WO2019051094A1 - ANTIGEN PRESENTATION POLYPEPTIDES AND METHODS OF USE - Google Patents

ANTIGEN PRESENTATION POLYPEPTIDES AND METHODS OF USE Download PDF

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Publication number
WO2019051094A1
WO2019051094A1 PCT/US2018/049760 US2018049760W WO2019051094A1 WO 2019051094 A1 WO2019051094 A1 WO 2019051094A1 US 2018049760 W US2018049760 W US 2018049760W WO 2019051094 A1 WO2019051094 A1 WO 2019051094A1
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Prior art keywords
polypeptide
mhc class
terminus
amino acid
immunomodulatory
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PCT/US2018/049760
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English (en)
French (fr)
Inventor
Ronald D. SEIDEL, III
Rodolfo J. CHAPARRO
John F. ROSS
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Cue Biopharma Inc
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Cue Biopharma Inc
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Priority to KR1020207009763A priority Critical patent/KR20200064083A/ko
Priority to CA3071881A priority patent/CA3071881A1/en
Priority to CN201880068747.7A priority patent/CN111278864A/zh
Priority to EP18854499.3A priority patent/EP3678677A4/en
Priority to JP2020513914A priority patent/JP2021500855A/ja
Application filed by Cue Biopharma Inc filed Critical Cue Biopharma Inc
Priority to AU2018328283A priority patent/AU2018328283A1/en
Publication of WO2019051094A1 publication Critical patent/WO2019051094A1/en
Priority to IL272332A priority patent/IL272332A/en
Priority to US16/789,057 priority patent/US20200172595A1/en
Anticipated expiration legal-status Critical
Priority to US18/103,809 priority patent/US20240034767A1/en
Priority to JP2024023312A priority patent/JP2024063050A/ja
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

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
  • Cell surface display of an MHC together with a peptide fragment also known as a T cell epitope, provides the underlying scaffold surveilled by T cells, allowing for specific recognition.
  • 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 human leukocyte antigens
  • HLA-DM HLA-DMA and HLA-DMB that encode HLA-DM a chain and HLA-DM ⁇ chain, respectively
  • HLA-DO HLA-DOA and HLA-DOB that encode HLA- DO a chain and HLA-DO ⁇ chain, respectively
  • HLA-DP HLA-DPA and HLA-DPB that encode HLA-DP a chain and HLA-DP ⁇ chain, respectively
  • HLA-DQ HLA-DQA and HLA- DQB that encode HLA-DQ a chain and HLA-DQ ⁇ chain, respectively
  • HLA-DR HLA- DRA and HLA-DRB that encode HLA-DR a chain and HLA-DR ⁇ chain, respectively).
  • the present disclosure provides antigen-presenting polypeptides, including single-chain antigen- presenting polypeptides and multimeric antigen-presenting polypeptides.
  • the present disclosure provides nucleic acids comprising nucleotide sequences encoding antigen-presenting polypeptides of the present disclosure, as well as cells genetically modified with the nucleic acids.
  • An antigen-presenting polypeptide of the present disclosure is useful for modulating activity of a T cell.
  • the present disclosure provides methods of modulating activity of a T cell.
  • FIG. 1 provides a schematic depiction of MHC Class II alpha- and beta-chains with a peptide.
  • FIG. 2A-2C provide schematic depictions of examples of antigen-presenting polypeptides
  • FIG. 3A-3B provide a schematic depiction of an example of an antigen-presenting polypeptide (FIG. 3A); and a crystal structure of the human Class II MHC protein HLA-DR1 complexed with an influenza virus peptide (FIG. 3B).
  • FIG. 4A-4C depict gel analysis (FIG. 4A), expression levels (FIG. 4B), and descriptions (FIG.
  • FIG. 5A-5B provide schematic depictions of APPs without immunomodulatory (MOD)
  • FIG. 5 A polypeptides
  • FIG. 5B The unmarked rectangle in FIG. 5A represents a dimerization domain (e.g., a bZIP polypeptide).
  • the arrows pointing to the dashed lines indicate possible positions of a MOD polypeptide(s).
  • FIG. 6 provides an amino acid sequence of an HLA Class II DRA a chain.
  • FIG. 7A-7J provide amino acid sequences of HLA Class II DRB1 ⁇ chains.
  • FIG. 8A-8C provide amino acid sequences of HLA Class II DRB3 ⁇ chains.
  • FIG. 9 provides an amino acid sequence of an HLA Class II DRB4 ⁇ chain.
  • FIG. 10 provides an amino acid sequence of an HLA Class II DRB5 ⁇ chain.
  • FIG. 11 provides an amino acid sequence of an HLA Class II DMA a chain.
  • FIG. 12 provides an amino acid sequence of an HLA Class II DMB ⁇ chain.
  • FIG. 13 provides an amino acid sequence of an HLA Class II DOA a chain.
  • FIG. 14 provides an amino acid sequence of an HLA Class II DOB ⁇ chain.
  • FIG. 15 provides an amino acid sequence of an HLA Class II DPA1 a chain.
  • FIG. 16 provides an amino acid sequence of an HLA Class II DPB1 ⁇ chain.
  • FIG. 17 provides an amino acid sequence of an HLA Class II DQA1 a chain.
  • FIG. 18 provides an amino acid sequence of an HLA Class II DQA2 a chain.
  • FIG. 19A-19B provide amino acid sequences of HLA Class II DQB 1 ⁇ chains.
  • FIG. 20A-20B provide amino acid sequence of HLA Class II DQB2 ⁇ chains.
  • FIG. 21A-21G provide amino acid sequences of immunoglobulin Fc polypeptides.
  • FIG. 22A-22L provide schematic depictions of exemplary multimeric T-cell modulatory
  • TMAPPs antigen-presenting polypeptides
  • FIG. 23A-23I provide schematic depictions of exemplary single -chain TMAPPs of the present disclosure.
  • FIG. 24 depicts production of exemplary APPs of the present disclosure.
  • FIG. 25A-25B provide the amino acid sequence (FIG. 25 A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 25B) encoding same.
  • FIG. 26A-26B provide the amino acid sequence (FIG. 26A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 26B) encoding same.
  • FIG. 27A-27B provide the amino acid sequence (FIG. 27 A) of an exemplary single-chain APP, and a nucleotide sequence (FIG. 27B) encoding same.
  • FIG. 28A-28B provide the amino acid sequence (FIG. 28A) of an exemplary single-chain
  • FIG. 29A-29B provide the amino acid sequence (FIG. 29 A) of an exemplary single-chain
  • FIG. 30A-30B provide the amino acid sequence (FIG. 30A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 30B) encoding same.
  • FIG. 31A-31B provide the amino acid sequence (FIG. 31 A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 3 IB) encoding same.
  • FIG. 32A-32B provide the amino acid sequence (FIG. 32A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 32B) encoding same.
  • FIG. 33A-33B provide the amino acid sequence (FIG. 33A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 33B) encoding same.
  • FIG. 34A-34B provide the amino acid sequence (FIG. 34A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 34B) encoding same.
  • FIG. 35A-35B provide the amino acid sequence (FIG. 35A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 35B) encoding same.
  • FIG. 36 provides a schematic depiction of an exemplary TMAPP of the present disclosure, and provides gel analysis of expression.
  • FIG. 37A and 37B provide the amino acid sequence (FIG. 37A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 37B) encoding same.
  • FIG. 38A and 38B provide the amino acid sequence (FIG. 38 A) of an exemplary polypeptide chain of a multimeric TMAPP, and a nucleotide sequence (FIG. 38B) encoding same.
  • FIG. 39 depicts production of an exemplary APP of the present disclosure.
  • 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 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. To determine sequence identity, 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. Bioi. 215:403-10.
  • 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 e.g. with reference to binding of a T-cell modulatory
  • 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.
  • Non- covalent binding interactions are generally characterized by a dissociation constant (KD) of less than 10 6 M, less than 10 7 M, less than 10 8 M, less than 10 9 M, less than 10 10 M, less than 10 11 M, less than 10 12 M, less than 10 13 M, less than 10 14 M, or less than 10 15 M.
  • KD dissociation constant
  • Binding refers to the strength of non-covalent binding, increased binding affinity being correlated with a lower KD-
  • Specific binding generally refers to binding with an affinity of at least about 10 7 M or greater, e.g. , 5x 10 7 M, 10 8 M, 5 x 10 8 M, 10 9 M, and greater.
  • Non-specific binding generally refers to binding (e.g., the binding of a ligand to a moiety other than its designated binding site or receptor) with an affinity of less than about 10 7 M (e.g. , binding with an affinity of 10 6 M, 10 s M, 10 4 M).
  • binding between a TCR and a peptide/MHC complex can be in the range of from 1 ⁇ to 100 ⁇ , or from 100 ⁇ to 1 mM.
  • Covalent binding or “covalent bond,” as used herein, refers to the formation of one or more covalent chemical binds between two different molecules.
  • immunological synapse or “immune synapse” as used herein generally refers to the natural interface between two interacting immune cells of an adaptive immune response including, e.g., the interface between an antigen-presenting cell (APC) or target cell and an effector cell, e.g., a lymphocyte, an effector T cell, a natural killer cell, and the like.
  • An immunological synapse between an APC and a T cell is generally initiated by the interaction of a T cell antigen receptor and major histocompatibility complex molecules, e.g., as described in Bromley et al., Annu Rev Immunol. 2001 ;19:375-96; the disclosure of which is incorporated herein by reference in its entirety.
  • 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, 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, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
  • a co- stimulatory polypeptide also encompasses, inter alia, an antibody that specifically binds with a cognate co-stimulatory molecule present on a T cell, such as, but not limited to, IL-2, CD27, CD28, 4- IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.
  • IL-2 IL-2
  • LFA-1 lymphocyte function-associated antigen- 1
  • an “immunomodulatory polypeptide” (also referred to herein as a "MOD”) specifically binds a cognate co-immunomodulatory polypeptide on a T cell.
  • An "immunomodulatory domain" ("MOD) of a TMAPP of the present disclosure binds a
  • cognate co-immunomodulatory polypeptide which may be present on a target T cell.
  • Heterologous means a nucleotide or polypeptide that is not found in the native nucleic acid or protein, respectively.
  • Recombinant means that a particular nucleic acid (DNA or RNA) is the
  • 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 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 (KD).
  • Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1,000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
  • Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.
  • nM nanomolar
  • pM picomolar
  • fM femtomolar
  • the term "avidity" 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.
  • Specific binding refers to binding with an affinity of at least about 10 7 M or greater, e.g., 5x 10 7 M, 10 s M, 5 x 10 s M, and greater.
  • Nonspecific binding refers to binding with an affinity of less than about 10 7 M, e.g., binding with an affinity of 10 6 M, 10 s M, 10 4 M, etc.
  • treatment used herein to 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; 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
  • the present disclosure provides antigen-presenting polypeptides, including single-chain antigen- presenting polypeptides and multimeric antigen-presenting polypeptides.
  • the present disclosure provides nucleic acids comprising nucleotide sequences encoding antigen-presenting polypeptides of the present disclosure, as well as cells genetically modified with the nucleic acids.
  • An antigen-presenting polypeptide of the present disclosure is useful for modulating activity of a T cell.
  • the present disclosure provides methods of modulating activity of a T cell.
  • An antigen-presenting polypeptide (APP) of the present disclosure can be a single-chain
  • a TMAPP of the present disclosure forms a higher order complex; for example, in some cases a TMAPP of the present disclosure forms a homodimer.
  • APP includes: a multimeric APP; a single -chain APP; a multimeric TMAPP; and a single-chain TMAPP. The term further includes higher-order complexes of an APP.
  • the present disclosure provides antigen-presenting polypeptides (APPs), including single -chain APPs and multimeric APPs.
  • APPs antigen-presenting polypeptides
  • Naturally occurring Class II MHC polypeptides comprise an a chain and a ⁇ chain.
  • Class II MHC polypeptides include human leukocyte antigen (HLA) a- and ⁇ -chains.
  • MHC Class II polypeptides include MCH Class II DP a and ⁇ polypeptides, DM a and ⁇ polypeptides, DOA a and ⁇ polypeptides, DOB a and ⁇ polypeptides, DQ a and ⁇ polypeptides, and DR a and ⁇ polypeptides.
  • a "Class II MHC polypeptide” can comprise a class II MHC a chain polypeptide, a class II MHC ⁇ chain polypeptide, or only a portion of a class II MHC a or ⁇ chain polypeptide.
  • a "Class II MHC polypeptide” can be a polypeptide that includes: i) only the al domain of a class II MHC a chain polypeptide; ii) only the a2 domain of a class II MHC a chain; iii) only the al domain and an a2 domain of a class II MHC a chain; iv) only the ⁇ domain of a class II MHC ⁇ chain; v) only the ⁇ 2 domain of a class II MHC ⁇ chain; vi) only the ⁇ domain and the ⁇ 2 domain of a class II MHC ⁇ chain; vii) the al domain of a class II MHC a chain, the ⁇ domain of a class II MHC ⁇ chain, and the ⁇ 2 domain of a class II MHC; and the like.
  • Class II MHC polypeptides include allelic forms.
  • the HLA locus is highly polymorphic in
  • DRB 7 alleles 10 DRB 8 alleles, 1 DRB9 alleles, 34 DQA1 alleles, 83 DQB 1 alleles, 23 DPA1,
  • Class II MHC polypeptide includes allelic forms of any known Class II MHC polypeptide.
  • an APP of the present disclosure comprises two polypeptide chains.
  • the two polypeptide chains are covalently linked to one another, e.g., via a disulfide bond.
  • the two polypeptide chains are not covalently linked to one another.
  • the two polypeptide chains are not covalently linked to one another; and in some of these cases, each of the two polypeptide chains comprises a member of a dimerization pair. Examples of multimeric APPs of the present disclosure are depicted schematically in FIG. 2A and FIG. 2B.
  • an antigen-presenting multimeric polypeptide (multimeric APP) of the present disclosure comprises: a) a first polypeptide comprising, in order from N- terminus to C-terminus: i) an MHC Class II al polypeptide; and ii) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a T-cell receptor (TCR); ii) an MHC Class II ⁇ polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide.
