referred to as CD122: and a gamma chain (IL-2R ⁇ ; also referred to as CD132).
  • Amino acid sequences of human IL-2R ⁇ , IL2R ⁇ , and IL-2R ⁇ can be as follows.
  • Human IL-2R ⁇ (SEQ ID NO: 102) ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC QCTSSATRNT TKQVTPQPEE QKERKTTEMQ SPMQPVDQAS LPGHCREPPP WENEATERIY HFVVGQMVYY QCVQGYRALH RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL ISVLLLSGLT WQRRQRKSRR TI.
  • Human IL-2R ⁇ (SEQ ID NO: 103) VNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ VHAWPDRRRW NOTCELLPVS QASWACNLIL GAPDSQKLTT VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNI SWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLKQKQ EWICLETLTP DTQYEFQVRV KPLQGEFTTW SPWSQPLAFR TKPAALGKDT IPWLGHLLVG LSGAFGFIIL VYLLINCRNT GPWLKKVLKC NTPDPSKFFS QLSSEHGGDV QKWLSSPFPS SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS SNHSLTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP TGSSP
  • Human IL-2R ⁇ (SEQ ID NO: 104) LNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLPLPEV QCFVFNVEYM NCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPREPRR QATQMLKLQN LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR TDWDHSWTEQ SVDYRHKFSL PSVDGQKRYT FRVRSRFNPL CGSAQHWSEW SHPIHWGSNT SKENPFLFAL EAVVISVGSM GLIISLLCVY FWLERTMPRI PTLKNLEDLV TEYHGNFSAW SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASP CNQHSPYWAP PCYTLKPET.
  • a “cognate co-immunomodulatory polypeptide” is an IL-2R comprising polypeptides comprising the amino acid sequences of human IL-2R ⁇ , human IL-2R ⁇ , and IL-2R ⁇ shown above.
  • a variant IL-2 polypeptide exhibits reduced binding affinity to IL-2R, compared to the binding affinity of an IL-2 polypeptide comprising a wild-type IL-2 polypeptide (e.g., a wild-type IL-2 polypeptide having the amino acid sequence shown above).
  • a variant IL-2 polypeptide binds IL-2R with a binding affinity that is at least 10% less, at least 15% less, at least 20% less, at least 25%, at least 30% less, at least 35% less, at least 40% less, at least 45% less, at least 50% less, at least 55% less, at least 60% less, at least 65% less, at least 70% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, at least 95% less, or more than 95% less, than the binding affinity of an IL-2 polypeptide comprising the amino acid sequence APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:101) for an IL-2R (e.g.,
  • a variant IL-2 polypeptide has a binding affinity to IL-2R that is from 100 nM to 100 ⁇ M.
  • a variant IL-2 polypeptide has a binding affinity for IL-2R (e.g., an IL-2R comprising human IL-2R ⁇ , human IL-2R ⁇ , and IL-2R ⁇ having the amino acid sequences shown above) that is from about 100 nM to 150 nM, from about 150 nM to about 200 nM, from about 200 nM to about 250 nM, from about 250 nM to about 300 nM, from about 300 nM to about 350 nM, from about 350 nM to about 400 nM, from about 400 nM to about 500 nM, from about 500 nM to about 600 nM, from about 600 nM to about 700 nM, from about 700 nM to about 800 nM, from about 800 nM to about 900 nM, from about 900
  • a variant IL-2 polypeptide has a single amino acid substitution, or from 2-10 amino acid substitutions compared to the IL-2 amino acid sequence APTSSSTKKT QLQLEHLLLD LQMILNGINN YKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:101).
  • Suitable IL-2 variants include a polypeptide that comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of the following amino acid sequences:
  • a suitable variant IL-2 polypeptide comprises the amino acid sequence: APTSSSTKKT QLQLEALLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:121), i.e., the variant IL-2 polypeptide has the amino acid sequence of wild-type IL-2 but with H16A and F42A substitutions (shown in bold).
  • At least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure comprises the amino acid sequence of a wild-type TGF- ⁇ polypeptide. In other instances, at least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure is a variant TGF- ⁇ polypeptide. Wild-type TGF- ⁇ and variant TGF- ⁇ polypeptides bind to TGF receptor.
  • the immunomodulatory polypeptide present in a TMMP of the present disclosure is a TGF- ⁇ polypeptide.
  • Amino acid sequences of TGF- ⁇ polypeptides are known in the art.
  • the immunomodulatory polypeptide present in a TMMP of the present disclosure is a TGF- ⁇ 1 polypeptide.
  • immunomodulatory polypeptide present in a TMMP of the present disclosure is a TGF- ⁇ 2 polypeptide.
  • immunomodulatory polypeptide present in a TMMP of the present disclosure is a TGF- ⁇ 3 polypeptide.
  • a suitable TGF- ⁇ polypeptide can comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the mature form of a human TGF- ⁇ 1 polypeptide, a human TGF- ⁇ 2 polypeptide, or a human TGF- ⁇ 3 polypeptide.
  • a suitable TGF- ⁇ polypeptide can have a length of from about 100 amino acids to about 125 amino acids; for example, a suitable TGF- ⁇ polypeptide can have a length of from about 100 amino acids to about 105 amino acids, from about 105 amino acids to about 110 amino acids, from about 110 amino acids to about 115 amino acids, from about 115 amino acids to about 120 amino acids, or from about 120 amino acids to about 125 amino acids.
  • a suitable TGF- ⁇ 1 polypeptide can comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following TGF- ⁇ 1 amino acid sequence: AL DTNYCFSSTE KNCCVRQLYI DFRKDLGWKW IHEPKGYHAN FCLGPCPYIW SLDTQYSKVL ALYNQHNPGA SAAPCCVPQA LEPLPIVYYV GRKPKVEQLS NMIVRSCKCS (SEQ ID NO:122); or the foregoing sequence comprising a C77S substitution; where the TGF- ⁇ 1 polypeptide has a length of about 112 amino acids.
  • a suitable TGF- ⁇ 2 polypeptide can comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following TGF- ⁇ 2 amino acid sequence: ALDAAYCF RNVQDNCCLR PLYIDFKRDL GWKWIHEPKG YNANFCAGAC PYLWSSDTQH SRVLSLYNTI NPEASASPCC VSQDLEPLTI LYYIGKTPKI EQLSNMIVKS CKCS (SEQ ID NO:123); or the foregoing sequence comprising a C79S substitution, where the TGF- ⁇ 2 polypeptide has a length of about 112 amino acids.
  • a suitable TGF- ⁇ 3 polypeptide can comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the following TGF- ⁇ 3 amino acid sequence: ALDTNYCFRN LEENCCVRPL YIDFRQDLGW KWVHEPKGYY ANFCSGPCPY LRSADTTHST VLGLYNTLNP EASASPCCVP QDLEPLTILY YVGRTPKVEQ LSNMVVKSCK CS (SEQ ID NO:124); or the foregoing sequence comprising a C77S substitution, where the TGF- ⁇ 3 polypeptide has a length of about 112 amino acids.
  • At least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure comprises the amino acid sequence of a wild-type Fas ligand (FasL) polypeptide.
  • at least one of the one or more immunomodulatory polypeptides present in a TMMP of the present disclosure is a variant FasL polypeptide. Wild-type FasL and variant FasL polypeptides bind to Fas receptor (FasR). FasL is also known as CD95L or CD178.
  • Human FasL polypeptide can have the following amino acid sequence: MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP PPPLPPPPPP PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK L (SEQ ID NO:125).
  • a FasL polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 150 amino acids to about 175, from about 175 amino acids to about 200 amino acids, from about 200 amino acids to about 250 amino acids, or from about 250 amino acids to about 281 amino acids, of the following amino acid sequence: MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRP GQRRPPPPPP PPPLPPPPPPPPLPPLPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE LAELRESTSQ MHTASSLEKQ IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVY
  • a FasL polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: IGHPSPPPEK KELRKVAHLT GKSNSRSMPL EWEDTYGIVL LSGVKYKKGG LVINETGLYF VYSKVYFRGQ SCNNLPLSHK VYMRNSKYPQ DLVMMEGKMM SYCTTGQMWA RSSYLGAVFN LTSADHLYVN VSELSLVNFE ESQTFFGLYK (SEQ ID NO:237); and has a length of about 150, 151, or 152 amino acids.
  • a FasL polypeptide suitable for inclusion in a TMMP of the present disclosure comprises an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: QLFHLQKELAELRESTSQMHTASSLEKQIGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTY GIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGK MMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL (SEQ ID NO:126); and can have a length of about 179 amino acids.
  • a FasR polypeptide can comprise an amino acid sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MLGIWTLLPL VLTSVARLSS KSVNAQVTDI NSKGLELRKT VTTVETQNLE GLHHDGQFCH KPCPPGERKA RDCTVNGDEP DCVPCQEGKE YTDKAHFSSK CRRCRLCDEG HGLEVEINCT RTQNTKCRCK PNFFCNSTVC EHCDPCTKCE HGIIKECTLT SNTKCKEEGS RSNLGWLCLL LLPIPLIVWV KRKEVQKTCR KHRKENQGSH ESPTLNPETV AINLSDVDLS KYITTIAGVM TLSQVKGFVR KNGVNEAKID EIKNDNVQDT AEQKVQLLRN WHQLHGKKEA YDTLIKDLKK AN
  • a TMMP of the present disclosure comprises a heterodimer (e.g., comprises two heterodimers), where the heterodimer comprises a first polypeptide chain and a second polypeptide chain that are disulfide bonded to one another.
  • TMMPs that are Disulfide Bonded Via a Cys in the MHC Class II Alpha Chain and a Cys in the MHC Class II Beta Chain
  • a TMMP of the present disclosure comprises a heterodimer comprising a first polypeptide chain comprising: i) a T1D peptide; and ii) a first MHC class II polypeptide; and b) a second polypeptide comprising a second MHC class II polypeptide, where the first and/or the second polypeptides comprise one or more immunomodulatory polypeptides, optionally wherein the first and/or the second polypeptide comprises an Ig Fc polypeptide; where the first MHC class II polypeptide comprises a substitution of an amino acid (other than a Cys) with a Cys (a “first Cys”), and the second MHC class II polypeptide comprises a substitution of an amino acid (other than a Cys) with a Cys (a “second” Cys), where the first polypeptide and the second polypeptide are joined by a disulfide bond formed between the first Cys and the second Cys.
  • the first MHC class II polypeptide can be a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 14 A ; and having an amino acid substitution selected from P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C.
  • the second MHC class II polypeptide can be a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 13 A ; and having an amino acid substitution selected from E3C, E4C, F12C, G28C, D29C, 172C, K75C, T80C, P81C, 182C, T93C, N94C, and S95C.
  • the first MHC class II polypeptide is a DRB MHC class II polypeptide comprising a substitution at a residue selected from the group consisting of P5C, H33C, G151C, and W153
  • the second MHC class II polypeptide is a DRA MHC class II polypeptide comprising a substitution selected from the group consisting of P81C, I82C, and D29C.
  • the MHC class II ⁇ chain polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG.
  • the polypeptide comprises a Cys at a residue position selected from the group consisting of 5, 33, 151, and 153; and the MHC class II ⁇ chain polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 13 A , where the polypeptide comprises a Cys at a residue selected from the group consisting of 81, 82, and 29.
  • the disulfide can be formed between one of the specific pairs of Cys residues in Table 3, below:
  • the DRB MHC class II polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 14 A
  • the DRA MHC class II polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG.