  • TCR T-cell receptor
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; and ii) an MHC Class II (x2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a T-cell receptor (TCR); ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; and ii) an MHC Class II (x2 polypeptide
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; and ii) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a T-cell receptor (TCR); ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) an immunoglobulin (Ig) Fc polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; and ii) an MHC Class II a2 polypeptide
  • a second polypeptide comprising, in order
  • the second polypeptide comprises a linker between the peptide antigen and the MHC Class II ⁇ polypeptide. In some cases, the second polypeptide comprises a linker between the MHC Class II ⁇ polypeptide and the immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • an antigen-presenting multimeric polypeptide (a multimeric APP) of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II a2 polypeptide; and iii) a first member of a dimerizer pair; and b) a second polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) a second member of the dimerizer pair.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC
  • the first and the second members of the dimerizer pair bind to one another non-covalently. In some cases, the first and the second members of the dimerizer pair bind to one another non-covalently without the need for a dimerization agent. In some cases, the first and the second members of the dimerizer pair bind to one another non-covalently in the presence of a dimerizer agent.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C- terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II a2 polypeptide; and iii) a first member of a dimerizer pair; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) a second member of the dimerizer pair; and v) an immunoglobulin or non- immunoglobulin scaffold polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C- terminus: i) an MHC Class II al polypeptide;
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II a2 polypeptide; and iii) a first member of a dimerizer pair; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) a second member of the dimerizer pair; and v) an Ig Fc polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II a2 polypeptide; and iii) a first leucine zipper polypeptide; and b) a second polypeptide comprising, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) a second leucine zipper polypeptide; and v) an Ig Fc polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II
  • the second polypeptide comprises a linker between the peptide antigen and the MHC Class II ⁇ polypeptide. In some cases, the second polypeptide comprises a linker between the MHC Class II ⁇ polypeptide and the second member of the dimerizing pair. In some cases, the first polypeptide comprises a linker between the MHC Class II a2 polypeptide and the first member of the dimerizing pair.
  • an antigen-presenting multimeric polypeptide comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) a first member of a dimerizing pair; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II ⁇ 2 polypeptide; and ii) a second member of the dimerizing pair.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) a first member of a dimerizing pair; vi) an immunoglobulin or non-immunoglobulin scaffold polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II ⁇ 2 polypeptide; and ii) a second member of the dimerizing pair.
  • a first polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epit
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) a first member of a dimerizing pair; vi) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II ⁇ 2 polypeptide; and ii) a second member of the dimerizing pair.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g.
  • an APP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) a first leucine zipper polypeptide; vi) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C- terminus: i) an MHC Class II ⁇ 2 polypeptide; and ii) a second leucine zipper polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g.,
  • the first polypeptide comprises a linker between the peptide antigen and the MHC Class II ⁇ polypeptide. In some cases, the first polypeptide comprises a linker between the MHC Class II ⁇ polypeptide and the MHC Class II al polypeptide. In some cases, the first polypeptide comprises a linker between the MHC Class II a2 polypeptide and the first member of the dimerizing pair. In some cases, the second polypeptide comprises a linker between the MHC Class II ⁇ 2 polypeptide and the second member of the dimerizing pair.
  • an APP of the present disclosure is a single polypeptide chain. Examples are depicted schematically in FIG. 2C and FIG. 5A.
  • an APP e.g., a single-chain APP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an MHC Class II al polypeptide; and v) an MHC Class II a2 polypeptide.
  • a peptide antigen an "epitope”
  • an APP of the present disclosure comprises, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; and vi) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a peptide antigen an "epitope”
  • an APP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; and vi) an Ig Fc polypeptide.
  • the APP comprises a linker between the peptide antigen and the MHC Class II ⁇ polypeptide.
  • the APP comprises a linker between the MHC Class II ⁇ 2 polypeptide and the MHC Class II al polypeptide. In some cases, the APP comprises a linker between the MHC Class II (x2 polypeptide and the immunoglobulin or non-immunoglobulin scaffold.
  • an APP of the present disclosure comprises, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) an MHC Class II ⁇ 2 polypeptide.
  • a peptide antigen an "epitope”
  • an APP of the present disclosure comprises, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; and vi) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a peptide antigen an "epitope”
  • an APP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; and vi) an Ig Fc polypeptide.
  • the APP comprises a linker between the peptide antigen and the MHC Class II ⁇ polypeptide.
  • the APP comprises a linker between the MHC Class II ⁇ polypeptide and the MHC Class II al polypeptide. In some cases, the APP comprises a linker between the MHC Class II a2 polypeptide and the MHC Class II ⁇ 2 polypeptide. In some cases, the APP comprises a linker between the MHC Class II ⁇ 2 polypeptide and the Ig or non-Ig scaffold.
  • a single-chain APP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA ⁇ polypeptide; iii) an HLA al polypeptide; iv) an HLA a2 polypeptide; v) an HLA ⁇ 2 polypeptide; and vi) an Ig Fc polypeptide.
  • a single -chain APP of the present disclosure can comprise, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA DRB1 ⁇ polypeptide; iii) an HLA DRA al polypeptide; iv) an HLA DRA a2 polypeptide; v) an HLA DRB ⁇ 2 polypeptide; and vi) an IgGl Fc polypeptide.
  • the epitope is a hemagglutinin epitope (PKYVKQNTLKLAT; SEQ ID NO: 19). In other instances, the epitope is not PKYVKQNTLKLAT (SEQ ID NO: 19);
  • the epitope is substituted with a different epitope.
  • the single-chain polypeptide comprises the 1559 amino acid sequence depicted in FIG. 27A, without the leader peptide and without the C-terminal linker and histidine tag.
  • the single -chain polypeptide comprises amino acids 21-700 of the amino acid sequence depicted in FIG. 27A.
  • MHC Class II alpha chains comprise an al domain and an a2 domain.
  • the al domain and the a2 domain present in an antigen-presenting cell are from the same MHC Class II a chain polypeptide.
  • the al domain and the a2 domain present in an antigen- presenting cell are from two different MHC Class II a chain polypeptides.
  • MHC Class II alpha chains suitable for inclusion in an APP e.g., a multimeric APP; a single- chain APP; a multimeric TMAPP; a single-chain TMAPP) 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 an APP 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 al domain suitable for inclusion in an APP 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 al domain suitable for inclusion in an APP 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 a2 domain suitable for inclusion in an APP 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 a2 domain suitable for inclusion in an APP 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.
  • a suitable MHC Class II a chain polypeptide is a DRA polypeptide.
  • a DRA DRA
  • 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:20), or an allelic variant thereof.
  • a suitable DRA al domain comprises an amino acid sequence having at least 85%, 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:21); 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 al domain can comprise the following amino acid sequence: VIIQAEFYLN
  • a suitable DRA a2 domain comprises an amino acid sequence having at least 85%, 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:22); 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 a chain polypeptide is a DMA polypeptide.
  • a DMA polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 27-217 of the DMA amino acid sequence depicted in FIG. 11.
  • the DMA polypeptide has a length of about 191 amino acids (e.g., 188, 189, 190, 191, 192, or 193 amino acids).
  • a "DMAA polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DMAA polypeptide comprises the following amino acid sequence: VPEA PTPMWPDDLQ NHTFLHTVYC QDGSPSVGLS EAYDEDQLFF FDFSQNTRVP RLPEFADWAQ EQGDAPAILF DKEFCEWMIQ QIGPKLDGKI PVSRGFPIAE VFTLKPLEFG KPNTLVCFVS NLFPPMLTVN WQHHSVPVEG FGPTFVSAVD GLSFQAFSYL NFTPEPSDIF SCIVTHEIDR YTAIAYW (SEQ ID NO:23), or an allelic variant thereof.
  • a suitable DMA al domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: VPEA PTPMWPDDLQ NHTFLHTVYC QDGSPSVGLS EAYDEDQLFF FDFSQNTRVP RLPEFADWAQ EQGDAPAILF DKEFCEWMIQ QIGPKLDGKI PVSR (SEQ ID NO:24); and can have a length of about 98 amino acids (e.g., 94, 95, 96, 97, 98, 99, 100, or 101 amino acids).
  • a suitable DMA al domain can comprise the following amino acid sequence: VPEA PTPMWPDDLQ NHTFLHTVYC QDGSPSVGLS EAYDEDQLFF FDFSQNTRVP RLPEFADWAQ EQGDAPAILF DKEFCEWMIQ QIGPKLDGKI PVSR (SEQ ID NO:24), or a naturally-occurring allelic variant thereof.
  • a suitable DMA a2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: GFPIAE VFTLKPLEFG KPNTLVCFVS NLFPPMLTVN WQHHSVPVEG FGPTFVSAVD GLSFQAFSYL NFTPEPSDIF SCIVTHEIDR YTAIAYW
  • a suitable DMA a2 domain can comprise the following amino acid sequence: GFPIAE VFTLKPLEFG KPNTLVCFVS NLFPPMLTVN WQHHSVPVEG
  • FGPTFVSAVD GLSFQAFSYL NFTPEPSDIF SCIVTHEIDR YTAIAYW (SEQ ID NO:25), or a naturally-occurring allelic variant thereof.
  • a suitable MHC Class II a chain polypeptide is a DOA polypeptide.
  • a DOA is a polypeptide.
  • polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 26-204 of the DOA amino acid sequence depicted in FIG. 13.
  • the DOA polypeptide has a length of about 179 amino acids (e.g., 175, 176, 177, 178, 179, 180, 181, or 182 amino acids).
  • a "DOA polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DOA polypeptide comprises the following amino acid sequence: TKADH MGSYGPAFYQ SYGASGQFTH EFDEEQLFSV DLKKSEAVWR LPEFGDFARF DPQGGLAGIA AIKAHLDILV ERSNRSRAIN VPPRVTVLPK SRVELGQPNI LICIVDNIFP PVINITWLRN GQTVTEGVAQ TSFYSQPDHL FRKFHYLPFV PSAEDVYDCQ VEHWGLDAPL LRHW (SEQ ID NO:26), or an allelic variant thereof.
  • a suitable DOA al domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: TKADH MGSYGPAFYQ SYGASGQFTH EFDEEQLFSV
  • a suitable DOA al domain can comprise the following amino acid sequence: TKADH MGSYGPAFYQ SYGASGQFTH EFDEEQLFSV DLKKSEAVWR LPEFGDFARF DPQGGLAGIA AIKAHLDILV ERSNRSRAIN (SEQ ID NO:27), or a naturally-occurring allelic variant.
  • a suitable DOA a2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: VPPRVTVLPK SRVELGQPNI LICIVDNIFP PVINITWLRN
  • a suitable DOA a2 domain can comprise the following amino acid sequence: VPPRVTVLPK SRVELGQPNI LICIVDNIFP PVINITWLRN GQTVTEGVAQ TSFYSQPDHL FRKFHYLPFV PSAEDVYDCQ VEHWGLDAPL LRHW (SEQ ID NO:28), or a naturally- occurring allelic variant thereof.
  • a suitable MHC Class II a chain polypeptide is a DPAl polypeptide.
  • a DPAl polypeptide can have at least 85%, 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 DPAl amino acid sequence depicted in FIG. 15.
  • the DPAl polypeptide has a length of about 181 amino acids (e.g., 178, 179, 180, 181, 182, 183, or 184 amino acids).
  • a "DPAl polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DPAl 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:29), or an allelic variant thereof.
  • a suitable DPAl al domain comprises an amino acid sequence having at least 85%, 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
  • a suitable DPAl al domain can comprise the following amino acid sequence: AIKADHVSTY AAFVQTHRPT GEFMFEFDED EMFYVDLDKK ETVWHLEEFG QAFSFEAQGG LANIAILNNN LNTLIQRSNH TQATN (SEQ ID NO:30), or a naturally- occurring allelic variant.
  • a suitable DPAl a2 domain comprises an amino acid sequence having at least 85%, 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:31); and can have a length of about 97 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).
  • a suitable DPAl a2 domain can comprise the following amino acid sequence: DPPEV TVFPKEPVEL GQPNTLICHI DKFFPPVLNV TWLCNGELVT EGVAESLFLP RTDYSFHKFH YLTFVPSAED FYDCRVEHWG LDQPLLKHW (SEQ ID NO: 31), or a naturally-occurring allelic variant thereof.
  • a suitable MHC Class II a chain polypeptide is a DQAl polypeptide.
  • a DQAl polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 24-204 of the DQAl amino acid sequence depicted in FIG. 17.
  • the DQAl polypeptide has a length of about 181 amino acids (e.g., 177, 178, 179, 180, 181, 182, or 183 amino acids).
  • a "DQAl polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DQAl 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:32), or an allelic variant thereof.
  • a suitable DQAl al domain comprises an amino acid sequence having at least 85%, 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:33); and can have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids).
  • a suitable DQAl al domain can comprise the following amino acid sequence:
  • a suitable DQAl a2 domain comprises an amino acid sequence having at least 85%, 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:34); 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 a2 domain can comprise the following amino acid sequence: EVPEVTVFSK SPVTLGQPNT LICLVDNIFP PVVNITWLSN GQSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDQPL LKHW (SEQ ID NO:34), or a naturally-occurring allelic variant thereof.
  • a suitable MHC Class II a chain polypeptide is a DQA2 polypeptide.
  • DQA2 polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 24-204 of the DQA2 amino acid sequence depicted in FIG. 18.
  • the DQA2 polypeptide has a length of about 181 amino acids (e.g., 177, 178, 179, 180, 181, 182, or 183 amino acids).
  • a "DQA2 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DQA2 polypeptide comprises the following amino acid sequence: EDIVADH VASYGVNFYQ SHGPSGQYTH EFDGDEEFYV DLETKETVWQ LPMFSKFISF DPQSALRNMA VGKHTLEFMM RQSNSTAATN EVPEVTVFSK FPVTLGQPNT LICLVDNIFP PVVNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO:35), or an allelic variant thereof.
  • a suitable DQA2 al domain comprises an amino acid sequence having at least 85%, 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 VASYGVNFYQ SHGPSGQYTH EFDGDEEFYV DLETKETVWQ LPMFSKFISF DPQSALRNMA VGKHTLEFMM RQSNSTAATN (SEQ ID NO:36); and can have a length of about 87 amino acids (e.g., 84, 85, 86, 87, 88, or 89 amino acids).
  • a suitable DQA2 al domain can comprise the following amino acid sequence:
  • a suitable DQA2 a2 domain comprises an amino acid sequence having at least 85%, 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 FPVTLGQPNT LICLVDNIFP PVVNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO:37); and can have a length of about 94 amino acids (e.g., 91, 92, 93, 94, 95, 96, or 97 amino acids).
  • a suitable DQA2 a2 domain can comprise the following amino acid sequence: EVPEVTVFSK FPVTLGQPNT LICLVDNIFP PVVNITWLSN GHSVTEGVSE TSFLSKSDHS FFKISYLTFL PSADEIYDCK VEHWGLDEPL LKHW (SEQ ID NO:37), or a naturally-occurring allelic variant thereof.
  • MHC Class II beta chains
  • MHC Class II beta chains comprise a ⁇ domain and a ⁇ 2 domain.
  • the ⁇ domain and the ⁇ 2 domain present in an antigen-presenting cell are from the same MHC Class II ⁇ chain polypeptide.
  • the ⁇ domain and the ⁇ 2 domain present in an antigen-presenting cell are from two different MHC Class II ⁇ chain polypeptides.
  • MHC Class II beta chains suitable for inclusion in an APP e.g., a multimeric APP; a single -chain APP; a multimeric TMAPP; a single -chain TMAPP) of the present disclosure lack a signal peptide.
  • An MHC Class II beta chain suitable for inclusion in an APP 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 an APP 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 ⁇ domain suitable for inclusion in an APP 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 ⁇ domain suitable for inclusion in an APP 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 an APP 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 an APP 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.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB1 polypeptide.
  • DRB 1 polypeptide can have at least 85%, 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 amino acid sequence depicted in any one of FIG. 7A-7J.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB l polypeptide.
  • a DRB l polypeptide can have at least 85%, 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 DRB 1 amino acid sequence depicted in FIG. 7A.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRBl polypeptide.
  • a DRBl polypeptide can have at least 85%, 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 DRBl amino acid sequence depicted in FIG. 7B.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB l polypeptide.
  • a DRBl polypeptide can have at least 85%, 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 DRB 1 amino acid sequence depicted in FIG. 7C.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB l polypeptide.
  • a DRBl polypeptide can have at least 85%, 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 DRBl amino acid sequence depicted in FIG. 7D.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRBl polypeptide.
  • a DRB l polypeptide can have at least 85%, 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 DRBl amino acid sequence depicted in FIG. 7E.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB l polypeptide.
  • a DRB l polypeptide can have at least 85%, 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 DRB l amino acid sequence depicted in FIG. 7F.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRBl polypeptide.
  • a DRBl polypeptide can have at least 85%, 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 DRBl amino acid sequence depicted in FIG. 7G.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB l polypeptide.
  • a DRBl polypeptide can have at least 85%, 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 DRB 1 amino acid sequence depicted in FIG. 7H.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRBl polypeptide.
  • a DRBl polypeptide can have at least 85%, 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 DRBl amino acid sequence depicted in FIG. 71.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRBl polypeptide.
  • a DRB l polypeptide can have at least 85%, 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 DRBl amino acid sequence depicted in FIG. 7J. In some cases, the DRBl polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).
  • a "DRB l polypeptide" includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DRB l polypeptide comprises the following amino acid sequence: DTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAE YWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRVYPEVTVYPAKTQPLQHHNLLV CSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQNGDWTFQTLVMLETVPRSGEVYTCQ VEHPSLTSPLTVEWRARSESAQSK (SEQ ID NO:38), or an allelic variant thereof.
  • a suitable DRB l ⁇ domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • DTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAE YWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRV (SEQ ID NO:39); and can have a length of about 95 amino acids (e.g., 92, 93, 94, 95, 96, 97, or 98 amino acids).
  • a suitable DRB l ⁇ domain can comprise the following amino acid sequence:
  • a suitable DRB l ⁇ 2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • YPEVTVYPAKTQPLQHHNLLVCSVNGFYPGSIEVRWFRNGQEEKTGVVSTGLIQNGDW TFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSK (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 DRBl ⁇ 2 domain can comprise the following amino acid sequence:
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB3 polypeptide.
  • DRB3 polypeptide can have at least 85%, 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 DRB3 amino acid sequence depicted in any one of FIG. 8A-8C.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB3 polypeptide.
  • a DRB3 polypeptide can have at least 85%, 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 DRB3 amino acid sequence depicted in FIG. 8 A.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB3 polypeptide.
  • a DRB3 polypeptide can have at least 85%, 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 DRB3 amino acid sequence depicted in FIG. 8B.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB3 polypeptide.
  • a DRB3 polypeptide can have at least 85%, 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 DRB3 amino acid sequence depicted in FIG. 8C.
  • the DRB3 polypeptide has a length of about 198 amino acids (e.g., 195, 196, 197, 198, 199, 200, 201, or 202 amino acids).
  • 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:41), or an allelic variant thereof.
  • a suitable DRB3 ⁇ domain comprises an amino acid sequence having at least 85%, 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:42); and can have a length of about 95 amino acids (e.g., 93, 94, 95, 96, 97, or 98 amino acids).
  • a suitable DRB3 ⁇ domain can comprise the following amino acid sequence: DTRPRFLELR KSECHFFNGT ERVRYLDRYF HNQEEFLRFD SDVGEYRAVT ELGRPVAESW NSQKDLLEQK RGRVDNYCRH NYGVGESFTV QRRV
  • a suitable DRB3 ⁇ 2 domain comprises an amino acid sequence having at least 85%, 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:43); 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:43), or a naturally-occurring allelic variant thereof.
  • DRB4
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB4 polypeptide.
  • DRB4 polypeptide can have at least 85%, 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. 9. .
  • 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” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable CDR4 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:44), or an allelic variant thereof.
  • a suitable DRB4 ⁇ domain comprises an amino acid sequence having at least 85%, 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
  • a suitable DRB4 ⁇ domain can comprise the following amino acid sequence: T VLSSPLALAG DTQPRFLEQA
  • a suitable DRB4 ⁇ 2 domain comprises an amino acid sequence having at least 85%, 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:46); 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:46), or a naturally-occurring allelic variant thereof.
  • DRB5 QPKVTV YPSKTQPLQH HNLLVCSVNG FYPGSIEVRW FRNGQEEKAG VVSTGLIQNG DWTFQTLVML ETVPRSGEVY TCQVEHPSMM SPLTVQWSAR SESAQSK (SEQ ID NO:46), or a naturally-occurring allelic variant thereof.
  • a suitable MHC Class II ⁇ chain polypeptide is a DRB5 polypeptide.
  • DRB5 polypeptide can have at least 85%, 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. 10. In some cases, 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" 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
  • a suitable DRB5 ⁇ domain comprises an amino acid sequence having at least 85%, 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
  • a suitable DRB5 ⁇ domain can comprise the following amino acid sequence: M VLSSPLALAG DTRPRFLQQD
  • KYECHFFNGT ERVRFLHRDI YNQEEDLRFD SDVGEYRAVT ELGRPDAEYW NSQKDFLEDR RAAVDTYCRH NYGVGESFTV QRRV (SEQ ID NO:48), or a naturally- occurring allelic variant.
  • a suitable DRB5 ⁇ 2 domain comprises an amino acid sequence having at least 85%, 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:49); 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
  • a suitable MHC Class II ⁇ chain polypeptide is a DMB polypeptide.
  • DMB polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 19-207 of the DMB amino acid sequence depicted in FIG. 12. In some cases, the DMB polypeptide has a length of about 189 amino acids (e.g., 187, 188, 189, 190, or 191 amino acids).
  • a "DMB polypeptide" includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DMB polypeptide comprises the following amino acid sequence: GG FVAHVESTCL LDDAGTPKDF TYCISFNKDL LTCWDPEENK MAPCEFGVLN SLANVLSQHL NQKDTLMQRL RNGLQNCATH TQPFWGSLTN RTRPPSVQVA KTTPFNTREP VMLACYVWGF YPAEVTITWR KNGKLVMPHS SAHKTAQPNG DWTYQTLSHL ALTPSYGDTY TCVVEHTGAP EPILRDW (SEQ ID NO:50), or an allelic variant thereof.
  • a suitable DMB ⁇ domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: GG FVAHVESTCL LDDAGTPKDF TYCISFNKDL
  • a suitable DMB ⁇ domain can comprise the following amino acid sequence: GG FVAHVESTCL LDDAGTPKDF TYCISFNKDL LTCWDPEENK MAPCEFGVLN SLANVLSQHL NQKDTLMQRL RNGLQNCATH TQPFWGSLTN RT (SEQ ID NO:51), or a naturally-occurring allelic variant.
  • a suitable DMB ⁇ 2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: RPPSVQVA KTTPFNTREP VMLACYVWGF
  • a suitable DMB ⁇ 2 domain can comprise the following amino acid sequence: RPPSVQVA KTTPFNTREP VMLACYVWGF
  • a suitable MHC Class II ⁇ chain polypeptide is a DOB polypeptide.
  • DOB polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 27-214 of the DOB amino acid sequence depicted in FIG. 14. In some cases, the DOB polypeptide has a length of about 188 amino acids (e.g., 186, 187, 188, 189, or 190 amino acids).
  • a "DOB polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DOB polypeptide comprises the following amino acid sequence: TDSP EDFVIQAKAD CYFTNGTEKV QFVVRFIFNL EEYVRFDSDV GMFVALTKLG QPDAEQWNSR LDLLERSRQA VDGVCRHNYR LGAPFTVGRK VQPEVTVYPE
  • a suitable DOB ⁇ domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: TDSP EDFVIQAKAD CYFTNGTEKV QFVVRFIFNL EEYVRFDSDV GMFVALTKLG QPDAEQWNSR LDLLERSRQA VDGVCRHNYR LGAPFTVGRK (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 DOB ⁇ domain can comprise the following amino acid sequence: TDSP EDFVIQAKAD CYFTNGTEKV QFVVRFIFNL EEYVRFDSDV GMFVALTKLG QPDAEQWNSR LDLLERSRQA VDGVCRHNYR LGAPFTVGRK (SEQ ID NO:54), or a naturally-occurring allelic variant.
  • a suitable DOB ⁇ 2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: VQPEVTVYPE RTPLLHQHNL LHCSVTGFYP
  • a suitable DOB ⁇ 2 domain can comprise the following amino acid sequence: VQPEVTVYPE RTPLLHQHNL LHCSVTGFYP GDIKIKWFLN GQEERAGVMS TGPIRNGDWT FQTVVMLEMT PELGHVYTCL VDHSSLLSPV SVEW (SEQ ID NO:55); and can have a length of about 94 amino acids (e.g., 92, 93, 94, 95, 96, or 97 amino acids).
  • a suitable DOB ⁇ 2 domain can comprise the following amino acid sequence: VQPEVTVYPE RTPLLHQHNL LHCSVTGFYP GDIKIKWFLN GQEERAGVMS TGPIRNGDWT FQTVVMLEMT PELGHVYTCL VDHSSLLSPV SVEW
  • a suitable MHC Class II ⁇ chain polypeptide is a DPB1 polypeptide.
  • DPB 1 polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 30-215 of the DPB1 amino acid sequence depicted in FIG. 16. In some cases, the DPB1 polypeptide has a length of about 186 amino acids (e.g., 184, 185, 186, 187, or 188 amino acids).
  • a "DPB 1 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DPB 1 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:56), or an allelic variant thereof.
  • a suitable DPB 1 ⁇ domain comprises an amino acid sequence having at least 85%, 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:57); and can have a length of about 92 amino acids (e.g., 90,
  • a suitable DPB1 ⁇ domain can comprise the following amino acid sequence: R ATPENYLFQG RQECYAFNGT QRFLERYIYN REEFARFDSD
  • VGEFRAVTEL GRPAAEYWNS QKDILEEKRA VPDRMCRHNY ELGGPMTLQR R (SEQ ID NO:57), or a naturally-occurring allelic variant.
  • a suitable DPB 1 ⁇ 2 domain comprises an amino acid sequence having at least 85%, 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:58); and can have a length of about 94 amino acids (e.g.,
  • a suitable DPB1 ⁇ 2 domain can comprise the following amino acid sequence: VQPRVNVSP SKKGPLQHHN LLVCHVTDFY PGSIQVRWFL
  • a suitable MHC Class II ⁇ chain polypeptide is a DQB l polypeptide.
  • DQBl polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 33-220 of the DQB l amino acid sequence depicted in FIG. 19A or FIG. 19B.
  • the DQB l polypeptide has a length of about 188 amino acids (e.g., 186, 187, 188, 190, 191, or 192 amino acids).
  • a "DQBl polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DQBl 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:59), or an allelic variant thereof.
  • a suitable DQBl ⁇ domain comprises an amino acid sequence having at least 85%, 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:60); and can have a length of about 94 amino acids (e.g., 92, 93, 94, 95, or 96 amino acids).
  • a suitable DQB1 ⁇ domain can comprise the following amino acid sequence: RDSPEDFV FQFKGMCYFT NGTERVRLVT RYIYNREEYA RFDSDVGVYR AVTPQGRPDA EYWNSQKEVL EGTRAELDTV CRHNYEVAFR GILQRR (SEQ ID NO: 60), or a naturally-occurring allelic variant.
  • a suitable DQB1 ⁇ 2 domain comprises an amino acid sequence having at least 85%, 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:61); 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: 61), or a naturally-occurring allelic variant thereof.
  • a suitable MHC Class II ⁇ chain polypeptide is a DQB2 polypeptide.
  • DQB2 polypeptide can have at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with amino acids 33-215 of the DQB2 amino acid sequence depicted in FIG. 20A or FIG. 20.
  • the DQB2 polypeptide has a length of about 182 amino acids (e.g., 175, 176, 177, 178, 179, 180, 181, or 182 amino acids).
  • a "DQB2 polypeptide” includes allelic variants, e.g., naturally occurring allelic variants.
  • a suitable DQB2 polypeptide comprises the following amino acid sequence: DFLVQFK GMCYFTNGTE RVRGVARYIY NREEYGRFDS DVGEFQAVTE LGRSIEDWNN YKDFLEQERA AVDKVCRHNY EAELRTTLQR QVEPTVTISP SRTEALNHHN LLVCSVTDFY PAQIKVRWFR NDQEETAGVV STSLIRNGDW TFQILVMLEI TPQRGDIYTC QVEHPSLQSP ITVEW (SEQ ID NO:62), or an allelic variant thereof.
  • a suitable DQB2 ⁇ domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DFLVQFK GMCYFTNGTE RVRGVARYIY NREEYGRFDS DVGEFQAVTE LGRSIEDWNN YKDFLEQERA AVDKVCRHNY EAELRTTLQR QVEPTV
  • a suitable DQB2 ⁇ domain can comprise the following amino acid sequence: DFLVQFK GMCYFTNGTE RVRGVARYIY NREEYGRFDS DVGEFQAVTE LGRSIEDWNN YKDFLEQERA AVDKVCRHNY EAELRTTLQR QVEPTV (SEQ ID NO:63), or a naturally-occurring allelic variant.
  • a suitable DQB2 ⁇ 2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: TISP SRTEALNHHN LLVCSVTDFY PAQIKVRWFR NDQEETAGVV STSLIRNGDW TFQILVMLEI TPQRGDIYTC QVEHPSLQSP ITVEW (SEQ ID NO:64); and can have a length of about 94 amino acids (e.g., 92 93, 94, 95, 96, or 97 amino acids).
  • a suitable DQB2 ⁇ 2 domain can comprise the following amino acid sequence: TISP SRTEALNHHN LLVCSVTDFY PAQIKVRWFR NDQEETAGVV STSLIRNGDW TFQILVMLEI TPQRGDIYTC QVEHPSLQSP ITVEW (SEQ ID NO:64), or a naturally- occurring allelic variant thereof.
  • An APP of the present disclosure can comprise an immunoglobulin or non-immunoglobulin scaffold.