  • ⁇ 3 chain polypeptide and ⁇ chain polypeptides are joined by a disulfide formed between a pair of Cys residues selected from the group consisting of: ⁇ 3 chain polypeptide residue 5 and ⁇ chain polypeptide residue 81; ⁇ 3 chain polypeptide residue ⁇ 3 and ⁇ chain polypeptide residue 81; ⁇ 3 chain polypeptide residue 33 and ⁇ chain polypeptide residue 82; ⁇ 3 chain polypeptide residue 151 and ⁇ chain polypeptide residue 29; and ⁇ 3 chain polypeptide residue 153 and ⁇ chain polypeptide residue 29.
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a P5C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a DRA MHC class II polypeptide comprising a P81C substitution; and (optionally) iii) an Ig Fc polypeptide.
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a P5C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class II polypeptide comprising a P81C substitution; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a P5C substitution
  • a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class
  • the one or more immunomodulatory polypeptides can be at Position 2, 4 or 5 as those positions are described above and depicted schematically in FIG. 5 .
  • the first polypeptide and the second polypeptide are linked via a disulfide bond between the Cys at residue 5 in the DRB MHC class II polypeptide and the Cys at residue 81 in the DRA MHC class II polypeptide.
  • linkers that do not comprise a Cys optionally can be used to connect the components of the first or second polypeptide.
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, a TGF- ⁇ polypeptide or variant thereof, an IL-2 polypeptide or variant thereof, or a FasL polypeptide or variant thereof.
  • the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 14 B , and comprises a Cys at position 5.
  • the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 13 J , and comprises a Cys at position 81.
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising an H33C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a DRA MHC class II polypeptide comprising a P81C substitution; and (optionally) iii) an Ig Fc polypeptide.
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising an H33C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class II polypeptide comprising a P81C substitution; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides. (See FIG. 5 , MOD Position 3).
  • the one or more immunomodulatory polypeptides can be at Position 2, 4 or 5 as those positions are described above and depicted schematically in FIG. 5 .
  • the first polypeptide and the second polypeptide are linked via a disulfide bond between the Cys at residue 33 in the DRB MHC class II polypeptide and the Cys at residue 81 in the DRA MHC class II polypeptide.
  • linkers that do not comprise a Cys can be used to connect the components of the first or second polypeptide.
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, a TGF- ⁇ polypeptide or variant thereof, an IL-2 polypeptide or variant thereof, or a FasL polypeptide or variant thereof.
  • the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14 G , and comprises a Cys at position 33.
  • the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 13 J , and comprises a Cys at position 81.
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising an H33C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a DRA MHC class II polypeptide comprising an I82C substitution; and (optionally) iii) an Ig Fc polypeptide.
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising an H33C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class II polypeptide comprising an I82C substitution; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising an H33C substitution
  • a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class II polypeptid
  • the one or more immunomodulatory polypeptides can be at Position 2, 4 or 5 as those positions are described above and depicted schematically in FIG. 5 .
  • the first polypeptide and the second polypeptide are linked via a disulfide bond between the Cys at residue 33 in the DRB MHC class II polypeptide and the Cys at residue 82 in the DRA MHC class II polypeptide.
  • linkers that do not comprise a Cys optionally can be used to connect the components of the first or second polypeptide.
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, a TGF- ⁇ polypeptide or variant thereof, an IL-2 polypeptide or variant thereof, or a FasL polypeptide or variant thereof.
  • the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14 G , and comprises a Cys at position 33.
  • the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 13 K , and comprises a Cys at position 82.
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO: 88; GAD65 555-567; F557I).
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a G151C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a DRA MHC class II polypeptide comprising a D29C substitution; and (optionally) iii) an Ig Fc polypeptide.
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a G151C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class II polypeptide comprising a D29C substitution; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a G151C substitution
  • a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class
  • the one or more immunomodulatory polypeptides can be at Position 2, 4 or 5 as those positions are described above and depicted schematically in FIG. 5 .
  • the first polypeptide and the second polypeptide are linked via a disulfide bond between the Cys at residue 151 in the DRB MHC class II polypeptide and the Cys at residue 29 in the DRA MHC class II polypeptide.
  • linkers that do not comprise a Cys optionally can be used to connect the components of the first or second polypeptide.
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, a TGF- ⁇ polypeptide or variant thereof, an IL-2 polypeptide or variant thereof, or a FasL polypeptide or variant thereof.
  • the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14 H , and comprises a Cys at position 151.
  • the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 13 F , and comprises a Cys at position 29.
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a W513C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) one or more immunomodulatory polypeptides; ii) a DRA MHC class II polypeptide comprising a D29C substitution; and (optionally) iii) an Ig Fc polypeptide.
  • a TMMP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a W513C substitution; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) a DRA MHC class II ⁇ chain polypeptide comprising a D29C substitution; ii) an Ig Fc polypeptide; and iii) one or more immunomodulatory polypeptides.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a DRB MHC class II polypeptide comprising a W513C substitution
  • a second polypeptide comprising, in order form N-terminus to C-terminus: i) a
  • the one or more immunomodulatory polypeptides can be at Position 2, 4 or 5 as those positions are described above and depicted schematically in FIG. 5 .
  • the first polypeptide and the second polypeptide are linked via a disulfide bond between the Cys at residue 153 in the DRB MHC class II polypeptide and the Cys at residue 29 in the DRA MHC class II polypeptide.
  • linkers that do not comprise a Cys optionally can be used to connect the components of the first or second polypeptide.
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, a TGF- ⁇ polypeptide or variant thereof, an IL-2 polypeptide or variant thereof, or a FasL polypeptide or variant thereof.
  • the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the DRB MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14 J , and comprises a Cys at position 153.
  • the DRA MHC class II polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence depicted in FIG. 13 F , and comprises a Cys at position 29.
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • TMMPs that are Disulfide Bonded Via a Cys in a Linker Between the T1D Peptide and the First MHC Class II Polypeptide and a Cys in the Second MHC Class II Polypeptide
  • a TMMP of the present disclosure comprises a heterodimer comprising a first polypeptide chain comprising: i) a T1D peptide; ii) a peptide linker comprising a Cys; and iii) a first MHC class II polypeptide; and b) a second polypeptide comprising a second MHC class II polypeptide, where the first and/or the second polypeptides comprise one or more immunomodulatory polypeptides, optionally wherein the first or the second polypeptide comprises an Ig Fc polypeptide, where the second MHC class II polypeptide comprises a substitution of an amino acid (other than a Cys) with a Cys; and where the first and the second polypeptides are linked via a disulfide bond between the Cys in the peptide linker and the Cys provided by the substitution in the second MHC class II polypeptide.
  • Cys-containing linkers between the T1D peptide and a DRB MHC class II polypeptide and Cys residues in the DRA MHC class II polypeptide that can form a disulfide bond are provided in Table 4, below:
  • the DRB MHC class II polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14 A .
  • the DRA MHC class II polypeptide can comprise an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 13 A , and can comprise a Cys at, e.g., position 72 or position 75.
  • n 0, 1, 2, 3, 4 or more, with 2 or 3 typically being used to create a total linker length of 15 or 20 amino acids, although longer lengths may be possible.
  • linkers that do not comprise a Cys optionally can be used to connect the other components of the first or second polypeptide.
  • the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, a TGF- ⁇ polypeptide or variant thereof, an IL-2 polypeptide or variant thereof, or a FasL polypeptide or variant thereof.
  • the one or more immunomodulatory polypeptides can be at Position 1, Position 2, Position 3, Position 4, or Position 5; where the immunomodulatory polypeptide positions are described above and depicted schematically in FIG. 5 .
  • TMMPs described above in which the first and the second polypeptides are linked via a disulfide bond between the Cys in the peptide linker and the Cys provided by the substitution in the second MHC class II polypeptide, and wherein the first polypeptide is a DRB MHC class II polypeptide (i.e., a DR ⁇ chain polypeptide), and the second polypeptide is a DRA MHC class II polypeptide (i.e., a DR ⁇ chain polypeptide).
  • DRB MHC class II polypeptide i.e., a DR ⁇ chain polypeptide
  • DRA MHC class II polypeptide i.e., a DR ⁇ chain polypeptide
  • a TMMP of the present disclosure comprises a heterodimer comprising one of the following five first polypeptides and one of the following two second polypeptides to create a heterodimer having one or more immunomodulatory polypeptides at MOD position 1 or 3:
  • each of the above combinations of first and second polypeptides for making a TMMP is thus expressly disclosed, i.e., a1-b1, a2-b1, a3-b1, a4-b1, a5-b1, a1-b2, a2-b2, a3-b2, a4-b2 and a5-b2, a1-b3, a2-b3, a3-b3, a4-b3, a5-b3, and a1-b4, a2-b4, a3-b4, a4-b4, and a5-b4.
  • the one or more immunomodulatory polypeptides in the second polypeptide can be at Position 2, Position 4, or Position 5, which are described above and depicted schematically in FIG. 5 .
  • the Ig Fc polypeptide is a human IgG1 Fc polypeptide comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • the one or more immunomodulatory polypeptides is a PD-L1 polypeptide or variant thereof, a TGF- ⁇ polypeptide or variant thereof, an IL-2 polypeptide or variant thereof, or a FasL polypeptide or variant thereof.
  • the PD-L1 polypeptide comprises the amino acid sequence
  • the IL-2 polypeptide comprises the following amino acid sequence: APTSSSTKKT QLQLEALLLD LQMILNGINN YKNPKLTRML TAKFYMPKKA TELKHLQCLEEELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNRWITFCQSIIS TLT (SEQ ID NO:121), where the IL-2 polypeptide is a variant comprising H16A and F42A substitutions relative to wild-type IL-2.
  • a TMMP in accordance with the above embodiments can comprise the combination of first polypeptide chain a3 and second polypeptide chain b2, wherein
  • a TMMP of the present disclosure can comprise: a) a first polypeptide comprising the amino acid sequence of construct “2938” depicted in FIG. 15 A ; and b) a second polypeptide comprising the amino acid sequence of construct “3893” depicted in FIG. 15 B .
  • the present disclosure provides an antigen-presenting polypeptide (APP) that comprises a heterodimer (e.g., comprises a heterodimer or a homodimer of two heterodimers), where the heterodimer comprises: a) a first polypeptide comprising a T1D peptide; and ii) a first MHC class II polypeptide; and b) a second polypeptide comprising a second MHC class II polypeptide, where the heterodimer does not include an immunomodulatory polypeptide, where the first polypeptide and the second polypeptide are disulfide linked to one another, optionally where the first or the second polypeptide comprises an Ig Fc polypeptide.
  • a heterodimer e.g., comprises a heterodimer or a homodimer of two heterodimers
  • the heterodimer comprises: a) a first polypeptide comprising a T1D peptide; and ii) a first MHC class II polypeptid
  • An APP of the present disclosure is depicted schematically in FIG. 16 A- 16 C .
  • An APP of the present disclosure is useful for diagnostic applications and therapeutic applications.
  • the APP when used for diagnostic applications, the APP also can comprise a detectable label so that binding of the APP to a target T cell is detected by detecting the detectable label.
  • the APP comprises an Ig Fc polypeptide
  • an APP typically will be a homodimer comprising two heterodimers that are joined by disulfide bonds that spontaneously form between the two Ig Fc polypeptides.
  • the APP comprises a suitable T1D peptides as described above.
  • the first MHC class II polypeptide is an MHC class II ⁇ chain polypeptide such as a DRB MHC class II polypeptide.
  • the second MHC class II polypeptide is an MHC class II ⁇ chain polypeptide such as a DRA MHC class II polypeptide.
  • Suitable Ig Fc polypeptides are as described above.
  • the Ig Fc polypeptide is one that induces cell lysis through activation of complement-dependent cytotoxicity (CDC).
  • the Ig Fc polypeptide is one that can elicit antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • the Ig Fc polypeptide binds complement or a complement component.
  • the Ig Fc polypeptide is one that substantially does not induce cell lysis through activation of CDC.
  • the Ig Fc polypeptide is one that that induces cell lysis through activation of CDC, and/or elicits ADCC
  • the APP can bind to and destroy the target T cell.
  • An APP used for diagnostic purposes typically will have an Ig Fc polypeptide is one that substantially does not induce cell lysis through activation of CDC or elicit ADCC.
  • an APP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; and ii) a first MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “first Cys”); and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “second Cys”); and ii) an Ig Fc polypeptide, where the first polypeptide and the second polypeptide are disulfide bonded via the first Cys and the second Cys.
  • the first MHC class II polypeptide can be a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 14 A ; and having an amino acid substitution selected from P5C, F7C, Q10C, N19C, G20C, H33C, G151C, D152C, and W153C.
  • the second MHC class II polypeptide can be a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 13 A ; and having an amino acid substitution selected from E3C, E4C, F12C, G28C, D29C, 172C, K75C, T80C, P81C, 182C, T93C, N94C, and S95C.
  • the Ig Fc polypeptide induces cell lysis through activation of complement-dependent cytotoxicity (CDC) and/or elicits antibody-dependent cellular cytotoxicity (ADCC).
  • the Ig Fc polypeptide is a variant that substantially does not induce cell lysis through activation of CDC, e.g., an IgG1 Fc polypeptide comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • an APP of the present disclosure comprises a heterodimer comprising: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a T1D peptide; ii) a linker comprising a Cys (a “linker Cys”), and iii) a first MHC class II polypeptide; and b) a second polypeptide comprising, in order form N-terminus to C-terminus: i) a second MHC class II polypeptide comprising a substitution of an amino acid (other than a Cys) with a Cys (a “second Cys”); and ii) an Ig Fc polypeptide, where the first polypeptide and the second polypeptide are disulfide bonded via the linker Cys and the second Cys.
  • the first MHC class II polypeptide can be a DRB MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% amino acid sequence identity to the amino acid sequence depicted in FIG. 14 A .
  • the second MHC class II polypeptide can be a DRA MHC class II polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 13 A ; and having an amino acid substitution selected from I72C or K75C.
  • the Ig Fc polypeptide induces cell lysis through activation of complement-dependent cytotoxicity (CDC) and/or elicits antibody-dependent cellular cytotoxicity (ADCC).
  • the Ig Fc polypeptide is a variant that substantially does not induce cell lysis through activation of CDC, e.g., an IgG1 Fc polypeptide comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • Cys-containing linkers between the T1D peptide and a DRB MHC class II polypeptide and Cys residues in the DRA MHC class II polypeptide that can form a disulfide bond are provided in the Table 5, below:
  • n 0, 1, 2, 3, 4 or more, with 2 or 3 typically being used to create a total linker length of 15 or 20 amino acids, although longer lengths may be possible.
  • linkers that do not comprise a Cys optionally can be used to connect the other components of second polypeptide.
  • the Ig Fc polypeptide induces cell lysis through activation of complement-dependent cytotoxicity (CDC) and/or elicits antibody-dependent cellular cytotoxicity (ADCC).
  • the Ig Fc polypeptide is present and is a human IgG1 Fc polypeptide, optionally comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:94; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • DRB MHC class II polypeptide i.e., a DR ⁇ chain polypeptide
  • DRA MHC class II polypeptide i.e., a DR ⁇ chain polypeptide
  • an APP of the present disclosure comprises a heterodimer comprising one of the following five first polypeptides and one of the following two second polypeptides:
  • linkers that do not comprise a Cys optionally can be used to connect the components of the second polypeptide (e.g., a GGSAAAGG (SEQ ID NO:83) linker or AAAGG (SEQ ID NO:82) linker can be used between the DRA MHC class II polypeptide and the Ig Fc polypeptide).
  • the Ig Fc polypeptide is a human IgG1 Fc polypeptide comprising L234A and L235A substitutions (L14A and L15A of the amino acid sequence depicted in FIG. 12 A ).
  • the Ig Fc polypeptide induces cell lysis through activation of complement-dependent cytotoxicity (CDC) and/or elicits antibody-dependent cellular cytotoxicity (ADCC).
  • the T1D peptide is a proinsulin peptide.
  • the T1D peptide is a proinsulin peptide selected from SLQPLALEGSLQKRG (SEQ ID NO:94; proIns 76-90), SLQPLALEGSLQSRG (SEQ ID NO:90; proIns 76-90; K88S), and GAGSLQPLALEGSLQKRG (SEQ ID NO:93; proIns 73-90).
  • the T1D peptide is a GAD peptide.
  • the T1D peptide is a GAD peptide selected from NFFRMVISNPAAT (SEQ ID NO:87; GAD65 555-567) and NFIRMVISNPAAT (SEQ ID NO:88; GAD65 555-567; F557I).
  • a TMMP in accordance with the above embodiments can comprise the combination of first polypeptide chain a3 and second polypeptide chain b2, wherein
  • a disulfide-bonded APP of the present disclosure will in some cases exhibit greater stability than a control non-disulfide-bonded APP.
  • An example of a control APP is an APP comprising first and second polypeptide chains 2744 and 2932 (depicted in FIG. 15 AA and FIG. 15 Z , respectively), or 2639 and 2932 (depicted in FIG. 15 C and FIG. 15 Z , respectively).
  • a disulfide-bonded APP of the present disclosure will in some cases exhibit at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 50%, at least 75%, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or greater than 100-fold, greater stability in the PBS buffer solution containing 500 mM NaCl (described above) in vitro over a specified period of time and at a specified temperature (e.g., in a solution at a temperature of from 37° C. to 42° C.
  • a disulfide-bonded APP of the present disclosure will in some cases exhibit greater expression than a control non-disulfide-bonded APP.
  • a disulfide-bonded APP of the present disclosure will in some cases exhibit both greater stability and greater expression than a control non-disulfide-bonded APP.
  • an APP of the present disclosure is expressed (produced) in a Chinese hamster ovary (CHO) cell in vitro at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than 100-fold, higher than the expression level of a control APP lacking the at least one disulfide bond when expressed under the same conditions and in the same CHO cells.
  • Expression levels can be determined by: i) producing the APP in a CHO cell in vitro; and ii) determining the amount of APP produced by the mammalian cell.
  • the APP can be isolated from the CHO cells and/or from culture medium in which the CHO cells are cultured, where isolation of the APP can be carried out by affinity chromatography, e.g., on a Protein A column, a Protein G column, or the like.
  • An example of a suitable mammalian cell is a CHO cell; e.g., an Expi-CHO-STM cell (e.g., ThermoFisher Scientific, Catalog #A29127).
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2986, depicted in FIG. 15 I , comprising an F7C substitution (Cys-7) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 2983, depicted in FIG. 15 H , comprising a P81C substitution (Cys-81) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-7 and Cys-81.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2987, depicted in FIG. 15 J , comprising an P5C substitution (Cys-5) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 2983, depicted in FIG. 15 H , comprising a P81C substitution (Cys-81) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-5 and Cys-81.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3027, depicted in FIG. 15 K , comprising an H33C substitution (Cys-33) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 2983, depicted in FIG. 15 H , comprising a P81C substitution (Cys-81) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-33 and Cys-81.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2988, depicted in FIG. 15 M , comprising an N19C substitution (N19C) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 2984, depicted in FIG. 15 L , comprising an E4C substitution (Cys-4) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-19 and Cys-4.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3032, depicted in FIG. 15 N , comprising a G20C substitution (Cys-20) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 2984, depicted in FIG. 15 L , comprising an E4C substitution (Cys-4) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-20 and Cys-4.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2989, depicted in FIG. 15 P , comprising a Q56C substitution (Cys-156) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 2985, depicted in FIG. 15 O , comprising a T93C substitution (Cys-93) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-156 and Cys-93.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3030, depicted in FIG. 15 Q , comprising a W153C substitution (Cys-153) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 2985, depicted in FIG. 15 O , comprising a T93C substitution (Cys-93) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-153 and Cys-93.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2986, depicted in FIG. 15 I , comprising an F7C substitution (Cys-7) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3018, depicted in FIG. 15 R , comprising an F12C substitution (Cys-12) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-7 and Cys-12.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3026, depicted in FIG. 15 S , comprising a Q10C substitution (Cys-10) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3018, depicted in FIG. 15 R , comprising an F12C substitution (Cys-12) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-10 and Cys-12.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2986, depicted in FIG. 15 I , comprising an F7C substitution (Cys-7) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3019, depicted in FIG. 15 T , comprising a T80C substitution (Cys-80) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-7 and Cys-80.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2987, depicted in FIG. 15 J , comprising a P5C substitution (Cys-5) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3019, depicted in FIG. 15 T , comprising a T80C substitution (Cys-80) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-5 and Cys-80.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3027, depicted in FIG. 15 K , comprising an I82C substitution (Cys-82) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3019, depicted in FIG. 15 T , comprising a T80C substitution (Cys-80) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-82 and Cys-80.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2986, depicted in FIG. 15 I , comprising an F7C substitution (Cys-7) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3018, depicted in FIG. 15 R , comprising an F12C substitution (Cys-12) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-7 and Cys-12.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3026, depicted in FIG. 15 S , comprising a Q10C substitution (Cys-10) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3018, depicted in FIG. 15 R , comprising an F12C substitution (Cys-12) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-10 and Cys-12.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2986, depicted in FIG. 15 I , comprising an F7C substitution (Cys-7) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3019, depicted in FIG. 15 T , comprising a T80C substitution (Cys-80) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-7 and Cys-80.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2987, depicted in FIG. 15 J , comprising a P5C substitution (Cys-5) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3019, depicted in FIG. 15 T , comprising a T80C substitution (Cys-80) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-5 and Cys-80.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3027, depicted in FIG. 15 K , comprising an I82C substitution (Cys-82) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3019, depicted in FIG. 15 T , comprising a T80C substitution (Cys-80) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-82 and Cys-80.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2986, depicted in FIG. 15 I , comprising an F7C substitution (Cys-7) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3020, depicted in FIG. 15 U , comprising an I82C substitution (Cys-82) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-7 and Cys-82.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2987, depicted in FIG. 15 J , comprising a P5C substitution (Cys-5) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3020, depicted in FIG. 15 U , comprising an I82C substitution (Cys-82) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-5 and Cys-82.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3027, depicted in FIG. 15 V , comprising an H33C substitution (Cys-33) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3020, depicted in FIG. 15 U , comprising an I82C substitution (Cys-82) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-33 and Cys-82.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3028, depicted in FIG. 15 X , comprising a G151C substitution (Cys-151) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3021, depicted in FIG. 15 W , comprising a G28C substitution (Cys-28) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-151 and Cys-28.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3029, depicted in FIG. 15 Y , comprising a D152C substitution (Cys-152) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3021, depicted in FIG. 15 W , comprising a G28C substitution (Cys-28) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-152 and Cys-28.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3030, depicted in FIG. 15 Q , comprising a W153C substitution (Cys-153) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3021, depicted in FIG. 15 W , comprising a G28C substitution (Cys-28) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-153 and Cys-28.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3028, depicted in FIG. 15 X , comprising a G151C substitution (Cys-151) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3022, depicted in FIG. 15 BB , comprising a D29C substitution (Cys-29) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-151 and Cys-29.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3029, depicted in FIG. 15 Y , comprising a D152C substitution (Cys-152) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3022, depicted in FIG. 15 BB , comprising a D29C substitution (Cys-29) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-152 and Cys-29.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3030, depicted in FIG. 15 Q , comprising a W153C substitution (Cys-153) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3022, depicted in FIG. 15 BB , comprising a D29C substitution (Cys-29) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-153 and Cys-29.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2989, depicted in FIG. 15 P , comprising a Q156C substitution (Cys-156) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3023, depicted in FIG. 15 CC , comprising an N94C substitution (Cys-94) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-156 and Cys-94.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3031, depicted in FIG. 15 DD , comprising an N120C substitution (Cys-120) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3023, depicted in FIG. 15 CC , comprising an N94C substitution (Cys-94) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-120 and Cys-94.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2989, depicted in FIG. 15 P , comprising a Q156C substitution (Cys-156) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3024, depicted in FIG. 15 EE , comprising an S95C substitution (Cys-95) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-156 and Cys-95.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3031, depicted in FIG. 15 DD , comprising an N120C substitution (Cys-120) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3024, depicted in FIG. 15 EE , comprising an S95C substitution (Cys-95) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-120 and Cys-95.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 2988, depicted in FIG. 15 M , comprising an N19C substitution (Cys-19) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3025, depicted in FIG. 15 FF , comprising an E3C substitution (Cys-3) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-19 and Cys-3.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising the amino acid sequence of construct 3032, depicted in FIG. 15 N , comprising a G20C substitution (Cys-20) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 14 A ; and b) a second polypeptide comprising the amino acid sequence of construct 3025, depicted in FIG. 15 FF , comprising an E3C substitution (Cys-3) in the MHC class II ⁇ chain relative to the MHC class II ⁇ chain sequence depicted in FIG. 13 A , where the first polypeptide and the second polypeptide are covalently linked to one another via a disulfide bond between Cys-20 and Cys-3.