  • An APP polypeptide of the present disclosure, whether multimeric or monomeric, 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.
  • Suitable XTEN polypeptides include, e.g., those disclosed in WO 2009/023270, WO
  • Suitable albumin polypeptides include, e.g., human serum albumin.
  • Suitable scaffold polypeptides will in some cases be a 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.
  • 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
  • an APP of the present disclosure can comprise an Ig Fc polypeptide.
  • the first and/or the second polypeptide chain of a multimeric polypeptide comprises an Fc polypeptide.
  • an APP of the present disclosure is a monomeric polypeptide and comprises an Ig Fc polypeptide.
  • the Fc polypeptide can be a human IgGl 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. 21A-21G.
  • 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 IgGl Fc polypeptide depicted in FIG. 21A.
  • 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 IgGl Fc polypeptide depicted in FIG. 21 A; and comprises a substitution of N77; e.g., the Fc polypeptide comprises an N77A substitution.
  • 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. 21A; 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. 21A.
  • 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. 21A; 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. 21 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. 21B; 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. 21B.
  • 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. 21C; 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. 21C.
  • 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. 21C; 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 100-327 of the human IgG4 Fc polypeptide depicted in FIG. 21C.
  • the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21 A (human IgGl Fc). In some cases, the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21 A (human IgGl Fc), except for a substitution of N297 with an amino acid other than asparagine. In some cases, the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21C (human IgGl Fc comprising an N297A substitution). In some cases, the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG.
  • the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21 A (human IgGl Fc), except for a substitution of L235 with an amino acid other than leucine.
  • the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21E. In some cases, the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 2 IF. In some cases, the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21G (human IgGl Fc comprising an L234A substitution and an L235A substitution). In some cases, the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG.
  • the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21 A (human IgGl Fc), except for substitutions at L234 and L235 with amino acids other than leucine.
  • the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21A (human IgGl Fc), except for substitutions at L234 and L235 with amino acids other than leucine, and a substitution of P331 with an amino acid other than proline.
  • the Fc polypeptide present in an APP of the present disclosure comprises the amino acid sequence depicted in FIG. 21B (human IgGl Fc comprising L234F, L235E, and P331S substitutions). In some cases, the Fc polypeptide present in an APP of the present disclosure is an IgGl Fc polypeptide that comprises L234A and L235A substitutions.
  • an APP of the present disclosure can include a linker peptide interposed between, e.g., an epitope and an MHC polypeptide; between an MHC polypeptide and an Ig Fc polypeptide; between a first MHC polypeptide polypeptide and a second MHC polypeptide; etc.
  • 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.
  • Exemplary linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , (GSGGS) n (SEQ ID NO:66) and (GGGS) n (SEQ ID NO:67), 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:68), GGSGG (SEQ ID NO:69), GSGSG (SEQ ID NO:70), GSGGG (SEQ ID NO:71), GGGSG (SEQ ID NO:72), GSSSG (SEQ ID NO:73), and the like.
  • Exemplary linkers can include, e.g., Gly(Ser4)n, (SEQ ID NO:344) 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:74), where n is 4.
  • a linker comprises the amino acid sequence (GSSSS)n (SEQ ID NO:74), where n is 5.
  • Exemplary linkers can include, e.g., (GlyGlyGlyGlySer)n (SEQ ID NO:75), 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:75), where n is 1.
  • a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:345), where n is 2. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:346), where n is 3. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:347), where n is 4. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:348), where n is 5. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:349), where n is 6.
  • a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:350), where n is 7. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:351), where n is 8. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:352), where n is 9. In some cases, a linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO:353), where n is 10. In some cases, a linker comprises the amino acid sequence AAAGG (SEQ ID NO:76).
  • a linker polypeptide present in an APP of the present disclosure includes a cysteine residue that can form a disulfide bond with a cysteine residue present in a second polypeptide of the APP.
  • a suitable linker comprises the amino acid sequence GCGASGGGGSGGGGS (SEQ ID NO:77).
  • a peptide epitope (also referred to herein as a "peptide antigen” or “epitope-presenting peptide” or “epitope”) present in an APP of the present disclosure presents an epitope to a TCR on the surface of a T cell.
  • An epitope-presenting peptide can have a length of from about 4 amino acids to about 25 amino acids, e.g., the epitope can have a length of from 4 amino acids (aa) to 10 aa, from 10 aa to 15 aa, from 15 aa to 20 aa, or from 20 aa to 25 aa.
  • an epitope present in an APP 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.
  • an epitope-presenting peptide present in an APP 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.
  • An epitope-presenting peptide present in an APP of the present disclosure is specifically bound by a T-cell, i.e., the epitope is specifically bound by an epitope-specific T cell.
  • An epitope-specific T cell binds an epitope-presenting peptide having a reference amino acid sequence, but does not substantially bind an epitope that differs from the reference amino acid sequence.
  • an epitope-specific T cell binds an epitope-presenting peptide having a reference amino acid sequence, and binds an epitope that differs from the reference amino acid sequence, if at all, with an affinity that is less than 10 6 M, less than 10 s M, or less than 10 4 M.
  • An epitope-specific T cell can bind an epitope-presenting peptide for which it is specific with an affinity of at least 10 7 M, at least 10 s M, at least 10 9 M, or at least 10 10 M.
  • Suitable epitope-presenting peptides include, but are not limited to, epitope-presenting peptides present in a cancer-associated antigen.
  • Cancer-associated antigens include, but are not limited to, a-folate receptor; carbonic anhydrase IX (CAIX); CD19; CD20; CD22; CD30; CD33; CD44v7/8; carcinoembryonic antigen (CEA); epithelial glycoprotein-2 (EGP-2); epithelial glycoprotein-40 (EGP-40); folate binding protein (FBP); fetal acetylcholine receptor;
  • ganglioside antigen GD2 Her2/neu; IL-13R-a2; kappa light chain; LeY; LI cell adhesion molecule; melanoma-associated antigen (MAGE); MAGE-A1 ; mesothelin; MUC1 ; NKG2D ligands; oncofetal antigen (h5T4); prostate stem cell antigen (PSCA); prostate-specific membrane antigen (PS MA); tumor-associate glycoprotein-72 (TAG-72); and vascular endothelial growth factor receptor-2 (VEGF-R2).
  • the epitope is a human papilloma virus E7 antigen epitope; see, e.g., Ramos et al. (2013) /.
  • a suitable peptide epitope is a peptide fragment of from about 4 amino acids to about 20 amino acids (e.g., 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, or 20 aa) in length of a MUC1 polypeptide, a human papillomavirus (HPV) E6 polypeptide, an LMP2 polypeptide, an HPV E7 polypeptide, an epidermal growth factor receptor (EGFR) vIII polypeptide, a HER-2/neu polypeptide, a melanoma antigen family A, 3 (MAGE A3) polypeptide, a p53 polypeptide, a mutant p53 polypeptide, an NY-ESO-1 poly
  • MUCl (GenBank CAA56734); LMP2 (GenBank CAA47024); HPV E6 (GenBank AAD33252); HPV E7 (GenBank AHG99480); EGFRvIII (GenBank NP_001333870); HER-2/neu (GenBank AAI67147); MAGE-A3 (GenBank AAH11744); p53 (GenBank BAC16799); NY-ESO-1 (GenBank CAA05908); PSMA (GenBank AAH25672); CEA (GenBank AAA51967);
  • NP_001888 AKAP-4 (GenBank NP_003877); SSX2 (GenBank CAA60111); XAGE1 (GenBank NP_001091073; XP_001125834; XP_001125856; and XP_001125872); B7H3 (GenBank NP_001019907; XP_947368; XP_950958; XP_950960; XP_950962; XP_950963; XP_950965; and XP_950967); LGMN1 (GenBank NP_001008530); TIE-2 (GenBank
  • NP_000450 PAGE4 (GenBank NP_001305806); VEGFR2 (GenBank NP_002244); MAD-CT- 1 (GenBank NP_005893 NP_056215); FAP (GenBank NP_004451); PDGF (GenBank NP_002600); MAD-CT-2 (GenBank NP_001138574); FOSL (GenBank NP_005429); and WT-1 (GenBank NP_000369).
  • These polypeptides are also discussed in, e.g., Cheever et al. (2009) Clin. Cancer Res. 15:5323, and references cited therein; Wagner et al. (2003) /. Cell. Sci.
  • the epitope is HPV16E7/82-90 (LLMGTLGIV; SEQ ID NO:78). In some cases, the epitope is HPV16E7/86-93 (TLGIVCPI; SEQ ID NO:79). In some cases, the epitope is HPV16E7/11-20 (YMLDLQPETT; SEQ ID NO:80). In some cases, the epitope is
  • HPV16E7/11-19 (YMLDLQPET; SEQ ID NO:81). See, e.g., Ressing et al. ((1995) /. Immunol. 154:5934) for additional suitable HPV epitopes.
  • the peptide epitope is an epitope associated with or present in a "self antigen (an autoantigen).
  • Autoantigens include, e.g., aggrecan, alanyl-tRNA syntetase (PL-12), alpha beta crystallin, alpha fodrin (Sptan 1), alpha-actinin, al antichymotrypsin, al antitripsin, al microglobulin, alsolase, aminoacyl-tRNA synthetase, an amyloid, an annexin, an apolipoprotein, aquaporin, bactericidal/permeability-increasing protein (BPI), ⁇ -globin precursor BP1, ⁇ -actin, ⁇ -lactoglobulin A, ⁇ -2-glycoprotein I, .beta.2-microglobulin, a blood group antigen, C reactive protein (CRP), calmodulin, calreticulin, cardiolipin, catalase,
  • C reactive protein
  • amidated gliadin peptides DGPs
  • gp210 nuclear envelope protein GP2 (major zymogen granule membrane glycoprotein), a glutenin, glycoprotein gpIIb/IIIa, glial fibrillary acidic protein (GFAP), glycated albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), haptoglobin A2, heat shock proteins, hemocyanin, heparin, a histone, histidyl-tRNA synthetase (Jo-1), a hordein, hyaluronidase, immunoglobulins, insulin, insulin receptor, an integrin, interstitial retinol-binding protein 3, intrinsic factor, Ku (p70/p80), lactate dehydrogenase, laminin, liver cytosol antigen type 1 (LCI), liver/kidney microsomal antigen 1 (LKM1), lysozyme, mela
  • gamma collagen threonyl-tRNA synthetase (PL-7), tissue transglutaminase, thyroid peroxidase, thyroglobulin, thyroid stimulating hormone receptor, transferrin, triosephosphate isomerase, tubulin, tumor necrosis alpha, topoisomerase, Ul-dnRNP 68/70 kDa, Ul-snRNP A, Ul-snRNP C, U-snRNP B/B', ubiquitin, vascular endothelial growth factor, vimentin, and vitronectin.
  • PL-7 threonyl-tRNA synthetase
  • tissue transglutaminase thyroid peroxidase
  • thyroglobulin thyroid stimulating hormone receptor
  • transferrin triosephosphate isomerase
  • tubulin tumor necrosis alpha
  • topoisomerase Ul-dnRNP 68/70 kDa
  • Ul-snRNP A Ul
  • 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). See, e.g., Mallone et al.
  • 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 epitope -presenting peptide for inclusion in an antigen- presenting polypeptide of the present disclosure can be an epitope-presenting peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned TID-associated antigens.
  • an epitope-presenting peptide is proinsulin 73-90 (GAGSLQPLALEGSLQKR; SEQ ID NO: 82).
  • an epitope-presenting peptide is the following insulin (InsA (1-15) peptide: GIVDQCCTSICSLYQ (SEQ ID NO:83).
  • an epitope-presenting peptide is the following insulin (InsA(l-15; D4E) peptide: GIVEQCCTSICSLYQ (SEQ ID NO:84).
  • an epitope-presenting peptide is the following GAD65 (555-567) peptide; NFFRMVISNPAAT (SEQ ID NO:85).
  • an epitope-presenting peptide is the following GAD65 (555-567; F557I) peptide; NFIRMVISNPAAT (SEQ ID NO: 86).
  • an epitope-presenting peptide is the following islet antigen 2 (IA2) peptide: SFYLKNVQTQETRTLTQFHF (SEQ ID NO: 87).
  • Antigens associated with Grave's disease include, for example, thyroglobulin, thyroid peroxidase, and thyrotropin receptor (TSH-R).
  • a suitable epitope-presenting peptide for inclusion in an A ⁇ ⁇ of the present disclosure can be an epitope-presenting peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned Grave's disease- associated antigens.
  • Antigens associated with autoimmune polyendocrine syndrome include, 17-alpha
  • a suitable epitope-presenting peptide for inclusion in an antigen-presenting polypeptide of the present disclosure can be an epitope-presenting peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned autoimmune polyendocrine syndrome- associated antigens.
  • Antigens associated with rheumatoid arthritis include, e.g., collagen, vimentin, aggregan, and fibrinogen.
  • a suitable epitope-presenting peptide for inclusion in an antigen-presenting polypeptide of the present disclosure can be an epitope-presenting peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned rheumatoid arthritis- associated antigens.
  • Antigens associated with Parkinson's disease include, e.g., a-synuclein.
  • a suitable epitope-presenting peptide for inclusion in an APP of the present disclosure can be an epitope - presenting peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned Parkinson's disease-associated antigens.
  • Antigens associated with multiple sclerosis include, e.g., myelin basic protein,
  • a suitable epitope-presenting peptide for inclusion in an APP of the present disclosure can be an epitope-presenting peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned multiple sclerosis-associated antigens.
  • Antigens associated with celiac disease include, e.g., tissue transglutaminase and gliadin.
  • a suitable epitope-presenting peptide for inclusion in an APP of the present disclosure can be an epitope-presenting peptide of from 4 amino acids to about 25 amino acids in length of any one of the aforementioned celiac-associated antigens.
  • Other antigens associated with celiac disease include, e.g., secalins, hordeins, avenins, and glutenins.
  • secalins include rye secalins.
  • hordeins include barley hordeins.
  • Examples of glutenins include wheat glutenins. See, e.g., U.S. 2016/0279233.
  • an APP of the present disclosure is a T-cell modulatory antigen- presenting polypeptide (TMAPP).
  • TMAPP T-cell modulatory antigen-presenting polypeptide
  • the present disclosure provides TMAPPs.
  • a TMAPP of the present disclosure comprises two polypeptide chains and is sometimes referred to herein as a "multimeric T-cell modulatory antigen-presenting polypeptide.”
  • a TMAPP of the present disclosure comprises a single polypeptide chain.
  • a TMAPP of the present disclosure is also referred to as a "synTac polypeptide.”