  • the present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a TMPP or an APP of the present disclosure.
  • the present disclosure provides a single nucleic acid comprising nucleotide sequences encoding both the first polypeptide and the second polypeptide of a TMPP or APP of the present disclosure.
  • the present disclosure provides: a) a first nucleic acid comprising a nucleotide sequence encoding the first polypeptide of a TMMP or APP of the present disclosure; and b) a second nucleic acid comprising a nucleotide sequence encoding the second polypeptide of a TMMP or APP of the present disclosure.
  • the nucleic acid is a recombinant expression vector; thus, the present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a TMPP or APP of the present disclosure.
  • the discussion, below, of nucleic acids refers to nucleic acids encoding TMPPs of the present disclosure; however, the discussion applies as well to nucleic acids encoding APPs of the present disclosure.
  • nucleic acids comprising nucleotide sequences encoding a TMMP or APP of the present disclosure.
  • the individual polypeptide chains of a TMMP of the present disclosure are encoded in separate nucleic acids.
  • nucleotide sequences encoding the separate polypeptide chains of a TMMP or APP of the present disclosure are operably linked to transcriptional control elements, e.g., promoters, such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
  • the present disclosure provides a first nucleic acid and a second nucleic acid, where the first nucleic acid comprises a nucleotide sequence encoding the first polypeptide of a TMMP or APP of the present disclosure, and where the second nucleic acid comprises a nucleotide sequence encoding the second polypeptide of the TMMP or APP.
  • the nucleotide sequences encoding the first and the second polypeptides are operably linked to transcriptional control elements.
  • the transcriptional control element is a promoter that is functional in a eukaryotic cell.
  • the nucleic acids are present in separate expression vectors.
  • the nucleotide sequences encoding the first and the second polypeptides are operably linked to transcriptional control elements.
  • the transcriptional control element is a promoter that is functional in a eukaryotic cell.
  • the nucleic acids are present in separate expression vectors.
  • the present disclosure provides a nucleic acid comprising nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP or APP of the present disclosure.
  • the nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP or APP of the present disclosure includes a proteolytically cleavable linker interposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide.
  • the nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP or APP of the present disclosure includes an internal ribosome entry site (IRES) interposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide.
  • the nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP or APP of the present disclosure includes a ribosome skipping signal (or cis-acting hydrolase element, CHYSEL) interposed between the nucleotide sequence encoding the first polypeptide and the nucleotide sequence encoding the second polypeptide.
  • nucleic acids examples include nucleic acids, where a proteolytically cleavable linker is provided between nucleotide sequences encoding the first polypeptide and the second polypeptide of a TMMP or APP of the present disclosure; in any of these embodiments, an IRES or a ribosome skipping signal can be used in place of the nucleotide sequence encoding the proteolytically cleavable linker.
  • a first nucleic acid (e.g., a recombinant expression vector, an mRNA, a viral RNA, etc.) comprises a nucleotide sequence encoding a first polypeptide chain of a TMMP or APP of the present disclosure
  • a second nucleic acid (e.g., a recombinant expression vector, an mRNA, a viral RNA, etc.) comprises a nucleotide sequence encoding a second polypeptide chain of a TMMP or APP of the present disclosure.
  • the nucleotide sequence encoding the first polypeptide, and the second nucleotide sequence encoding the second polypeptide are each operably linked to transcriptional control elements, e.g., promoters, such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
  • promoters such as promoters that are functional in a eukaryotic cell, where the promoter can be a constitutive promoter or an inducible promoter.
  • the present disclosure provides recombinant expression vectors comprising nucleic acids of the present disclosure.
  • the recombinant expression vector is a non-viral vector.
  • the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus construct (see, e.g., U.S. Pat. No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, a non-integrating viral vector, etc.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus; and the like.
  • a retroviral vector e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus
  • suitable expression vectors are known to those of skill in the art, and many are commercial
  • any of a number of suitable transcription and translation control elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).
  • a nucleotide sequence encoding a TMMP or APP of the present disclosure is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.
  • a control element e.g., a transcriptional control element, such as a promoter.
  • the transcriptional control element may be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell).
  • a nucleotide sequence encoding a DNA-targeting RNA and/or a site-directed modifying polypeptide is operably linked to multiple control elements that allow expression of the nucleotide sequence encoding a DNA-targeting RNA and/or a site-directed modifying polypeptide in both prokaryotic and eukaryotic cells.
  • eukaryotic promoters include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector may also include appropriate sequences for amplifying expression.
  • the present disclosure provides a genetically modified host cell, where the host cell is genetically modified with a nucleic acid(s) of the present disclosure.
  • Suitable host cells include eukaryotic cells, such as yeast cells, insect cells, and mammalian cells.
  • the host cell is a cell of a mammalian cell line.
  • Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.
  • Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No.
  • Vero cells NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLL3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
  • BHK cells e.g., ATCC No. CCL10
  • PC12 cells ATCC No. CRL1721
  • COS cells COS-7 cells
  • RAT1 cells RAT1 cells
  • mouse L cells ATCC No. CCLL3
  • human embryonic kidney (HEK) cells ATCC No. CRL1573)
  • HLHepG2 cells and the like.
  • Genetically modified host cells can be used to produce a TMMP or an APP of the present disclosure.
  • a genetically modified host cell can be used to produce a TMMP of the present disclosure, or an APP of the present disclosure.
  • An expression vector(s) comprising nucleotide sequences encoding the polypeptide(s) is/are introduced into a host cell, generating a genetically modified host cell, which genetically modified host cell produces the polypeptide(s).
  • the present disclosure provides methods of producing TMMP or an APP of the present disclosure.
  • the methods generally involve culturing, in a culture medium, a host cell (e.g., a genetically modified host cell of the present disclosure) that is genetically modified with a recombinant expression vector(s) comprising a nucleotide sequence(s) encoding the TMMP or the APP; and isolating the TMMP or the APP from the genetically modified host cell and/or the culture medium.
  • a host cell e.g., a genetically modified host cell of the present disclosure
  • a recombinant expression vector(s) comprising a nucleotide sequence(s) encoding the TMMP or the APP
  • isolating the TMMP or the APP from the genetically modified host cell and/or the culture medium.
  • the individual polypeptide chains of a TMMP or APP of the present disclosure are encoded in separate recombinant expression vectors.
  • Isolation of the TMMP or the APP from the expression host cell e.g., from a lysate of the expression host cell
  • the culture medium in which the host cell is cultured can be carried out using standard methods of protein purification.
  • a lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography (e.g., size exclusion chromatography), gel electrophoresis, affinity chromatography, or other purification technique.
  • HPLC high performance liquid chromatography
  • exclusion chromatography e.g., size exclusion chromatography
  • gel electrophoresis e.g., affinity chromatography
  • affinity chromatography e.g., affinity chromatography
  • purification technique e.g., affinity chromatography
  • the TMMP or APP is purified, e.g., a composition is generated that comprises at least 80% by weight, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, of the TMMP or APP in relation to contaminants related to the method of preparation of the product and its purification.
  • the percentages can be based upon total protein.
  • the TMMP or APP can be purified using an immobilized binding partner of the affinity tag.
  • the TMMP or the APP can be isolated from genetically modified mammalian host cell and/or from culture medium in which the mammalian cells are cultured, where isolation of the TMMP or APP can be carried out by affinity chromatography, e.g., on a Protein A column, a Protein G column, or the like.
  • An example of a suitable mammalian cell is a CHO cell; e.g., an Expi-CHO-STM cell (e.g., ThermoFisher Scientific, Catalog #A29127).
  • the first and second polypeptides will self-assemble into heterodimers by spontaneously forming disulfide bonds between the above-discussed Cys residues in the first and second polypeptides.
  • both heterodimers include Ig Fc polypeptides
  • disulfide bonds will spontaneously form between the respective Ig Fc polypeptides to covalently link the two heterodimers to one another (depicted schematically in FIG. 1 E ).
  • compositions including pharmaceutical compositions, comprising a TMMP or an APP of the present disclosure.
  • compositions including pharmaceutical compositions, comprising a nucleic acid or a recombinant expression vector of the present disclosure.
  • the discussion, below, of compositions refers to compositions comprising a TMMP of the present disclosure; however, the discussion applies equally to an APP of the present disclosure.
  • compositions Comprising a TMMP or an APP
  • a composition of the present disclosure can comprise, in addition to a TMMP of the present disclosure, or an APP of the present disclosure, one or more of: a salt, e.g., NaCl, MgCl 2 , KCl, MgSO 4 , etc.; a buffering agent, e.g., a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Twe
  • composition may comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein.
  • Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, “Remington: The Science and Practice of Pharmacy”, 19 th Ed. (1995), or latest edition, Mack Publishing Co; A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3 rd ed. Amer. Pharmaceutical Assoc.
  • a pharmaceutical composition can comprise: i) a TMMP of the present disclosure; and ii) a pharmaceutically acceptable excipient.
  • a pharmaceutical composition can comprise: i) an APP of the present disclosure; and ii) a pharmaceutically acceptable excipient.
  • a subject pharmaceutical composition will be suitable for administration to a subject, e.g., will be sterile.
  • a subject pharmaceutical composition will be suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and/or other toxins.
  • the protein compositions may comprise other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, hydrochloride, sulfate salts, solvates (e.g., mixed ionic salts, water, organics), hydrates (e.g., water), and the like.
  • compositions may include aqueous solution, powder form, granules, tablets, pills, suppositories, capsules, suspensions, sprays, and the like.
  • the composition may be formulated according to the various routes of administration described below.
  • a formulation can be provided as a ready-to-use dosage form, or as non-aqueous form (e.g. a reconstitutable storage-stable powder) or aqueous form, such as liquid composed of pharmaceutically acceptable carriers and excipients.
  • the protein-containing formulations may also be provided so as to enhance serum half-life of the subject protein following administration.
  • the protein may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum half-life.
  • liposomes A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
  • the preparations may also be provided in controlled release or slow-release forms.
  • a composition of the present disclosure comprises: a) a TMMP of the present disclosure; and b) saline (e.g., 0.9% NaCl).
  • the composition is sterile.
  • the composition is suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and/or other toxins.
  • the present disclosure provides a composition comprising: a) a TMMP of the present disclosure; and b) saline (e.g., 0.9% NaCl), where the composition is sterile and is free of detectable pyrogens and/or other toxins.
  • formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • a subject pharmaceutical composition can be present in a container, e.g., a sterile container, such as a syringe.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.
  • concentration of a TMMP of the present disclosure in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient's needs.
  • the present disclosure provides a container comprising a composition of the present disclosure, e.g., a liquid composition.
  • the container can be, e.g., a syringe, an ampoule, and the like.
  • the container is sterile. In some cases, both the container and the composition are sterile.
  • compositions Comprising a Nucleic Acid or a Recombinant Expression Vector
  • compositions e.g., pharmaceutical compositions, comprising a nucleic acid or a recombinant expression vector of the present disclosure.
  • a wide variety of pharmaceutically acceptable excipients is known in the art and need not be discussed in detail herein.
  • Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds 7 th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3 rd ed. Amer. Pharmaceutical Assoc.
  • a composition of the present disclosure can include: a) one or more nucleic acids or one or more recombinant expression vectors comprising nucleotide sequences encoding a TMMP or an APP of the present disclosure; and b) one or more of: a buffer, a surfactant, an antioxidant, a hydrophilic polymer, a dextrin, a chelating agent, a suspending agent, a solubilizer, a thickening agent, a stabilizer, a bacteriostatic agent, a wetting agent, and a preservative.
  • Suitable buffers include, but are not limited to, (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N′3-propanesulfonic acid (EPPS or HEPPS), glycylglycine, N-2-hydroxyehtylpiperazine-N′-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS), piperazine-N,N′-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid) TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulf
  • a pharmaceutical formulation of the present disclosure can include a nucleic acid or recombinant expression vector of the present disclosure in an amount of from about 0.001% to about 90% (w/w).
  • “subject nucleic acid or recombinant expression vector” will be understood to include a nucleic acid or recombinant expression vector of the present disclosure.
  • a subject formulation comprises a nucleic acid or recombinant expression vector of the present disclosure.
  • a subject nucleic acid or recombinant expression vector can be admixed, encapsulated, conjugated or otherwise associated with other compounds or mixtures of compounds; such compounds can include, e.g., liposomes or receptor-targeted molecules.
  • a subject nucleic acid or recombinant expression vector can be combined in a formulation with one or more components that assist in uptake, distribution and/or absorption.
  • a subject nucleic acid or recombinant expression vector composition can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • a subject nucleic acid or recombinant expression vector composition can also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • a formulation comprising a subject nucleic acid or recombinant expression vector can be a liposomal formulation.
  • liposome means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that can interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH sensitive or negatively charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes can be used to deliver a subject nucleic acid or recombinant expression vector.
  • Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • compositions of the present disclosure may also include surfactants.
  • surfactants used in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860.
  • various penetration enhancers are included, to effect the efficient delivery of nucleic acids.
  • penetration enhancers also enhance the permeability of lipophilic drugs.
  • Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein by reference in its entirety.
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Suitable oral formulations include those in which a subject antisense nucleic acid is administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Suitable surfactants include, but are not limited to, fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Suitable bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860.
  • Also suitable are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts.
  • An exemplary suitable combination is the sodium salt of lauric acid, capric acid, and UDCA.
  • Further penetration enhancers include, but are not limited to, polyoxyethylene-9-lauryl ether, and polyoxyethylene-20-cetyl ether.
  • Suitable penetration enhancers also include propylene glycol, dimethylsulfoxide, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol, and AZONETM.
  • a TMMP of the present disclosure is useful for modulating an activity of a T cell.
  • the present disclosure provides methods of modulating an activity of a T cell, the methods generally involving contacting a target T cell with a TMMP of the present disclosure.
  • An APP of the present disclosure is useful for various research, therapeutic, and diagnostic purposes.
  • an APP of the present disclosure can be used to label, directly or indirectly, an antigen-specific T cell.
  • An APP of the present disclosure can provide effector functions in an antigen-specific manner.
  • the present disclosure provides a method of selectively modulating the activity of an epitope-specific T cell, the method comprising contacting the T cell with a TMMP of the present disclosure, where contacting the T cell with a TMMP of the present disclosure selectively modulates the activity of the epitope-specific T cell.
  • the contacting occurs in vivo. In some cases, the contacting occurs in vitro.
  • a TMMP of the present disclosure reduces activity of an autoreactive T cell and/or an autoreactive B cell. In some cases, a TMMP of the present disclosure increases the number and/or activity of a regulatory T cell (Treg), resulting in reduced activity of an autoreactive T cell and/or an autoreactive B cell.
  • Treg regulatory T cell
  • the T cell being contacted with a TMMP of the present disclosure is a regulatory T cell (Treg).
  • Tregs are CD4 + , FOXP3 + , and CD25 + . Tregs can suppress autoreactive T cells.
  • a method of the present disclosure activates Tregs, thereby reducing autoreactive T cell activity.
  • the present disclosure provides a method of increasing proliferation of Tregs, the method comprising contacting Tregs with a TMMP of the present disclosure, where the contacting increases proliferation of Tregs.
  • the present disclosure provides a method of increasing the number of Tregs in an individual, the method comprising administering to the individual a TMMP of the present disclosure, where the administering results in an increase in the number of Tregs in the individual.
  • the number of Tregs can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold.
  • the present disclosure provides a method of detecting an antigen-specific T-cell.
  • the methods comprise contacting a T cell with an APP of the present disclosure; and detecting binding of the APP to the T cell.
  • the present disclosure provides a method of detecting an antigen-specific T cell, the method comprising contacting a T cell with an APP of the present disclosure, wherein binding of the APP to the T cell indicates that the T cell is specific for the epitope present in the APP.
  • the APP comprises a detectable label.
  • Suitable detectable labels include, but are not limited to, a radioisotope, a fluorescent polypeptide, or an enzyme that generates a fluorescent product, and an enzyme that generates a colored product.
  • binding of the APP to the T cell is detected by detecting the detectable label.
  • an APP of the present disclosure comprises a detectable label suitable for use in in vivo imaging, e.g., suitable for use in positron emission tomography (PET), single photon emission tomography (SPECT), near infrared (NIR) optical imaging, x-ray imaging, computer-assisted tomography (CAT), or magnetic resonance imaging (MRI), or other in vivo imaging method.
  • PET positron emission tomography
  • SPECT single photon emission tomography
  • NIR near infrared
  • CAT computer-assisted tomography
  • MRI magnetic resonance imaging
  • gadolinium chelates e.g., gadolinium chelates with DTPA (diethylenetriamine penta-acetic acid), DTPA-bismethylamide (BMA), DOTA (dodecane tetraacetic acid), or HP-DO3A (1,4,7-tris(carboxymethyl)-10-(2′-hydroxypropyl)-1,4,7,10-tetraazacycl ododecane)
  • iron chelates magnesium chelates
  • manganese chelates copper chelates
  • chromium chelates iodine-based materials
  • radionuclides e.g., radionuclides.
  • Suitable radionuclides include, but are not limited to, 123 I, 125 I, 130 I, 131 I, 133 I, 135 I, 47 Sc, 72 As, 72 Se, 90 Y, 88 Y, 97 Ru, 100 Pd, 101 mRh, 119 Sb, 128 Ba, 197 Hg, 21 At, 212 Bi, 212 Pb, 109 Pd, 111 In, 67 Ga, 68 Ga, 64 Cu, 67 Cu, 75 Br, 77 Br, 99 mTc, 14 C, 13 N, 15 O, 32 P, 33 P, and 18 F.
  • the detectable label is a positron-emitting isotope such as 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 78 Br, 82 Rb, 86 Y, 90 Y, 22 Na, 26 Al, 40 K, 83 Sr, 89 Zr, or 124 I.
  • the detectable label is 64 Cu.
  • Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede
  • fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrapel, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.
  • Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, ⁇ -glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, glucose oxidase (GO), and the like.
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • GAL beta-galactosidase
  • glucose-6-phosphate dehydrogenase beta-N-acetylglucosaminidase
  • ⁇ -glucuronidase invertase
  • Xanthine Oxidase firefly luciferase
  • glucose oxidase GO
  • binding of the APP to the T cell is detected using a detectably labeled antibody specific for the APP.
  • An antibody specific for the APP can comprise a detectable label such as a radioisotope, a fluorescent polypeptide, or an enzyme that generates a fluorescent product, or an enzyme that generates a colored product.
  • the T cell being detected is present in a sample comprising a plurality of T cells.
  • a T cell being detected can be present in a sample comprising from 10 to 10 9 T cells, e.g., from 10 to 10 2 , from 10 2 to 10 4 , from 10 4 to 10 6 , from 10 6 to 10 7 , from 10 7 to 10 8 , or from 10 8 to 10 9 , or more than 10 9 , T cells.
  • the present disclosure provides treatment methods, the methods comprising administering to the individual an amount of a TMMP of the present disclosure, or one or more nucleic acids or expression vectors encoding the TMMP effective to selectively modulate the activity of an epitope-specific T cell in an individual and to treat the individual.
  • a treatment method of the present disclosure comprises administering to an individual in need thereof one or more recombinant expression vectors comprising nucleotide sequences encoding a TMMP of the present disclosure.
  • a treatment method of the present disclosure comprises administering to an individual in need thereof one or more mRNA molecules comprising nucleotide sequences encoding a TMMP of the present disclosure.
  • a treatment method of the present disclosure comprises administering to an individual in need thereof a TMMP of the present disclosure.
  • a TMMP of the present disclosure is useful for treating type 1 diabetes (T1D).
  • the present disclosure provides a method of selectively modulating the activity of an epitope-specific T cell in an individual, the method comprising administering to the individual an effective amount of a TMMP of the present disclosure, or one or more nucleic acids (e.g., expression vectors; mRNA; etc.) comprising nucleotide sequences encoding the TMMP, where the TMMP selectively modulates the activity of the epitope-specific T cell in the individual.
  • Selectively modulating the activity of an epitope-specific T cell can treat a disease or disorder in the individual.
  • the present disclosure provides a treatment method comprising administering to an individual in need thereof an effective amount of a TMMP of the present disclosure.
  • a treatment method of the present disclosure, comprising administering an effective amount of a TMMP of the present disclosure is suitable for treating T1D.
  • the immunomodulatory polypeptide is an inhibitory polypeptide, and a T TMMP of the present disclosure inhibits activity of the epitope-specific T cell.
  • the epitope is a self-epitope, and a TMMP of the present disclosure selectively inhibits the activity of a T cell specific for the self-epitope.
  • the present disclosure provides a method of treating T1D in an individual, the method comprising administering to the individual an effective amount of a TMMP of the present disclosure, or one or more nucleic acids comprising nucleotide sequences encoding the TMMP, where the TMMP comprises a T1D peptide (as described above), and where the TMMP comprises an inhibitory immunomodulatory polypeptide.