  • a TMAPP of the present disclosure comprises one or more immunomodulatory
  • a TMAPP of the present disclosure comprises a single
  • a TMAPP of the present disclosure comprises two or more immunomodulatory polypeptides (e.g., 2, 3, 4, or 5 immunomodulatory
  • a TMAPP of the present disclosure comprises two or more
  • TMAPP of the present disclosure comprises 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.
  • at least one of the two or more immunomodulatory polypeptides are present in the first polypeptide chain; and at least one of the two or more immunomodulatory polypeptides are present in the second polypeptide chain.
  • TMAPP of the present disclosure comprises two
  • the two immunomodulatory polypeptides have the same amino acid sequence, i.e., the TMAPP 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 TMAPP of the present disclosure modulates activity of a T cell.
  • a T cell In some cases, a T cell is a T cell that modulates activity of a T cell.
  • TMAPP of the present disclosure activates a CD8 + T cell response, e.g., a CD8 + T cell response to a cancer cell.
  • a TMAPP of the present disclosure reduces activity of an autoreactive T cell and/or an autoreactive B cell.
  • a TMAPP of the present disclosure increases the number and/or activity of a regulator T cell (Treg), resulting in reduced activity of an autoreactive T cell and/or an autoreactive B cell.
  • Treg regulator T cell
  • Immunomodulatory polypeptides that are suitable for inclusion in a TMAPP 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, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM.
  • TGF transforming growth factor-beta
  • an immunomodulatory polypeptide suitable for inclusion in a TMAPP of the present disclosure is a variant that comprises from 1 to 10 amino acid substitutions relative to a wild-type or naturally-occurring immunomodulatory polypeptide, and 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 TMAPP of the present disclosure comprises: i) a peptide epitope (a peptide
  • a TMAPP of the present disclosure can further include one or both of: a dimerizer polypeptide; and an immunoglobulin scaffold (e.g., an Ig Fc polypeptide) or a non-immunoglobulin scaffold.
  • a dimerizer polypeptide e.g., an Ig Fc polypeptide
  • a non-immunoglobulin scaffold e.g., an Ig Fc polypeptide
  • a TMAPP of the present disclosure comprises a single
  • a TMAPP of the present disclosure comprises 2 copies of an immunomodulatory polypeptide. In some cases, a TMAPP of the present disclosure comprises 3 copies of an immunomodulatory polypeptide. Where a TMAPP of the present disclosure comprises 2 or 3 copies of an immunomodulatory polypeptide, in some cases, the 2 or 3 copies are in tandem. Where a TMAPP 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 TMAPP of the present disclosure can include one or more linkers, where the one or more linkers are between one or more of: i) an MHC Class II polypeptide and an Ig Fc polypeptide, where such a linker is referred to herein as "LI"; ii) an immunomodulatory polypeptide and an MHC Class II polypeptide, where such a linker is referred to herein as "L2"; iii) a first immunomodulatory polypeptide and a second immunomodulatory polypeptide, where such a linker is referred to herein as "L3”; iv) a peptide antigen ("epitope") and an MHC Class II polypeptide; v) an MHC Class II polypeptide and a dimerization polypeptide (e.g., a first or a second member of a dimerizing pair); and vi) a dimerization polypeptide (e.g., a first or a second member of a dimerizing pair); and vi
  • an LI linker comprises (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • an L2 linker comprises (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • an L3 linker comprises (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; and iv) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class
  • the second polypeptide comprises, in order from N- terminus to C-terminus: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) an immunoglobulin or non-immunoglobulin scaffold polypeptide; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • the second polypeptide comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) an Ig Fc polypeptide; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • the second polypeptide comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) a first member of a dimerizer pair; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; iii) a second member of the dimerizer pair.
  • a first polypeptide compris
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) a first leucine zipper polypeptide; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) a second leucine zipper polypeptide.
  • the second polypeptide comprises, in order from N- terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) a second leucine zipper polypeptide.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) a first leucine zipper polypeptide; and vi) an Ig Fc polypeptide; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) a second leucine zipper polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.
  • the second polypeptide comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) a second leucine zipper polypeptide.
  • the TMAPP can include a single immunomodulatory polypeptide.
  • the TMAPP can include 2 copies of the immunomodulatory polypeptide; the 2 copies can be in tandem, or can be separated by a linker.
  • the TMAPP can include 3 copies of the immunomodulatory polypeptide; the 3 copies can be in tandem, or can be separated by a linker.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) a first leucine zipper polypeptide; and vi) an Ig Fc polypeptide; and b) a second polypeptide comprising: i) a first immunomodulatory polypeptide; ii) a second
  • the immunomodulatory polypeptide comprises, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide; ii) a second immunomodulatory polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) a second leucine zipper polypeptide.
  • the first and the second immunomodulatory polypeptides comprise the same amino acid sequences.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) an Ig Fc polypeptide; and b) a second polypeptide comprising: i) a first immunomodulatory polypeptide; ii) a second immunomodulatory polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by
  • the second polypeptide comprises, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide; ii) a second immunomodulatory polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide.
  • the first and the second immunomodulatory polypeptides comprise the same amino acid sequences.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; and iv) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) an Ig Fc polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II
  • the second polypeptide comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) an Ig Fc polypeptide.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; and iv) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising: i) a first immunomodulatory polypeptide; ii) a second immunomodulatory polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an Ig Fc polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by
  • the second polypeptide comprises, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide; ii) a second immunomodulatory polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an Ig Fc polypeptide.
  • the first and the second immunomodulatory polypeptides comprise the same amino acid sequence.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an
  • immunomodulatory polypeptide ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; and iv) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ 2 polypeptide; and iii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • the second polypeptide comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ 2 polypeptide; and iii) an Ig Fc polypeptide.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N- terminus to C-terminus: i) a first immunomodulatory polypeptide; ii) a second
  • immunomodulatory polypeptide iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; and v) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ 2 polypeptide; and iii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • the second polypeptide comprises, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ 2 polypeptide; and iii) an Ig Fc polypeptide.
  • the first and the second immunomodulatory polypeptides comprise the same amino acid sequence.
  • a TMAPP of the present disclosure comprises two immunomodulatory polypeptides
  • the first immunomodulatory polypeptide is linked to the second immunomodulatory polypeptide by a linker (an "L3" linker); e.g., a linker of from about 2 amino acids to 50 amino acids in length.
  • Suitable L3 linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "LI") between the MHC polypeptide and the Ig Fc polypeptide; where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; and iv) an immunoglobulin or non- immunoglobulin scaffold polypeptide; and b) a second polypeptide comprising, in order from N- terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II a 1 polypeptide; and iii) an MHC Class II (x2 polypeptide; and iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II a 1 poly
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; and iii) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) an immunoglobulin or non- immunoglobulin scaffold polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; and iii) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; and
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide; and v) a first member of a dimerizer pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) a second member of a dimerizer pair (e
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide; and v) and v) a first member of a dimerizer pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) a second member of a dimer
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; and iv) a first member of a dimerizer pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an immunoglobulin or non- immunoglobulin scaffold polypeptide; and v) a second member of a dimerizer pair (e.g.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; and iv) a first member of a dimerizer pair (e.g., a first leucine zipper polypeptide); and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide; and v) a second member of a dimerizer pair (e
  • the TMAPP can include 2 copies of the immunomodulatory polypeptide; the 2 copies can be in tandem, or can be separated by a linker. In any one of the above embodiments, the TMAPP can include 3 copies of the immunomodulatory polypeptide; the 3 copies can be in tandem, or can be separated by a linker. In some cases, the TMAPP comprises a linker (an "LI") between the MHC polypeptide and the Ig Fc polypeptide; where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; and ii) an MHC Class II a2 polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II a2 polypeptide; and iii) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g.,
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) an immunomodulatory polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II a2 polypeptide; and iii) an Ig Fc polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an immunomodulatory polypeptide; and v) a first member of a dimerizer pair; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II a2 polypeptide; and iii) a second member of the dimerizer pair.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an immunomodulatory polypeptide; and v) a first leucine zipper polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an MHC Class II al polypeptide; ii) an MHC Class II (x2 polypeptide; and iii) a second leucine zipper polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope" that is recognized (
  • the TMAPP can include a single immunomodulatory polypeptide.
  • the TMAPP can include 2 copies of the immunomodulatory polypeptide; the 2 copies can be in tandem, or can be separated by a linker.
  • the TMAPP can include 3 copies of the immunomodulatory polypeptide; the 3 copies can be in tandem, or can be separated by a linker.
  • the TMAPP comprises a linker (an "LI") between the MHC polypeptide and the Ig Fc polypeptide; where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; and iii) an MHC Class II a2 polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C- terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; and iv) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g.,
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; and iii) an MHC Class II ⁇ 2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; and iv) an Ig Fc polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) a first member of a dimerizer pair; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; and iv) a second member of the dimerizer pair.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; and iv) a first leucine zipper polypeptide; and b) a second polypeptide comprising, in order from N- terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; and iv) a second leucine zipper polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized
  • the TMAPP can include a single immunomodulatory polypeptide. In any one of the above embodiments, the TMAPP can include 2 copies of the immunomodulatory polypeptide; the 2 copies can be in tandem, or can be separated by a linker. In any one of the above embodiments, the TMAPP can include 3 copies of the
  • the TMAPP comprises a linker (an "LI") between the MHC polypeptide and the Ig Fc polypeptide; where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises two immunomodulatory polypeptides, in some cases, the two
  • immunomodulatory polypeptides are separated by a linker (an "L3); where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; and iv) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunogen (epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; and iv) an MHC Class II a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) an immunoglobulin or non- immunoglobulin scaffold polypeptide; and b) a second polypeptide comprising, in order from N- terminus to C-terminus: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C- terminus: i) an immunomodulatory polypeptide; and ii) an MHC Class II ⁇ 2 polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) a first member of a dimerizer pair; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) a second member of the dimerizer pair.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) a first leucine zipper polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) an MHC Class II ⁇ 2 polypeptide; and iii) a second leucine zipper polypeptide.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (
  • the TMAPP can include a single immunomodulatory polypeptide.
  • the TMAPP can include 2 copies of the immunomodulatory polypeptide; the 2 copies can be in tandem, or can be separated by a linker.
  • the TMAPP can include 3 copies of the immunomodulatory polypeptide; the 3 copies can be in tandem, or can be separated by a linker.
  • the TMAPP comprises a linker (an "LI") between the MHC polypeptide and the Ig Fc polypeptide; where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "L2") between the immunomodulatory polypeptide and the MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP to be administered to an individual in need thereof will generally not include a leader sequence or a histidine tag as depicted in the aforementioned figures.
  • a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a linker; iii) an HLA ⁇ polypeptide; iv) an HLA al polypeptide; v) an HLA a2 polypeptide; vi) a dimerizer polypeptide; and vii) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); ii) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); iii) an HLA ⁇ 2 polypeptide; and iv) a dimerizer polypeptide.
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a linker; iii) an HLA DRB 1 ⁇ polypeptide; iv) an HLA DRA al polypeptide; v) an HLA DRA a2 polypeptide; vi) a leucine zipper dimerizer polypeptide; and vii) an IgGl Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); iii) an HLA DRB
  • the epitope is a hemagglutinin epitope, e.g., PKYVKQNTLKLAT (SEQ ID NO: 19).
  • the variant IL-2 polypeptide comprises the following amino acid sequence:
  • the HLA-DRB 1 ⁇ polypeptide comprises the following amino acid sequence: DTRPRFLWQHKFECHFFNGTERVRLLERCIYNQEESVRFDSDVGEYRAVTELGRPDAEY WNSQKDLLEQRRAAVDTYCRHNYGVGESFTVQR (SEQ ID NO:89).
  • the HLA DRA al polypeptide comprises the following amino acid sequence
  • HLA DRA al polypeptide comprises the following amino acid sequence
  • the leucine zipper dimerizer polypeptide comprises the following amino acid sequence: LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGK (SEQ ID NO:92).
  • the IgGl Fc polypeptide comprises the following amino acid sequence: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:93).
  • the first polypeptide comprises the 1452 amino acid sequence depicted in FIG.
  • the first polypeptide comprises amino acids 21 to 628 of the 1452 amino acid sequence depicted in FIG. 26A.
  • the second polypeptide comprises the 1661 amino acid sequence depicted in FIG. 34A, without the leader sequence.
  • the second polypeptide comprises amino acids 21 to 491 of the amino acid sequence depicted in FIG. 34A.
  • the epitope of the first polypeptide is not
  • PKYVKQNTLKLAT (SEQ ID NO: 19), but instead is substituted with a different epitope.
  • a multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA ⁇ polypeptide; iii) an HLA al polypeptide; iv) an HLA a2 polypeptide; and v) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C- terminus: i) a first immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); ii) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); and iii) an HLA ⁇ 2 polypeptide.
  • a first immunomodulatory polypeptide e.g., a variant
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA DRB 1 ⁇ polypeptide; iii) an HLA DRA al polypeptide; iv) an HLA DRA (x2 polypeptide; and v) an IgGl Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); and iii) an HLA DRB1 ⁇ 2 polypeptide.
  • a first immunomodulatory polypeptide e.g., a variant
  • the epitope is a hemagglutinin epitope, e.g., PKYVKQNTLKLAT (SEQ ID NO: 19).
  • the HLA DRB1 ⁇ polypeptide comprises the following amino acid sequence:
  • the DRA al polypeptide comprises the following amino acid sequence:
  • the DRA al polypeptide comprises the following amino acid sequence:
  • the IgGl Fc polypeptide comprises the following amino acid sequence:
  • the variant IL-2 polypeptide comprises the following amino acid sequence:
  • the HLA DRB1 ⁇ 2 polypeptide comprises the following amino acid sequence: PKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGDWTF QTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSKM (SEQ ID NO:95).
  • the first polypeptide comprises the 1659 amino acid sequence depicted in FIG. 33A, without the leader peptide and without the C-terminal linker and histidine tag.
  • the first polypeptide comprises amino acids 21 to 591 of the 1659 amino acid sequence depicted in FIG. 33A.
  • the epitope is not PKYVKQNTLKLAT (SEQ ID NO: 19), but instead is substituted with a different epitope.
  • the second polypeptide comprises the 1664 amino acid sequence depicted in FIG. 35 A, without the leader sequence.
  • the second polypeptide comprises amino acids 21 to 429 of the 1664 amino acid sequence depicted in FIG. 35A.
  • a multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA ⁇ polypeptide; iii) an HLA al polypeptide; iv) an HLA a2 polypeptide; v) a dimerizer polypeptide; and vi) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant
  • immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide ii) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); iii) an HLA ⁇ 2 polypeptide; and iv) a dimerizer polypeptide.
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA DRB1 ⁇ polypeptide; iii) an HLA DRA al polypeptide; iv) an HLA DRA a2 polypeptide; v) a leucine zipper dimerizer polypeptide; and vi) an IgGl Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); iii) an HLA DRB1 ⁇ 2 polypeptide; and
  • the epitope is a cytomegalovirus (CMV) pp65 epitope (LPLKMLNIPSINVH; SEQ ID NO:96).