  • an “effective amount” of a TMMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of self-reactive (i.e., reactive with a T1D-associated antigen) CD4 + and/or CD8 + T cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to number of self-reactive T cells in the individual before administration of the TMMP, or in the absence of administration with the TMMP.
  • self-reactive i.e., reactive with a T1D-associated antigen
  • an “effective amount” of a TMMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces production of Th2 cytokines in the individual. In some cases, an “effective amount” of a TMMP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, ameliorates one or more symptoms associated with T1D in the individual.
  • the TMMP reduces the number of CD4 + self-reactive T cells (i.e., the number of CD4 + T cells reactive with a T1D-associated antigen), which in turn leads to a reduction in CD8 + self-reactive T cells. In some instances, the TMMP increases the number of CD4 + Tregs, which in turn reduces the number of CD4 + self-reactive T cells and/or CD8 + T self-reactive T cells.
  • a TMMP of the present disclosure is administered to an individual in need thereof, as the polypeptide per se.
  • one or more nucleic acids comprising nucleotide sequences encoding a TMMP is/are administering to an individual in need thereof.
  • one or more nucleic acids of the present disclosure e.g., one or more recombinant expression vectors of the present disclosure, is/are administered to an individual in need thereof.
  • the present disclosure also provides treatment methods, the methods comprising administering to the individual an amount of an APP of the present disclosure, or one or more nucleic acids or expression vectors encoding the APP effective to selectively engage with an epitope-specific T cell in an individual in order to treat the individual, e.g., by depleting epitope-specific T cells when the Ig Fc polypeptide is one that that induces cell lysis through activation of CDC, and/or elicits ADCC.
  • a treatment method of the present disclosure comprises administering to an individual in need thereof one or more recombinant expression vectors comprising nucleotide sequences encoding an APP of the present disclosure.
  • a treatment method of the present disclosure comprises administering to an individual in need thereof one or more mRNA molecules comprising nucleotide sequences encoding an APP of the present disclosure. In some cases, a treatment method of the present disclosure comprises administering to an individual in need thereof an APP of the present disclosure.
  • An APP of the present disclosure is useful for treating type 1 diabetes (T1D) by selectively engaging with an epitope-specific T cell in an individual, including by, e.g., depleting epitope-specific T cells when the Ig Fc polypeptide is one that that induces cell lysis through activation of CDC, and/or elicits ADCC.
  • the present disclosure provides a method of treating T1D in an individual, the method comprising administering to the individual an effective amount of an APP of the present disclosure, or one or more nucleic acids comprising nucleotide sequences encoding the APP, where the APP comprises a T1D peptide (as described above).
  • an “effective amount” of an APP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces the number of self-reactive (i.e., reactive with a T1D-associated antigen) CD4 + and/or CD8 + T cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, compared to number of self-reactive T cells in the individual before administration of the APP, or in the absence of administration with the APP.
  • self-reactive i.e., reactive with a T1D-associated antigen
  • an “effective amount” of an APP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, reduces production of Th2 cytokines in the individual. In some cases, an “effective amount” of an APP of the present disclosure is an amount that, when administered in one or more doses to an individual in need thereof, ameliorates one or more symptoms associated with T1D in the individual. In some instances, the APP reduces the number of CD4 + self-reactive T cells (i.e., the number of CD4 + T cells reactive with a T1D-associated antigen), which in turn leads to a reduction in CD8 + self-reactive T cells.
  • the APP increases the number of CD4 + Tregs, which in turn reduces the number of CD4 + self-reactive T cells and/or CD8 + T self-reactive T cells.
  • an APP of the present disclosure is administered to an individual in need thereof, as the polypeptide per se.
  • one or more nucleic acids comprising nucleotide sequences encoding an APP is/are administering to an individual in need thereof.
  • one or more nucleic acids of the present disclosure e.g., one or more recombinant expression vectors of the present disclosure, is/are administered to an individual in need thereof.
  • a suitable formulation comprises: a) a TMMP or APP of the present disclosure; and b) a pharmaceutically acceptable excipient.
  • a suitable formulation comprises: a) a nucleic acid comprising a nucleotide sequence encoding a TMMP or APP of the present disclosure; and b) a pharmaceutically acceptable excipient; in some instances, the nucleic acid is an mRNA.
  • a suitable formulation comprises: a) a first nucleic acid comprising a nucleotide sequence encoding the first polypeptide of a TMMP or APP of the present disclosure; b) a second nucleic acid comprising a nucleotide sequence encoding the second polypeptide of a TMMP or APP of the present disclosure; and c) a pharmaceutically acceptable excipient.
  • a suitable formulation comprises: a) a recombinant expression vector comprising a nucleotide sequence encoding a TMMP or APP of the present disclosure; and b) a pharmaceutically acceptable excipient.
  • a suitable formulation comprises: a) a first recombinant expression vector comprising a nucleotide sequence encoding the first polypeptide of a TMMP or APP of the present disclosure; b) a second recombinant expression vector comprising a nucleotide sequence encoding the second polypeptide of a TMMP or APP of the present disclosure; and c) a pharmaceutically acceptable excipient.
  • Suitable pharmaceutically acceptable excipients are described above.
  • a suitable dosage can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular polypeptide or nucleic acid to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently.
  • a TMMP or APP (whether as a single heterodimer or, as described above, as a homodimer comprising two heterodimers linked together) of the present disclosure may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g.
  • Amounts thus include from about 0.1 mg/kg body weight to about 0.5 mg/kg body weight, from about 0.5 mg/kg body weight to about 1 mg/kg body weight, from about 1.0 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg/kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight to about 40 mg/kg body weight, or from about 40 mg/kg body weight to about 50 mg/kg body weight. If the regimen is a continuous infusion, it can also be in the range of 1 ⁇ g to 10 mg per kilogram of body weight per minute.
  • TMMP or APP of the present disclosure is administered in maintenance doses, ranging from those recited above, i.e., 0.1 mg/kg body weight to about 0.5 mg/kg body weight, from about 0.5 mg/kg body weight to about 1 mg/kg body weight, from about 1.0 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg/kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg
  • dose levels can vary as a function of the specific TMMP or APP, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
  • multiple doses of a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure are administered.
  • the frequency of administration of a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure can vary depending on any of a variety of factors, e.g., severity of the symptoms, patient response, etc.
  • a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure is administered once per month, twice per month, three times per month, every other week (qow), one every three weeks, once every four weeks, once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).
  • the duration of administration of a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure can vary, depending on any of a variety of factors, e.g., patient response, etc.
  • a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure can be administered over a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years, or more, including continued administration for the patient's life.
  • a TMMP or APP of the present disclosure is administered in maintenance doses, ranging from those recited above, i.e., 0.1 mg/kg body weight to about 0.5 mg/kg body weight, from about 0.5 mg/kg body weight to about 1 mg/kg body weight, from about 1.0 mg/kg body weight to about 5 mg/kg body weight, from about 5 mg/kg body weight to about 10 mg/kg body weight, from about 10 mg/kg body weight to about 15 mg/kg body weight, from about 15 mg/kg body weight to about 20 mg/kg body weight, from about 20 mg/kg body weight to about 25 mg/kg body weight, from about 25 mg/kg body weight to about 30 mg/kg body weight, from about 30 mg/kg body weight to about 35 mg/kg body weight, from about 35 mg/kg body weight to about 40 mg/kg body weight, or from about 40 mg/kg body weight, or from about 40 mg/kg body weight, or from about 40 mg/kg body weight, or from about 40 mg/kg body weight, or from about 40 mg/kg body
  • An active agent (a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure) is administered to an individual using any available method and route suitable for drug delivery, including in vivo and in vitro methods, as well as systemic and localized routes of administration.
  • a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes.
  • routes of administration contemplated for use in a method of the present disclosure include, but are not necessarily limited to, enteral, parenteral, and inhalational routes.
  • routes of administration include intramuscular, intratracheal, intralymphatic, subcutaneous, intradermal, topical application, intravenous, intraarterial, rectal, nasal, oral, and other enteral and parenteral routes of administration. Of these, intravenous, intramuscular and subcutaneous may be more commonly employed. Routes of administration may be combined, if desired, or adjusted depending upon the TMMP or APP and/or the desired effect.
  • a TMMP or APP of the present disclosure, or a nucleic acid or recombinant expression vector of the present disclosure can be administered in a single dose or in multiple doses.
  • a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure is administered intravenously. In some cases, a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure is administered intramuscularly. In some cases, a TMMP or APP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure is administered intralymphatically. In some cases, a TMMP of the present disclosure, a nucleic acid of the present disclosure, or a recombinant expression vector of the present disclosure is administered subcutaneously.
  • Subjects suitable for treatment with a method of the present disclosure include individuals who have T1D, including individuals who have been diagnosed as having T1D, and individuals who have been treated for T1D but who failed to respond to the treatment. Suitable subjects also may include individuals who have been diagnosed as being likely to develop T1D or who have symptoms indicating the imminent onset of T1D.
  • the present disclosure provides a method of delivering an immunomodulatory polypeptide (such as a wild-type (wt) or variant of immunomodulatory polypeptide described herein, e.g., PD-L1, IL-2, TGF ⁇ , or FasL) to a selected T cell or a selected T cell population, e.g., in a manner such that a TCR specific for a given T1D epitope is targeted.
  • an immunomodulatory polypeptide such as a wild-type (wt) or variant of immunomodulatory polypeptide described herein, e.g., PD-L1, IL-2, TGF ⁇ , or FasL
  • the present disclosure provides a method of delivering a wt or variant immunomodulatory polypeptide disclosed herein selectively to a target T cell bearing a TCR specific for the epitope present in a TMMP of the present disclosure.
  • the term “selective delivery” means that the immunomodulatory polypeptide is not delivered to all T cells, but rather that the majority of T cells to which the immunomodulatory polypeptide is delivered are T cells specific for the T1D peptide, thereby minimizing the modulation of non-target T cells.
  • the non-target T cells bind the TMMP and, as a result, the immunomodulatory polypeptide is not delivered to the non-target T cells that do not bind the TMMP.
  • the method comprises contacting a population of T cells, in vivo or in vitro, with a TMMP of the present disclosure.
  • the population of T cells can be a mixed population that comprises: i) the target T cell; and ii) non-target T cells that are not specific for the epitope (e.g., T cells that are specific for an epitope(s) other than the epitope to which the epitope-specific T cell binds).
  • the epitope-specific T cell is specific for the T1D peptide epitope present in the TMMP binds to the TMMP.
  • Contacting the population of T cells with the TMMP thus delivers the immunomodulatory polypeptide present in the TMMP selectively to the T cell(s) that are specific for the epitope present in the TMMP.
  • the non-target T cells bind the TMMP and, as a result, the immunomodulatory polypeptide is not delivered to the non-target T cells that are not bound to the TMMP.
  • the population of T cells is in vivo.
  • the population of T cells is in vitro, and a biological response (e.g., T cell activation and/or expansion and/or phenotypic differentiation) of the target T cell population to the TMMP of the present disclosure is elicited in the context of an in vitro culture.
  • a mixed population of T cells can be obtained from an individual, and can be contacted with the TMMP in vitro. Such contacting can comprise single or multiple exposures of the population of T cells to a defined dose(s) and/or exposure schedule(s).
  • a mixed population of T cells can be peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • PBMC from a patient can be obtained by standard blood drawing and PBMC enrichment techniques before being exposed to 0.1-1000 nM of a TMMP of the present disclosure under standard lymphocyte culture conditions.
  • the abundance of target T cells in the in vitro culture can be monitored by specific peptide-MHC multimers and/or phenotypic markers and/or functional activity (e.g. cytokine ELISpot assays).