  • CMV cytomegalovirus
  • LLKMLNIPSINVH pp65 epitope
  • the HLA DRB ⁇ polypeptide comprises the following amino acid sequence:
  • the HLA DRA al polypeptide comprises the following amino acid sequence:
  • the HLA DRA a2 polypeptide comprises the following amino acid sequence:
  • the leucine zipper polypeptide comprises the following amino acid sequence:
  • the IgGl Fc polypeptide comprises the following amino acid sequence:
  • the variant IL-2 polypeptide comprises the following amino acid sequence:
  • the HLA DRB1 ⁇ 2 polypeptide comprises the following amino acid sequence: VEPKVTVYPSKTQPLQHHNLLVCSVSGFYPGSIEVRWFRNGQEEKAGVVSTGLIQNGD WTFQTLVMLETVPRSGEVYTCQVEHPSVTSPLTVEWRARSESAQSKM (SEQ ID NO:98).
  • the leucine zipper polypeptide comprises the following amino acid sequence: LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO:99).
  • the first polypeptide comprises the 1637 amino acid sequence depicted in FIG. 30A, without the leader sequence and without the C-terminal linker and histidine tag.
  • the first polypeptide comprises amino acids 21-629 of the 1637 amino acid sequence depicted in FIG. 30A.
  • the first polypeptide does not include the epitope LPLKMLNIPSINVH (SEQ ID NO:96); instead, the epitope is substituted with a different epitope.
  • the second polypeptide comprises the amino acid sequence depicted in FIG. 25A, but without the leader peptide.
  • the second polypeptide comprises amino acids 21-493 of the amino acid sequence depicted in FIG. 25A.
  • a multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA ⁇ polypeptide; iii) an HLA al polypeptide; iv) an HLA a2 polypeptide; v) a dimerizer polypeptide; and vi) an Ig Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); ii) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); iii) an HLA ⁇ 2 polypeptide
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA DRB1-4 ⁇ polypeptide; iii) an HLA DRA al polypeptide; iv) an HLA DRA a2 polypeptide; v) a leucine zipper dimerizer polypeptide; and vi) an IgGl Fc polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); iii) an HLA DRB1-4 ⁇ 2 polypeptide; and
  • the HLA DRA al polypeptide comprises the following amino acid sequence:
  • HLA DRA al polypeptide comprises the following amino acid sequence:
  • the leucine zipper polypeptide comprises the following amino acid sequence:
  • the IgGl Fc polypeptide comprises the following amino acid sequence:
  • the variant IL-2 polypeptide comprises the following amino acid sequence:
  • the HLA DRB1-4 ⁇ 2 polypeptide comprises the following amino acid sequence: VYPEVTVYPAKTQPLQHHNLLVCSVNGFYPASIEVRWFRNGQEEKTGVVSTGLIQNGD WTFQTLVMLETVPRSGEVYTCQVEHPSLTSPLTVEWRARSESAQSKM (SEQ ID NO:88), where the H16A and F42A substitutions are underlined.
  • the leucine zipper polypeptide comprises the following amino acid sequence: LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK (SEQ ID NO:99).
  • the first polypeptide comprises the amino acid sequence depicted in FIG. 31 A, without the leader peptide and without the C-terminal linker and histidine tag.
  • the first polypeptide comprises amino acids 21-633 of the amino acid sequence depicted in FIG. 31 A.
  • the epitope is not proinsulin 73-90
  • the second polypeptide comprises the amino acid sequence depicted in FIG. 32A, without the leader peptide.
  • the second polypeptide comprises amino acids 21-493 of the amino acid sequence depicted in FIG. 32A.
  • a multimeric TMAPP of the present disclosure comprises: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA ⁇ polypeptide; iii) an HLA al polypeptide; and iv) an HLA al polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); ii) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); iii) an HLA ⁇ 2 polypeptide; and iv) an Ig Fc polypeptide.
  • a first immunomodulatory polypeptide e.g., a variant immuno
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA DRB l ⁇ polypeptide; iii) an HLA DRA al polypeptide; and iv) an HLA DRA a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); iii) an HLA DRB l ⁇ 2 polypeptide; and iv) an IgG Fc polypeptide.
  • a first immunomodulatory polypeptide e.g., a
  • the multimeric TMAPP can include a variant IgG Fc polypeptide.
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA DRBl ⁇ polypeptide; iii) an HLA DRA al polypeptide; and iv) an HLA DRA a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); iii) an HLA DRBl ⁇ 2 polypeptide; and iv) an IgGl Fc polypeptid
  • the multimeric TMAPP can include one or more linkers.
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) a peptide linker; iii) an HLA DRBl ⁇ polypeptide; iv) a peptide linker; v) an HLA DRA al polypeptide; and vi) an HLA DRA a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); iii) a peptide linker; iv)
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C-terminus: i) an epitope; ii) the peptide linker (GGGGS) 3 (SEQ ID NO:346); iii) an HLA DRB 1 ⁇ polypeptide; iv) the peptide linker GGGGS (SEQ ID NO:75); v) an HLA DRA al polypeptide; and vi) an HLA DRA a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); ii) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); iii) the peptide
  • a multimeric TMAPP of the present disclosure can comprise: a) a first polypeptide comprising, in order from N-terminus to C- terminus: i) an epitope; ii) the peptide linker (GGGGS) 3 (SEQ ID NO:346); iii) an HLA DRB1 ⁇ polypeptide; iv) the peptide linker GGGGS (SEQ ID NO:75); v) an HLA DRA al polypeptide; and vi) an HLA DRA a2 polypeptide; and b) a second polypeptide comprising, in order from N-terminus to C-terminus: i) a first variant IL-2 polypeptide comprising H16A and F42A substitutions; ii) a second variant IL-2 polypeptide comprising H16A and F42A substitutions (e.g., where the first and the second variant IL-2 polypeptides comprise the same amino acid sequence); iii) the peptide linker
  • the HLA DRB1 ⁇ polypeptide comprises the following amino acid sequence:
  • HLA DRA al polypeptide comprises the following amino acid sequence:
  • the HLA DRA a2 polypeptide comprises the following amino acid sequence:
  • the HLA DRB1 ⁇ 2 polypeptide comprises the following amino acid sequence:
  • the first and the second immunomodulatory polypeptides are variant IL-2 polypeptides, both comprising the amino acid sequence:
  • the Fc polypeptide is an IgGl Fc polypeptide comprising L234A and L235A substitutions, and comprises the amino acid sequence:
  • a TMAPP of the present disclosure comprises: a) a first polypeptide comprising amino acids 21-328 of the amino acid sequence depicted in FIG.
  • a TMAPP of the present disclosure comprises: a) a first polypeptide encoded by the nucleotide sequence depicted in FIG. 37B; and b) a second polypeptide encoded by the nucleotide sequence depicted in FIG. 38B.
  • a TMAPP of the present disclosure comprises a single polypeptide chain.
  • Non-limiting examples are depicted schematically in FIG. 23A-23I.
  • a single- chain TMAPP of the present disclosure can include one or more linkers between any two adjacent polypeptides, e.g., between a peptide antigen and an immunomodulatory polypeptide, between an immunomodulatory polypeptide and an MHC Class II polypeptide, between two MHC Class II polypeptides, between an immunomodulatory polypeptide and an Ig Fc polypeptide, etc.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; and vi) an immunomodulatory polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) an immunomodulatory polypeptide; and vii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; and vi) 2 copies of an immunomodulatory polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) 2 copies of an immunomodulatory polypeptide; and v) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; and v) a second copy of the immunomodulatory polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C- terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; and vi) an MHC Class II ⁇ 2 polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; and vii) an immunoglobulin or non- immunoglobulin scaffold polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II (x2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; and vii) an Ig Fc polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; and vii) a second copy of the immunomodulatory polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C- terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a T
  • immunomodulatory polypeptide ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an MHC Class II ⁇ 2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope"
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; and vi) an immunomodulatory polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an immunomodulatory polypeptide; and vii) an immunoglobulin or non- immunoglobulin scaffold polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II ⁇ 2 polypeptide; iv) an MHC Class II al polypeptide; v) an MHC Class II a2 polypeptide; vi) an immunomodulatory polypeptide; and vii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • the TMAPP can comprise one or more linkers, where the linker may be between one or more of: i) the peptide antigen and the MHC Class II ⁇ polypeptide; ii) the MHC Class II ⁇ 2 polypeptide and the MHC Class II al polypeptide; iii) the MHC Class II a2 polypeptide and the immunomodulatory polypeptide; and iv) the immunomodulatory polypeptide and the Ig Fc polypeptide.
  • the linker may be between one or more of: i) the peptide antigen and the MHC Class II ⁇ polypeptide; ii) the MHC Class II ⁇ 2 polypeptide and the MHC Class II al polypeptide; iii) the MHC Class II a2 polypeptide and the immunomodulatory polypeptide; and iv) the immunomodulatory polypeptide and the Ig Fc polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; and v) an MHC Class II a2 polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; and vii) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; and vii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; and vii) a second copy of the
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a peptide antigen an "epitope"
  • a TMAPP of the present disclosure comprises, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an immunomodulatory polypeptide; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; and vi) an MHC Class II a2 polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; and vii) an immunoglobulin or non- immunoglobulin scaffold polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; and vii) an Ig Fc polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C- terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; and vii) a second copy of the immunomodulatory polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C- terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an immunoglobulin or non-immunoglobulin scaffold polypeptide.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an immunomodulatory polypeptide; ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a T
  • immunomodulatory polypeptide ii) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; iii) an MHC Class II ⁇ polypeptide; iv) an MHC Class II ⁇ 2 polypeptide; v) an MHC Class II al polypeptide; vi) an MHC Class II a2 polypeptide; vii) a second copy of the immunomodulatory polypeptide; and viii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope"
  • the TMAPP can comprise one or more linkers, where the linker may be between one or more of: i) the immunomodulatory polypeptide and the peptide antigen; ii) the peptide antigen and the MHC Class II ⁇ polypeptide; iii) the MHC Class II a2 polypeptide and the Ig Fc polypeptide; and iv) the MHC Class II a2 polypeptide and the second copy of the immunomodulatory polypeptide.
  • the linker may be between one or more of: i) the immunomodulatory polypeptide and the peptide antigen; ii) the peptide antigen and the MHC Class II ⁇ polypeptide; iii) the MHC Class II a2 polypeptide and the Ig Fc polypeptide; and iv) the MHC Class II a2 polypeptide and the second copy of the immunomodulatory polypeptide.
  • a TMAPP of the present disclosure comprises a single polypeptide chain.
  • a TMAPP of the present disclosure comprises a single polypeptide chain comprising: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; iv) an MHC Class II ⁇ polypeptide; and v) one or more immunomodulatory polypeptides.
  • a TMAPP of the present disclosure comprises a single polypeptide chain.
  • a TMAPP of the present disclosure comprises a single polypeptide chain comprising: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; iv) an MHC Class II ⁇ polypeptide; v) an MHC Class II ⁇ 2 polypeptide; and vi) one or more immunomodulatory polypeptides.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises a single polypeptide chain comprising: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; iv) an MHC Class II ⁇ polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) one or more immunomodulatory polypeptides; and vii) an Ig or a non-Ig scaffold polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises a single polypeptide chain comprising: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II al polypeptide; iii) an MHC Class II a2 polypeptide; iv) an MHC Class II ⁇ polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) one or more immunomodulatory polypeptides; and vii) a dimerizing polypeptide.
  • a peptide antigen an "epitope”
  • the TMAPP comprises a linker (an "LI") between an MHC polypeptide and an Ig Fc polypeptide; where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "L2") between an immunomodulatory polypeptide and an MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises two
  • the two immunomodulatory polypeptides are separated by a linker (an "L3); where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; and vi) one or more immunomodulatory polypeptides.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; and v) one or more immunomodulatory polypeptides.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) one or more immunomodulatory polypeptides; and vii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) a first immunomodulatory polypeptide; vii) a second immunomodulatory polypeptide; and viii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) a first immunomodulatory polypeptide; vi) a second immunomodulatory polypeptide; and vii) an Ig Fc polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N- terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) one or more immunomodulatory polypeptides; and vii) a dimerizing polypeptide.
  • a peptide antigen an "epitope”
  • a TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) a peptide antigen (an "epitope") that is recognized (e.g., is capable of being recognized and bound) by a TCR; ii) an MHC Class II ⁇ polypeptide; iii) an MHC Class II al polypeptide; iv) an MHC Class II a2 polypeptide; v) an MHC Class II ⁇ 2 polypeptide; vi) one or more
  • the TMAPP comprises a linker (an "LI") between an MHC polypeptide and an Ig Fc polypeptide; where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises a linker (an "L2") between an immunomodulatory polypeptide and an MHC polypeptide, where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • the TMAPP comprises two immunomodulatory polypeptides
  • the two immunomodulatory polypeptides are separated by a linker (an "L3); where exemplary suitable linkers include (GGGGS)n (SEQ ID NO:75), where n is 1, 2, 3, 4, 5, 6, 7, or 8.
  • a TMAPP to be administered to an individual in need thereof will generally not include a leader sequence or a histidine tag as depicted in the aforementioned figures.
  • a single -chain TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA ⁇ polypeptide; iii) an HLA al polypeptide; iv) an HLA a2 polypeptide; v) an HLA ⁇ 2 polypeptide; vi) an immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); and vii) an Ig Fc polypeptide.
  • an immunomodulatory polypeptide e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide
  • a single -chain TMAPP of the present disclosure can comprise, in order from N- terminus to C-terminus: i) an epitope; ii) an HLA DRB1 ⁇ polypeptide; iii) an HLA DRA al polypeptide; iv) an HLA DRA a2 polypeptide; v) an HLA DRB ⁇ 2 polypeptide; vi) an immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); and vii) an IgGl Fc polypeptide.
  • an immunomodulatory polypeptide e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions
  • the epitope is a hemagglutinin epitope (e.g., PK YVKQNTLKL AT ; SEQ ID NO: 19).
  • the HLA DRB 1 ⁇ polypeptide comprises the following amino acid sequence:
  • HLA DRA al polypeptide comprises the following amino acid sequence:
  • the HLA DRB ⁇ 2 polypeptide comprises the following amino acid sequence:
  • the variant IL-2 polypeptide comprises the following amino acid sequence:
  • the IgGl Fc polypeptide comprises the following amino acid sequence:
  • the single -chain polypeptide comprises the amino acid sequence depicted in FIG.
  • the single-chain polypeptide comprises amino acids 21-981 of the amino acid sequence depicted in FIG. 28A.