  • specific peptide-MHC multimers and/or phenotypic markers and/or functional activity e.g. cytokine ELISpot assays.
  • all or a portion of the population of activated and/or expanded target T cells is administered to the individual (the individual from whom the mixed population of T cells was obtained).
  • the population of T cells is in vitro.
  • a mixed population of T cells is obtained from an individual, and is contacted with a TMMP of the present disclosure in vitro.
  • Such contacting which can comprise single or multiple exposures of the T cells to a defined dose(s) and/or exposure schedule(s) in the context of in vitro cell culture, can be used to determine whether the mixed population of T cells includes T cells that are specific for the epitope presented by the TMMP.
  • the presence of T cells that are specific for the T1D peptide of the TMMP can be determined by assaying a sample comprising a mixed population of T cells, which population of T cells comprises T cells that are not specific for the epitope (non-target T cells) and may comprise T cells that are specific for the epitope (target T cells).
  • Known assays can be used to detect activation and/or proliferation of the target T cells, thereby providing an in vitro assay that can determine whether a particular TMMP possesses an epitope that binds to T cells present in the individual and thus whether the TMMP has potential use as a therapeutic composition for that individual.
  • Suitable known assays for detection of activation and/or proliferation of target T cells include, e.g., flow cytometric characterization of T cell phenotype and/or antigen specificity and/or proliferation.
  • Such an assay to detect the presence of epitope-specific T cells e.g., a companion diagnostic, can further include additional assays (e.g. effector cytokine ELISpot assays) and/or appropriate controls (e.g. antigen-specific and antigen-nonspecific multimeric peptide-HLA staining reagents) to determine whether the TMMP is selectively binding/activating and/or expanding the target T cell.
  • the present disclosure provides a method of detecting, in a mixed population of T cells obtained from an individual, the presence of a target T cell that binds an epitope of interest, the method comprising: a) contacting in vitro the mixed population of T cells with a TMMP of the present disclosure, wherein the multimeric polypeptide comprises the epitope of interest; and b) detecting activation and/or proliferation of T cells in response to said contacting, wherein activated and/or proliferated T cells indicates the presence of the target T cell.
  • all or a portion of the population of T cells comprising the activated/expanded T cells can be administered back to the individual as a therapy.
  • the population of T cells is in vivo in an individual.
  • a method of the present disclosure for selectively delivering an immunomodulatory polypeptide as described herein to an epitope-specific T cell comprises administering the TMMP to the individual.
  • the epitope-specific T cell to which an immunomodulatory polypeptide is being selectively delivered is also referred to herein as a “target T cell.”
  • the target T cell is a regulatory T cell (Treg).
  • the Treg inhibits or suppresses activity of an autoreactive T cell.
  • the present disclosure provides a method of delivering an inhibitory immunomodulatory polypeptide (such as wt or variant of PD-L1 or FasL) to a selected T cell or a selected T cell population, e.g., in a manner such that a TCR specific for a given epitope is targeted.
  • the present disclosure provides a method of delivering such an inhibitory immunomodulatory polypeptide selectively to a target T cell bearing a TCR specific for the epitope present in a TMMP of the present disclosure.
  • the method comprises contacting a population of T cells with a TMMP of the present disclosure.
  • the population of T cells can be a mixed population that comprises: i) the target T cell; and ii) non-target T cells that are not specific for the epitope (e.g., T cells that are specific for an epitope(s) other than the epitope to which the epitope-specific T cell binds).
  • the epitope-specific T cell is specific for the epitope-presenting peptide present in the TMMP, and binds to the TMMP. Contacting the population of T cells with the TMMP thus delivers the inhibitory immunomodulatory polypeptide present in the TMMP selectively to the T cell(s) that are specific for the T1D peptide present in the TMMP.
  • a TMMP of the present disclosure is contacted with a population of T cells comprising: i) a target T cell(s) that is specific for the epitope present in the TMMP; and ii) a non-target T cell(s), e.g., a T cell(s) that is specific for a second epitope(s) that is not the epitope present in the TMMP.
  • a target T cell(s) that is specific for the epitope present in the TMMP
  • a non-target T cell(s) e.g., a T cell(s) that is specific for a second epitope(s) that is not the epitope present in the TMMP.
  • Contacting the population results in selective delivery of an inhibitory immunomodulatory polypeptide(s) an inhibitory immunomodulatory polypeptide (such as wt or variant of PD-L1 or FasL) which is present in the TMMP, to the target T cell.
  • the non-target T cells bind the TMMP and, as a result, the immunomodulatory polypeptide is not delivered to the non-target T cells.
  • the population of T cells is in vitro.
  • the population of T cells is in vitro, and a biological response (e.g., T cell activation and/or expansion and/or phenotypic differentiation) of the target T cell population to the TMMP of the present disclosure is elicited in the context of an in vitro culture.
  • a mixed population of T cells can be obtained from an individual, and can be contacted with the TMMP in vitro. Such contacting can comprise single or multiple exposures of the population of T cells to a defined dose(s) and/or exposure schedule(s).
  • contacting results in selectively binding/activating and/or expanding target T cells within the population of T cells, and results in generation of a population of activated and/or expanded target T cells.
  • a mixed population of T cells can be peripheral blood mononuclear cells (PBMC).
  • PBMC from a patient can be obtained by standard blood drawing and PBMC enrichment techniques before being exposed to 0.1-1000 nM of a TMMP of the present disclosure under standard lymphocyte culture conditions.
  • the abundance of target T cells in the in vitro culture can be monitored by specific peptide-MHC multimers and/or phenotypic markers and/or functional activity (e.g. cytokine ELISpot assays).
  • specific peptide-MHC multimers and/or phenotypic markers and/or functional activity e.g. cytokine ELISpot assays.
  • all or a portion of the population of activated and/or expanded target T cells is administered to the individual (the individual from whom the mixed population of T cells was obtained).
  • the population of T cells is in vitro.
  • a mixed population of T cells is obtained from an individual, and is contacted with a TMMP of the present disclosure in vitro.
  • Such contacting which can comprise single or multiple exposures of the T cells to a defined dose(s) and/or exposure schedule(s) in the context of in vitro cell culture, can be used to determine whether the mixed population of T cells includes T cells that are specific for the epitope presented by the TMMP.
  • the presence of T cells that are specific for the T1D peptide epitope of the TMMP can be determined by assaying a sample comprising a mixed population of T cells, which population of T cells comprises T cells that are not specific for the epitope (non-target T cells) and may comprise T cells that are specific for the epitope (target T cells).
  • Known assays can be used to detect activation and/or proliferation of the target T cells, thereby providing an in vitro assay that can determine whether a particular TMMP possesses an epitope that binds to T cells present in the individual and thus whether the TMMP has potential use as a therapeutic composition for that individual.
  • Suitable known assays for detection of activation and/or proliferation of target T cells include, e.g., flow cytometric characterization of T cell phenotype and/or antigen specificity and/or proliferation.
  • Such an assay to detect the presence of epitope-specific T cells e.g., a companion diagnostic, can further include additional assays (e.g. effector cytokine ELISpot assays) and/or appropriate controls (e.g. antigen-specific and antigen-nonspecific multimeric peptide-HLA staining reagents) to determine whether the TMMP is selectively binding/activating and/or expanding the target T cell.
  • the present disclosure provides a method of detecting, in a mixed population of T cells obtained from an individual, the presence of a target T cell that binds an epitope of interest, the method comprising: a) contacting in vitro the mixed population of T cells with a TMMP of the present disclosure, wherein the multimeric polypeptide comprises the epitope of interest; and b) detecting activation and/or proliferation of T cells in response to said contacting, wherein activated and/or proliferated T cells indicates the presence of the target T cell.
  • all or a portion of the population of T cells comprising the activated/expanded T cells can be administered back to the individual as a therapy.
  • the population of T cells is in vivo in an individual.
  • a method of the present disclosure for selectively delivering an inhibitory immunomodulatory polypeptide (such as wt or variant of PD-L1 or FasL) to an epitope-specific T cell comprises administering the TMMP to the individual.
  • the epitope-specific T cell to which an inhibitory immunomodulatory polypeptide is being selectively delivered is also referred to herein as a “target T cell.”
  • the target T cell is a regulatory T cell (Treg).
  • the Treg inhibits or suppresses activity of an autoreactive T cell.
  • the target T cell is a CD4 + T cell.
  • the target T cell is a CD4 + T cell that is specific for an autoantigen.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • body disulfide set of constructs (“body disulfide” set) was made that included two heterodimers, each heterodimer including: a) a first polypeptide comprising: i) a peptide epitope (e.g., a proinsulin peptide or a GAD65 peptide); and ii) an hDRB1*0401 ⁇ chain polypeptide comprising an amino acid substitution providing a Cys (a “first Cys”); and b) a second polypeptide comprising: i) an hDRA1*0101 ⁇ chain polypeptide comprising an amino acid substitution providing a Cys (a “second Cys”); and ii) an Ig Fc polypeptide, where the first and second polypeptides are covalently linked to one another via a disulfide between the first Cys and the second Cys.
  • a first polypeptide comprising: i) a peptide epitope (e.g., a proinsulin
  • a second set of constructs (“linker disulfide set”) was made that included two heterodimers, each heterodimer including: a) a first polypeptide comprising: i) a peptide epitope (e.g., a proinsulin peptide or a GAD65 peptide); ii) a peptide linker comprising a Cys; and iii) an hDRB1*0401 ⁇ chain polypeptide; and b) a second polypeptide comprising: i) an hDRA1*0101 ⁇ chain polypeptide comprising an amino acid substitution providing a Cys; and ii) an Ig Fc polypeptide, where the first and second polypeptides are covalently linked to one another via a disulfide between the Cys in the peptide linker and the Cys provided by the amino acid substitution in the ⁇ chain.
  • a first polypeptide comprising: i) a peptide epitope (e.g.
  • the ability of the first and the second polypeptides to form disulfide-linked heterodimers was tested by running the constructs on sodium dodecyl sulfate (SDS) polyacrylamide gels (PAGE) containing dithiothreitol (reducing) or not containing dithiothreitol (non-reducing).
  • SDS sodium dodecyl sulfate
  • PAGE polyacrylamide gels
  • FIG. 17 A depicts various “body disulfide” and “linker disulfide” constructs, the position of the Cys residues forming a disulfide bond between the two polypeptides of a heterodimer, the yield from a Protein A column, and the percent monomer. Amino acid sequences of the various polypeptide chains of the constructs are depicted in FIG. 15 .
  • FIG. 17 B depicts expression level from the Protein A column.
  • FIG. 17 C shows reducing (lower panels) and non-reducing (upper panels) SDS-PAGE results for the various proteins.
  • FIG. 18 A- 18 B depict various “body disulfide” constructs and features of the two polypeptides of the heterodimers. Amino acid sequences of the various polypeptide chains of the constructs are depicted in FIG. 15 . The constructs were analyzed on reducing and non-reducing SDS-PAGE. The results are shown in FIG. 18 C .
  • TMMPs were generated that comprise: a) PD-L1 as the MOD, at various positions; b) GAD65 (555-567) as the T1D peptide; and c) MHC class II DRA ⁇ chain and DRB f chains. The two polypeptide chains of the heterodimers were not covalently linked. Amino acid sequences of the polypeptide chains of the constructs are depicted in FIG. 15 .
  • FIG. 19 A shows expression levels of the various constructs.
  • FIG. 19 B depicts reducing and non-reducing SDS-PAGE results.
  • FIG. 19 C depicts SEC data used to determine the % monomer.
  • FIG. 19 D shows expression levels determined by either Protein A column yields or SEC data.
  • FIG. 20 A- 20 B Features of the constructs are shown in FIG. 20 A- 20 B .
  • Production levels of the constructs are shown in FIG. 20 C .
  • Results of non-reducing and reducing SDS-PAGE analysis of the constructs are shown in FIG. 20 D .
  • TMMP 3005-2639 The in vitro stability of TMMP 3005-2639 was tested.
  • the % monomer was determined.
  • FIG. 21 A- 21 E show the % monomer at 9.5 mg/mL ( FIG. 21 A ), 1 mg/mL ( FIG. 21 B ), and 0.1 mg/mL ( FIG. 21 C ).
  • FIG. 30 D shows % monomer recovery at the various concentrations and temperatures.
  • the % monomer recovery data from FIG. 21 D are summarized in FIG. 21 E .
  • the data show that increased aggregation upon thermal stress is proportional to increased concentration and temperature.
  • the in vitro stability of disulfide-stabilized TMMP was tested.
  • the disulfide-stabilized TMMP was 3732-2639 ( FIG. 15 NN and FIG. 15 C ).
  • Control TMMPs in which the first and second polypeptides were not disulfide bonded, were also tested.
  • the control TMMPs were: i) 2792-2639 ( FIG. 1500 and FIG. 15 C ); and ii) 3005-2639 ( FIG. 15 D and FIG. 15 C ).
  • FIG. 22 A and FIG. 22 B The data are shown in FIG. 22 A and FIG. 22 B .
  • FIG. 22 A depicts the % monomer remaining after 5 days at 37° C.
  • FIG. 22 B depict the % monomer after 5 days at 4° C., 25° C., and 37° C.
  • the data indicate that disulfide linkage between the first polypeptide and the second polypeptide of a TMMP increases stability.
  • TMMPs in which the MOD (PD-L1) is at Position 1, and in which the disulfide bond was between i) a Cys in the linker between the proIns epitope (proIns 76-90 (K88S)) and the MHC class II chain; and ii) a Cys in the MHC class II ⁇ chain, was tested.
  • the TMMPs were: i) 3892-2640 ( FIG. 15 PP and FIG. 15 JJ ); ii) 3893-2938 ( FIG. 15 B and FIG. 15 A ); and iii) 3893-2640 ( FIG. 15 B and FIG. 15 JJ ).
  • a control TMMP, 3005-2639 FIG.
  • Expression levels of various TMMPs are shown in the table presented in FIG. 24 . It is noted that expression levels can vary with the conditions and scale employed. For example, while further expression results in a larger scale showed that 3003-2639 remained low, expression of 3005-2639 was able to achieve 30-40 mg/L expression titer.
  • TMMP 3005-2639 comprises two heterodimers; each heterodimer comprises: a) a 3005 polypeptide ( FIG. 15 D ); and b) a 2639 polypeptide ( FIG. 15 C ), where the 3005 polypeptide and the 2639 polypeptide are non-covalently associated.
  • TMMP 3892-2938 comprises two heterodimers; each heterodimer comprises: a) a 3893 polypeptide ( FIG. 15 B ); and b) a 2938 polypeptide ( FIG. 15 A ), where the 3893 polypeptide and the 2938 polypeptide are covalently linked via a disulfide bond.
  • PBMCs Peripheral blood mononuclear cells from multiple healthy or T1D patients were expanded with proinsulin (PI) 76-90K88S peptide and recombinant human IL-2 (rhIL-2) in RPMI+10% human serum for 14 days. Expanded PBMCs were washed and incubated (2e6 cells/well, 24 well plate) with increasing concentrations of indicated TMMPs overnight at 37° C., 5% CO 2 . The TMMP-treated cells were washed, and functional response of antigen-specific cells was assessed using an IFN ⁇ ELISPOT upon re-stimulation with PI 76-90K88S peptide. Additionally, the TMMP-treated, washed cells were cultured in presence of autologous PBMCs (1:1) and rhIL-2 to assess duration of suppressive effect. IFN ⁇ ELISPOT was performed at the indicated time points.
  • FIG. 25 and FIG. 26 The results are shown in FIG. 25 and FIG. 26 .
  • functional response of Proinsulin-specific CD4 + T cells were strongly suppressed by overnight treatment with 3005-2639 and 3893-2938 as compared to control molecules and remained suppressed 3-5 days post-treatment.
  • FIG. 26 the antigen-specific, PD-L1-dependent suppression of PI-specific CD4 + T cells was observed across multiple donors.
  • the ability of GAD65-PDL1 Pos3 (3005-2580) to suppress GAD65-specific CD4 cells was assessed in an overnight suppression assay.
  • the TMMP comprises two heterodimers; each heterodimer comprises: a) a 3005 polypeptide ( FIG. 15 D ); and b) a 2580 polypeptide ( FIG. 15 G ).
  • PBMCs from a T1D patient were expanded with GAD65 555-567F557I peptide and recombinant human IL-2 in RPMI+10% human serum for 14 days. Expanded PBMCs were washed and incubated (2e6 cells/well, 24 well plate) with increasing concentrations of indicated TMMPs overnight at 37° C., 5% CO 2 .
  • the TMMP-treated cells were washed, and functional response of antigen-specific cells was assessed using an IFN ⁇ ELISPOT upon re-stimulation with GAD65 555-567F557I peptide. Additionally, the TMMP-treated, washed cells were cultured in presence of autologous PBMCs (1:1) and rhIL-2 to assess duration of suppressive effect. IFN ⁇ ELISPOT was performed at the indicated time points.
  • Proins-PDL1 Pos3 3005-2639
  • Proins-PDL1 Pos 1 3893-2938
  • PBMCs from multiple healthy or T1D patients were expanded with PI 76-90K88S peptide and recombinant human IL-2 in RPMI+10% human serum for 14 days.
  • CD4 cells were purified by negative selection from expanded PBMCs and stimulated with PI 76-90K88S peptide loaded autologous dendritic cells (DCs) in presence of increasing concentrations of indicated TMMPs (DCs were prepared as follows autologous monocytes were isolated by negative selection and matured with IL4+ granulocyte-monocyte colony stimulating factor (GMCSF) for 24 hrs, followed by activation with TNF ⁇ , IL1 ⁇ , IL6, and PGE 2 for 24 hrs). After 5 days of culture, TMMP-treated cells were washed, and functional response of antigen-specific cells was assessed using an IFN ⁇ ELISPOT upon re-stimulation with PI 76-90K88S peptide. Additionally, the TMMP-treated, washed cells were cultured in presence of autologous PBMCs (1:1) and rhIL-2 to assess duration of suppressive effect. IFN ⁇ ELISPOT was performed at the indicated time points.
  • TMMP 3893-2938 A pharmacokinetic analysis of TMMP 3893-2938 was conducted. The effect of TMMP 3893-2938 on proinsulin-specific responses in vivo was assessed.
  • TMMP 3005-2639 ( FIGS. 15 C- 15 D ) and TMMP 3893-2938 ( FIG. 15 A- 15 B ); depicted schematically in FIG. 29 ) were assessed in female albino C57Bl/6 mice.
  • TMMPs were administered intravenously at 5 mg/kg, and serum samples were collected for up to 72 hours post-dose. The concentration of each TMMP in serum was determined relative to a standard curve using a ligand binding assay, in which each TMMP was captured by an anti-PDL1 antibody, and then detected using a sulfotagged anti-DR4 antibody.
  • the data are shown in FIG. 29 .
  • the data indicate that the pharmacokinetics of TMMP 3893-2938 are similar to those of TMMP 3005-2639.
  • mice The in vivo activity of Proins-DR4-PDL1 TMMP 3893-2938 was tested in human HLA-DRB1*04 transgenic mice.
  • the study design is depicted in FIG. 30 .
  • Mice were simultaneously immunized with Proins (PI; 76-90, K88S) and influenza HA (307-319) peptides in Complete Freund's Adjuvant (CFA) on Day 0.
  • CFA Complete Freund's Adjuvant
  • blood samples were drawn from mice, PBMCs purified, and seeded in an IL-2 ELISpot assay.
  • PBMCs were stimulated with PMA/ionomycin, an irrelevant HIV peptide, Proins (PI; 76-90, K88S), or HA (307-319) peptides.
  • the data are shown in FIG. 31 .
  • the frequencies of Proins- and HA-reactive T cells are shown for each animal. All animals demonstrated successful immunization with the relevant immunizing peptides. While variable per group, each animal exhibited similar functional responses to both Proins and HA peptide stimulation 11 days post-immunization.
  • mice were treated intravenously with (1) vehicle, or (2) 20 mg/kg of Proins-DR4-PDL1 IST-3005-2639, or (3) 20 mg/kg of Proins-DR4-PDL1 IST-3893-2938, or (4) 2 mg/kg of Proins-DR4-PDL1 IST-3893-2938, or (5) 0.2 mg/kg of Proins-DR4-PDL1 IST-3893-2938, or (6) 20 mg/kg of Proins-DR4-PDL1 IST-3893-2938 after being immunized with PI peptide alone.
  • blood samples were drawn from mice, PBMCs purified, and seeded in an IL-2 ELISpot assay. PBMCs were stimulated as above.
  • the data are shown in FIG. 32 .
  • the frequencies of Proins- and HA-reactive T cells are shown for each animal.
  • a single treatment with Proins-DR4-PDL1 TMMP (TMMP-3005-2639 or TMMP-3893-2938) on Day 11 post-immunization resulted in a dose-dependent reduction in the frequency of Proins-reactive T cells on Day 12 post-immunization.
  • This suppression of Proins-reactive T cells was antigen specific, as HA-reactive T cells were not suppressed.
  • TMMP 3893-2938 (in which the two polypeptides of the heterodimer disulfide linked) demonstrated comparable suppression to TMMP 3005-2639 (in which the two polypeptides of the heterodimer are non-covalently associated). Comparable suppression of proinsulin-specific responses was observed, regardless whether animals were co-immunized against HA. Proins-specific responses were suppressed in a dose-dependent manner by TMMP 3893-2938.
  • the results depicted in FIG. 32 were quantified by another means.
  • the data are shown in FIG. 33 .
  • the percent relative change in frequency of PI and HA reactive cells post-treatment (Day 12) is shown relative to the pre-treatment (Day 11) sample from each individual mouse.
  • This result demonstrates that a 20 mg/kg dose of TMMP 3005-2639 or TMMP 3892-2938 results in reduced frequency of PI-reactive cells while the frequency of HA-reactive cells continues to increase, confirming the antigen-specificity of this treatment effect.
  • cytokine-producing T cells were further demonstrated using intracellular cytokine staining and flow cytometry.
  • Spleens were harvested from all animals in the above experiment on Day 12 post-immunization (post-treatment). Spleens were then dissociated into single cell suspensions and stimulated with Proins (PI; 76-90, K88S) or HA (307-319) peptides and cytokine production was analyzed by flow cytometry.
  • Splenic CD4+ T cells from immunized animals produced IL-2 and/or IFN ⁇ plus TNF ⁇ upon stimulation with Proins or HA peptides.
  • treatment with Proins-DR4-PDL1 TMMP 3005-2639 or TMMP 3893-2938 reduced the frequency of splenic CD4+ T cells producing cytokine in response to Proins stimulation.
  • the frequency of HA-reactive CD4+ T cells was not altered by treatment with Proins-DR4-PDL1 TMMP.
  • ProIns-Specific T Cells are CD4 + , CD44 hi , and PD-1 + .

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