  • the single -chain polypeptide does not include a hemagglutinin epitope (e.g., PKY VKQNTLKL AT ; SEQ ID NO: 19); instead, the epitope is substituted with a different epitope.
  • a single -chain TMAPP of the present disclosure comprises, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA ⁇ polypeptide; iii) an HLA al polypeptide; iv) an HLA a2 polypeptide; v) a first immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co- immunomodulatory polypeptide); vi) a second immunomodulatory polypeptide (e.g., a variant immunomodulatory polypeptide with reduced affinity for its cognate co-immunomodulatory polypeptide); and vii) an Ig Fc polypeptide.
  • a single -chain TMAPP of the present disclosure can comprise, in order from N-terminus to C-terminus: i) an epitope; ii) an HLA DRB 1 ⁇ polypeptide; iii) an HLA DRA al polypeptide; iv) an HLA DRA (x2 polypeptide; v) a first immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); vi) a second immunomodulatory polypeptide (e.g., a variant IL-2 polypeptide comprising H16A and F42A substitutions); and vii) an IgGl Fc polypeptide.
  • an epitope ii) an HLA DRB 1 ⁇ polypeptide
  • iii) an HLA DRA al polypeptide iv) an HLA DRA (x2 polypeptide
  • a first immunomodulatory polypeptide e.g., a variant IL-2 polypeptide
  • the epitope is a hemagglutinin epitope (e.g., PKYVKQNTLKLAT; SEQ ID NO: 19).
  • the HLA DRB1 ⁇ polypeptide comprises the following amino acid sequence:
  • HLA DRA al polypeptide comprises the following amino acid sequence:
  • the HLA DRA a2 polypeptide comprises the following amino acid sequence:
  • the variant IL-2 polypeptide comprises the following amino acid sequence:
  • the IgGl Fc polypeptide comprises the following amino acid sequence:
  • the single -chain polypeptide comprises the amino acid sequence depicted in FIG. 29A, without the leader peptide and without the C-terminal linker and histidine tag.
  • the single-chain polypeptide comprises amino acids 21-876 of the amino acid sequence depicted in FIG. 29A.
  • Immunomodulatory polypeptides that are suitable for inclusion in a TMAPP of the present disclosure include, but are not limited to, IL-2, CD7, B7-1 (CD80), B7-2 (CD86), PD- Ll, 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 a 4-1BBL
  • 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 TMAPP 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 TMAPP 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 TMAPP 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 TMAPP 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.
  • immunomodulatory polypeptide present in a TMAPP 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 TMAPP 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 present in a TMAPP of the present disclosure includes a single amino acid substitution compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 2 amino acid substitutions (e.g., no more than 2 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 3 amino acid substitutions (e.g., no more than 3 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 4 amino acid substitutions (e.g., no more than 4 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 5 amino acid substitutions (e.g., no more than 5 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 6 amino acid substitutions (e.g., no more than 6 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 7 amino acid substitutions (e.g., no more than 7 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 8 amino acid substitutions (e.g., no more than 8 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 9 amino acid substitutions (e.g., no more than 9 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 10 amino acid substitutions (e.g., no more than 10 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 11 amino acid substitutions (e.g., no more than 11 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 12 amino acid substitutions (e.g., no more than 12 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 13 amino acid substitutions (e.g., no more than 13 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 14 amino acid substitutions (e.g., no more than 14 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 15 amino acid substitutions (e.g., no more than 15 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 16 amino acid substitutions (e.g., no more than 16 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 17 amino acid substitutions (e.g., no more than 17 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 18 amino acid substitutions (e.g., no more than 18 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 19 amino acid substitutions (e.g., no more than 19 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide. In some cases, a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure includes 20 amino acid substitutions (e.g., no more than 20 amino acid substitutions) compared to a corresponding reference (e.g., wild-type) immunomodulatory polypeptide.
  • TMAPP 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:
  • PD-L1 immunomodulatory polypeptide
  • PD1 cognate co-immunomodulatory polypeptide
  • IL-2 immunomodulatory polypeptide
  • IL-2 receptor cognate co- immunomodulatory polypeptide
  • CD80 immunomodulatory polypeptide
  • CD28 cognate co-immunomodulatory polypeptide
  • CD86 immunomodulatory polypeptide
  • CD28 cognate co-immunomodulatory polypeptide
  • Fas ligand immunomodulatory polypeptide
  • Fas cognate co-immunomodulatory polypeptide
  • ICOS-L immunomodulatory polypeptide
  • ICOS cognate co-immunomodulatory polypeptide
  • ICAM immunomodulatory polypeptide
  • LFA-1 cognate co-immunomodulatory polypeptide
  • CD30L immunomodulatory polypeptide
  • CD30 cognate co-immunomodulatory polypeptide
  • k CD40 (immunomodulatory polypeptide) and CD40L (cognate co-immunomodulatory polypeptide);
  • CD83 immunomodulatory polypeptide
  • CD83L cognate co-immunomodulatory polypeptide
  • HVEM immunomodulatory polypeptide
  • CD160 cognate co- immunomodulatory polypeptide
  • JAG1 CD339
  • Notch cognate co- immunomodulatory polypeptide
  • JAG1 immunomodulatory polypeptide
  • CD46 cognate co-immunomodulatory polypeptide
  • CD80 immunomodulatory polypeptide
  • CTLA4 cognate co-immunomodulatory polypeptide
  • CD86 immunomodulatory polypeptide
  • CTLA4 cognate co-immunomodulatory polypeptide
  • CD70 immunomodulatory polypeptide
  • CD27 cognate co-immunomodulatory polypeptide
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure has a binding affinity for a cognate co-immunomodulatory polypeptide that is from 100 nM to 100 ⁇ .
  • a variant immunomodulatory polypeptide present in a TMAPP 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
  • 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. Binding affinity between a TMAPP and its cognate co-immunomodulatory polypeptide can also be determined by BLI using purified TMAPP and the cognate co- immunomodulatory polypeptide. BLI methods are well known to those skilled in the art. See, e.g., Lad et al. (2015) /. Biomol. Screen. 20(4):498-507; and Shah and Duncan (2014) /. Vis. Exp. 18:e51383.
  • the specific and relative binding affinities described in this disclosure between an immunomodulatory polypeptide and its cognate co-immunomodulatory polypeptide, or between a synTac and its cognate co-immunomodulatory polypeptide, can be determined using the following procedures.
  • a BLI assay can be carried out using an Octet RED 96 (Pal ForteBio) instrument, or a similar instrument, as follows.
  • a TMAPP e.g., a TMAPP of the present disclosure; a control TMAPP (where a control TMAPP comprises a wild-type immunomodulatory polypeptide)
  • the immobilized TMAPP is the "target.” Immobilization can be effected by immobilizing a capture antibody onto the insoluble support, where the capture antibody immobilizes the TMAPP.
  • immobilization can be effected by immobilizing anti-Fc (e.g., anti-human IgG Fc) antibodies onto the insoluble support, where the immobilized anti-Fc antibodies bind to and immobilize the TMAPP (where the TMAPP comprises an IgFc polypeptide).
  • a co-immunomodulatory polypeptide is applied, at several different concentrations, to the immobilized TMAPP, and the instrument's response recorded.
  • Assays are conducted in a liquid medium comprising 25mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KC1, 0.1% bovine serum albumin, and 0.02% Tween 20 nonionic detergent.
  • Binding of the co-immunomodulatory polypeptide to the immobilized TMAPP is conducted at 30°C.
  • an anti-MHC Class II monoclonal antibody can be used as a positive control for binding affinity.
  • an anti-HLD-DR3 monoclonal antibody such as the 16-23 antibody (Sigma; also referred to as "16.23”; see, e.g., Pious et al. (1985) /. Exp. Med. 162: 1193; Mellins et al. (1991) /. Exp. Med. 174: 1607; ECACC hybridoma collection 16-23, ECACC 99043001) can be used as a positive control for binding affinity.
  • a pan-HLA Class II antibody such as the HKB1 antibody (Immunotools; Holte et al. (1989) Eur. J. Immunol. 19: 1221) can be used as a positive control for binding affinity.
  • a standard curve can be generated using serial dilutions of the anti-MHC Class II monoclonal antibody.
  • the co- immunomodulatory polypeptide, or the anti-MHC Class II mAb, is the "analyte.”
  • BLI analyzes the interference pattern of white light reflected from two surfaces: i) from the immobilized polypeptide ("target"); and ii) an internal reference layer.
  • a change in the number of molecules ("analyte”; e.g., co-immunomodulatory polypeptide; anti-HLA antibody) bound to the biosensor tip causes a shift in the interference pattern; this shift in interference pattern can be measured in real time.
  • the two kinetic terms that describe the affinity of the target/analyte interaction are the association constant (fa) and dissociation constant (fa). The ratio of these two terms (fa/ a ) gives rise to the affinity constant KD.
  • determining binding affinity between an immunomodulatory polypeptide e.g., IL-2 or an IL-2 variant
  • its cognate co-immunomodulatory polypeptide e.g., IL-2R
  • BLI binding affinity between an immunomodulatory polypeptide and its cognate co-immunomodulatory polypeptide
  • the assay is similar to that described above for the TMAPP.
  • a BLI assay can be carried out using an Octet RED 96 (Pal ForteBio) instrument, or a similar instrument, as follows.
  • a component immunomodulatory polypeptide of a TMAPP of the present disclosure e.g., a variant IL-2 polypeptide of the present disclosure
  • a control immunomodulatory polypeptide where a control immunomodulatory polypeptide comprises a wild-type immunomodulatory polypeptide, e.g. wild-type IL-2)
  • the immunomodulatory polypeptide is the "target.”
  • Immobilization can be effected by immobilizing a capture antibody onto the insoluble support, where the capture antibody immobilizes the immunomodulatory polypeptide.
  • an immuno-affinity tag e.g. FLAG, human IgG Fc
  • immobilization can be effected by immobilizing with the appropriate antibody to the immuno-affinity tag (e.g. anti- human IgG Fc) onto the insoluble support, where the immobilized antibodies bind to and immobilize the immunomodulatory polypeptide (where the immunomodulatory polypeptide comprises an IgFc polypeptide).
  • a co-immunomodulatory polypeptide (or polypeptides) is applied, at several different concentrations, to the immobilized immunomodulatory polypeptide, and the instrument's response recorded.
  • a co-immunomodulatory polypeptide (or polypeptides) is immobilized to the biosensor (e.g., for the IL-2 receptor heterotrimer, as a monomeric subunit, heterodimeric subcomplex, or the complete heterotrimer) and the immunomodulatory polypeptide is applied, at several different concentrations, to the immobilized coimmunomodulatory polypeptide(s), and the instrument's response is recorded.
  • Assays are conducted in a liquid medium comprising 25mM HEPES pH 6.8, 5% poly(ethylene glycol) 6000, 50 mM KC1, 0.1% bovine serum albumin, and 0.02% Tween 20 nonionic detergent. Binding of the co-immunomodulatory polypeptide to the immobilized
  • immunomodulatory polypeptide is conducted at 30°C.
  • BLI analyzes the interference pattern of white light reflected from two surfaces: i) from the immobilized polypeptide ("target”); and ii) an internal reference layer.
  • a change in the number of molecules (“analyte”; e.g., co- immunomodulatory polypeptide) bound to the biosensor tip causes a shift in the interference pattern; this shift in interference pattern can be measured in real time.
  • the two kinetic terms that describe the affinity of the target/analyte interaction are the association constant (fa) and dissociation constant (fa).
  • the ratio of these two terms gives rise to the affinity constant KD- Determining the binding affinity of both a wild-type immunomodulatory polypeptide (e.g., IL-2) for its receptor (e.g., IL-2R) and a variant immunomodulatory polypeptide (e.g., an IL-2 variant as disclosed herein) for its cognate co-immunomodulatory polypeptide (e.g., its receptor) (e.g., IL-2R) thus allows one to determine the relative binding affinity of the variant co- immunomodulatory polypeptide, as compared to the wild-type co-immunomodulatory polypeptide, for the cognate co-immunomodulatory polypeptide.
  • a wild-type immunomodulatory polypeptide e.g., IL-22
  • a variant immunomodulatory polypeptide e.g., an IL-2 variant as disclosed herein
  • co-immunomodulatory polypeptide e.g., its receptor
  • IL-2R cognate
  • the BLI assay is carried out in a multi-well plate. To run the assay, the plate layout is defined, the assay steps are defined, and biosensors are assigned in Octet Data Acquisition software. The biosensor assembly is hydrated. The hydrated biosensor assembly and the assay plate are equilibrated for 10 minutes on the Octet instrument. Once the data are acquired, the acquired data are loaded into the Octet Data Analysis software. The data are processed in the Processing window by specifying method for reference subtraction, y-axis alignment, inter-step correction, and Savitzky-Golay filtering.
  • KD values for each data trace can be averaged if within a 3-fold range. KD error values should be within one order of magnitude of the affinity constant values; R 2 values should be above 0.95. See, e.g., Abdiche et al. (2008) /. Anal. Biochem. 377:209.
  • TMAPP comprises a wild-type immunomodulatory polypeptide) to a cognate co- immunomodulatory polypeptide to ii) the binding affinity of a TMAPP of the present disclosure comprising a variant of the wild-type immunomodulatory polypeptide to the cognate co- immunomodulatory polypeptide, when measured by BLI (as described above), is at least 1.5: 1, at least 2: 1, at least 5: 1, at least 10:1, at least 15: 1, at least 20: 1, at least 25: 1, at least 50: 1, at least 100: 1, at least 500:1, at least 10 2 :1, at least 5 x 10 2 :1, at least 10 3 :1, at least 5 x 10 3 :1, at least 10 4 : 1 , at least 10 s : 1, or at least 10 6 :1.
  • the ratio of: i) the binding affinity of a control TMAPP (where the control TMAPP comprises a wild-type immunomodulatory polypeptide) to a cognate co-immunomodulatory polypeptide to ii) the binding affinity of a TMAPP of the present disclosure comprising a variant of the wild-type immunomodulatory polypeptide to the cognate co-immunomodulatory polypeptide, when measured by BLI, is in a range of from 1.5: 1 to 10 6 : 1 , e.g., from 1.5: 1 to 10: 1, from 10: 1 to 50: 1, from 50: 1 to 10 2 : 1 , from 10 2 :1 to 10 3 :1, froml0 3 : l to 10 4 : 1, from 10 4 :1 to 10 5 :1, or from 10 5 : 1 to 10 6 : 1.
  • the epitope present in a TMAPP of the present disclosure binds to a T-cell receptor
  • TCR on a T cell with an affinity of at least 100 ⁇ (e.g., at least 10 ⁇ , at least 1 ⁇ , at least 100 nM, at least 10 nM, or at least 1 nM).
  • the epitope present in a TMAPP of the present disclosure binds to a TCR on a T cell with an affinity of from about 10 4 M to about 5 x 10 4 M, from about 5 x 10 4 M to about 10 s M, from about 10 s M to 5 x 10 s M, from about 5 x 10 s M to 10 6 M, from about 10 6 M to about 5 x 10 6 M, from about 5 x 10 6 M to about 10 7 M, from about 10 7 M to about 5 x 10 7 M, from about 5 x 10 7 M to about 10 s M, or from about 10 s M to about 10 9 M.
  • the epitope present in a TMAPP of the present disclosure binds to a TCR on a T cell with an affinity of from about 1 nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 50 nM, from about 50 nM to about 100 nM, from about 0.1 ⁇ to about 0.5 ⁇ , from about 0.5 ⁇ to about 1 ⁇ , from about 1 ⁇ to about 5 ⁇ , from about 5 ⁇ to about 10 ⁇ , from about 10 ⁇ to about 25 ⁇ , from about 25 ⁇ to about 50 ⁇ , from about 50 ⁇ to about 75 ⁇ , from about 75 ⁇ to about 100 ⁇ .
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure has a binding affinity for a cognate co-immunomodulatory polypeptide that is from 1 nM to 100 nM, or from 100 nM to 100 ⁇ .
  • a variant immunomodulatory polypeptide present in a TMAPP 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 to about 1 ⁇ , to about 1 ⁇ to about 5 ⁇ , from about 5 ⁇ to about 10 ⁇ , from about 10 ⁇ to about 15 ⁇ , from about 15 ⁇ to about 20 ⁇ , from about 20 ⁇
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure has a binding affinity for a cognate co-immunomodulatory polypeptide that is from about 1 nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 50 nM, from about 50 nM to about 100 nM.
  • PD-Ll variants are from about 1 nM to about 5 nM, from about 5 nM to about 10 nM, from about 10 nM to about 50 nM, from about 50 nM to about 100 nM.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure is a variant PD-Ll polypeptide. Wild-type PD-Ll binds to PDl.
  • a wild-type human PD-Ll polypeptide can comprise the following amino acid
  • a wild-type human PD-Ll ectodomain can comprise the following amino acid
  • a wild-type PD-1 polypeptide can comprise the following amino acid sequence:
  • a variant PD-Ll polypeptide exhibits reduced binding affinity to PD-1
  • a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3 compared to the binding affinity of a PD-Ll polypeptide comprising the amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2.
  • a variant PD-Ll polypeptide of the present disclosure binds PD-1 (e.g., a PD-1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3) 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-Ll polypeptide comprising the amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2.
  • a variant PD-Ll polypeptide has a binding affinity to PD-lthat is from
  • a variant PD-Ll polypeptide of the present disclosure has a binding affinity to PD-1 that is from 100 nM to 100 ⁇ .
  • a variant PD-Ll polypeptide has a binding affinity for PDl (e.g., a PDl polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3) 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 PD-Ll polypeptide has a single amino acid substitution
  • a variant PD-Ll polypeptide has from 2 to 10 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2. In some cases, a variant PD-Ll polypeptide has 2 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2. In some cases, a variant PD-Ll polypeptide has 3 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2.
  • a variant PD-Ll polypeptide has 4 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2. In some cases, a variant PD-Ll polypeptide has 5 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2. In some cases, a variant PD-Ll polypeptide has 6 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2.
  • a variant PD-Ll polypeptide has 7 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2. In some cases, a variant PD-Ll polypeptide has 8 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2. In some cases, a variant PD-Ll polypeptide has 9 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2.
  • a variant PD-Ll polypeptide has 10 amino acid substitutions compared to the PD-Ll amino acid sequence set forth in SEQ ID NO: l or SEQ ID NO:2.
  • a suitable PD-L1 variant includes a polypeptide that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILWDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 108 ) , where X is any amino acid other than Asp. In some cases, X is Ala. In some cases, X is Arg.
  • a suitable PD-L1 variant includes a polypeptide that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILWDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIPGNI LNVSIKI (SEQ ID NO: 109), where X is any amino acid other than He. In some cases, X is Asp.
  • a suitable PD-L1 variant includes a polypeptide that comprises an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure is a variant CD80 polypeptide. Wild-type CD80 binds to CD28.
  • a wild-type amino acid sequence of the ectodomain of human CD80 can be as follows:
  • a wild-type CD28 amino acid sequence can be as follows: MLRLLLALNL FPS IQVTGNK I LVKQSPMLV AYDNAVNLS C KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDI Y FCKI EVMYPP PYLDNEKSNG T I I HVKGKHL CPSPLFPGPS KPFWVLVWG GVLACYS LLV TVAF I IFWVR SKRSRLLHS D YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS (SEQ ID NO:5).
  • a wild-type CD28 amino acid sequence can be as follows: MLRLLLALNL
  • a wild-type CD28 amino acid sequence can be as follows: MLRLLLALNL
  • a variant CD80 polypeptide exhibits reduced binding affinity to CD28, compared to the binding affinity of a CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4 for CD28.
  • a variant CD80 polypeptide binds CD28 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 CD80 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4 for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NO:5, 6, or 7).
  • a variant CD80 polypeptide has a binding affinity to CD28 that is from
  • a variant CD80 polypeptide of the present disclosure has a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7) 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 ⁇ , to about 1 ⁇ to about 5 ⁇ , from about 5 ⁇ to about 10
  • a variant CD80 polypeptide has a single amino acid substitution
  • a variant CD80 polypeptide has from 2 to 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 2 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 3 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 4 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4.
  • a variant CD80 polypeptide has 5 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 6 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 7 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 8 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 9 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4. In some cases, a variant CD80 polypeptide has 10 amino acid substitutions compared to the CD80 amino acid sequence set forth in SEQ ID NO:4.
  • Suitable CD80 variants include a polypeptide that comprises an amino acid sequence having 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 variant immunomodulatory polypeptide present in a T TMAPP of the present disclosure is a variant CD86 polypeptide. Wild-type CD86 binds to CD28.
  • amino acid sequence of the full ectodomain of a wild-type human CD 86 can be as follows:
  • amino acid sequence of the IgV domain of a wild-type human CD86 can be as follows:
  • a variant CD86 polypeptide exhibits reduced binding affinity to CD28, compared to the binding affinity of a CD 86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:9 for CD28.
  • a variant CD86 polypeptide binds CD28 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 CD86 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO:9 for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NO: 5, 6, or 7).
  • a variant CD86 polypeptide has a binding affinity to CD28 that is from
  • a variant CD 86 polypeptide of the present disclosure has a binding affinity for CD28 (e.g., a CD28 polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs:5, 6, or 7) 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 ⁇ , to about 1 ⁇ to about 5 ⁇ , from about 5 ⁇ to about 10 ⁇ , from about
  • a variant CD86 polypeptide has a single amino acid substitution
  • a variant CD 86 polypeptide has from 2 to 10 amino acid substitutions compared to the CD 86 amino acid sequence set forth in SEQ ID NO: 8. In some cases, a variant CD 86 polypeptide has 2 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8. In some cases, a variant CD86 polypeptide has 3 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8. In some cases, a variant CD86 polypeptide has 4 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8.
  • a variant CD86 polypeptide has 5 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8. In some cases, a variant CD86 polypeptide has 6 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8. In some cases, a variant CD86 polypeptide has 7 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8. In some cases, a variant CD86 polypeptide has 8 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8. In some cases, a variant CD86 polypeptide has 9 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8. In some cases, a variant CD86 polypeptide has 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:8.
  • a variant CD86 polypeptide has a single amino acid substitution
  • a variant CD 86 polypeptide has from 2 to 10 amino acid substitutions compared to the CD 86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD 86 polypeptide has 2 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD86 polypeptide has 3 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD86 polypeptide has 4 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9.
  • a variant CD86 polypeptide has 5 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD86 polypeptide has 6 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD86 polypeptide has 7 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD86 polypeptide has 8 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD86 polypeptide has 9 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9. In some cases, a variant CD86 polypeptide has 10 amino acid substitutions compared to the CD86 amino acid sequence set forth in SEQ ID NO:9.
  • Suitable CD86 variants include a polypeptide that comprises an amino acid sequence having 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:
  • MXRTSFDSDSWTLRLHNLQIKDKGLYQCI IHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 132), where X is any amino acid other than Asn. In some cases, X is Ala;
  • MNRTSFXSDSWTLRLHNLQIKDKGLYQCI IHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 133), where X is any amino acid other than Asp. In some cases, X is Ala;
  • MNRTSFDSDSXTLRLHNLQIKDKGLYQCI IHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 134), where X is any amino acid other than Trp. In some cases, X is Ala;
  • TSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 136), where X is any amino acid other than Val. In some cases, X is Ala;
  • MNRTSFDSDSWTLRLHNLQIKDKGLYQCI IHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 137), where X is any amino acid other than Val. In some cases, X is Ala;
  • TSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 138), where X is any amino acid other than Gin. In some cases, X is Ala;
  • MNRTSFDSDSWTLRLHNLQIKDKGLYQCI IHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 139), where X is any amino acid other than Gin. In some cases, X is Ala;
  • TSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP (SEQ ID NO: 140), where X is any amino acid other than Phe.
  • X is Ala;
  • APLKIQAYFNETADLPCQFANSQNQSLSELWXWQDQENLVLNEVYLGKEKFDSVHSKY MNRTSFDSDSWTLRLHNLQIKDKGLYQCI IHHKKPTGMIRIHQMNSELSVL (SEQ ID NO: 141), where X is any amino acid other than Phe.
  • X is Ala;
  • the first X is any amino acid other than Asn and the second X is any amino acid other than His.
  • the first and the second X are both Ala;
  • APLKIQAYFNETADLPCQFANSQNQSLSELWFWQDQENLVLNEVYLGKEKFDSVHSKY MX 1 RTSFX 2 SDSWTLRLHNLQIKDKGLYQCIIHX 3 KKPTGMIRIHQMNSELSVL (SEQ ID NO: 151), where Xi is any amino acid other than Asn, X 2 is any amino acid other than Asp, and X 3 is any amino acid other than His . In some cases, Xi is Ala, X 2 is Ala, and X 3 is Ala.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure is a variant 4-lBBL polypeptide. Wild-type 4-lBBL binds to 4-lBB (CD137).
  • a wild-type 4-lBBL amino acid sequence can be as follows: MEYASDASLD
  • a variant 4-lBBL polypeptide is a variant of the tumor necrosis factor
  • TNF homology domain
  • a wild-type amino acid sequence of the THD of human 4-lBBL can be, e.g., one of
  • a wild-type 4-1BB amino acid sequence can be as follows: MGNSCYNIVA
  • a variant 4-1BBL polypeptide exhibits reduced binding affinity to 4-1BB, compared to the binding affinity of a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 10-13.
  • a variant 4-1BBL polypeptide of the present disclosure binds 4-1BB 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 a 4-1BBL polypeptide comprising the amino acid sequence set forth in one of SEQ ID NOs: 10- 13 for a 4- IBB polypeptide (e.g., a 4-1BB polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14), when assayed under the same conditions.
  • 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%
  • a variant 4-1BBL polypeptide has a binding affinity to 4-1BB that is from
  • a variant 4-1BBL polypeptide has a binding affinity for 4-1BB (e.g., a 4-1BB polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14) 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 ⁇ , to about 1 ⁇ to about 5 ⁇ , from about 5 ⁇ to about 10 ⁇ , from about 10 ⁇ to about 15
  • 4-1BB e.g., a 4-1BB poly
  • a variant 4-lBBL polypeptide has a single amino acid substitution
  • a variant 4-lBBL polypeptide has from 2 to 10 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13.
  • a variant 4- 1BBL polypeptide has 2 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13.
  • a variant 4-lBBL polypeptide has 3 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13.
  • a variant 4-lBBL polypeptide has 4 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13. In some cases, a variant 4-lBBL polypeptide has 5 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13. In some cases, a variant 4-lBBL polypeptide has 6 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13. In some cases, a variant 4-lBBL polypeptide has 7 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13.
  • a variant 4-lBBL polypeptide has 8 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13. In some cases, a variant 4-lBBL polypeptide has 9 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13. In some cases, a variant 4-lBBL polypeptide has 10 amino acid substitutions compared to the 4-lBBL amino acid sequence set forth in one of SEQ ID NOs: 10-13.
  • Suitable 4-lBBL variants include a polypeptide that comprises an amino acid sequence having 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:
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 152), where X is any amino acid other than Lys. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWXLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 153), where X is any amino acid other than Gin. In some cases, X is Ala; [00321] PAGLLDLRQG XFAQLVAQNV LLIDGPLSWY SDPGLAGVSL TGGLSYKEDT
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 154), where X is any amino acid other than Met. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 155), where X is any amino acid other than Phe. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 156), where X is any amino acid other than Gin. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 157), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 158), where X is any amino acid other than Val. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 159), where X is any amino acid other than Gin. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 160), where X is any amino acid other than Asn. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 161), where X is any amino acid other than Val. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 162), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 163), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 164), where X is any amino acid other than He. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 165), where X is any amino acid other than Asp. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 166), where X is any amino acid other than Gly. In some cases, X is Ala;
  • GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 167), where X is any amino acid other than Pro. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 168), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 169), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 170), where X is any amino acid other than Trp. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 171), where X is any amino acid other than Tyr. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 172), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 173), where X is any amino acid other than Asp. In some cases, X is Ala; [00341] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDXGLAGVSL TGGLSYKEDT
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 174), where X is any amino acid other than Pro. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 175), where X is any amino acid other than Gly. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 176), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 177), where X is any amino acid other than Gly. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 178), where X is any amino acid other than Val. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 179), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 180), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 181), where X is any amino acid other than Thr. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 182), where X is any amino acid other than Gly. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 183), where X is any amino acid other than Gly. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 184), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 185), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 186), where X is any amino acid other than Tyr. In some cases, X is Ala;
  • GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 187), where X is any amino acid other than Glu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 188), where X is any amino acid other than Asp. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 189), where X is any amino acid other than Thr. In some cases, X is Ala;
  • XELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 190), where X is any amino acid other than Lys. In some cases, X is Ala;
  • KXLVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 191), where X is any amino acid other than Glu. In some cases, X is Ala;
  • KELVVAKAGV YYVXFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 192), where X is any amino acid other than Phe. In some cases, X is Ala;
  • KELVVAKAGV YYVFXQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:913), where X is any amino acid other than Phe. In some cases, X is Ala; [00361] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT
  • KELVVAKAGV YYVFFXLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 194), where X is any amino acid other than Gin. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQXELR RVVAGEGSGS GVSALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 195), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLXLR RVVAGEGSGS G VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 196), where X is any amino acid other than Glu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLEXR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 197), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELX RVVAGEGSGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 198), where X is any amino acid other than Arg. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR XVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 199), where X is any amino acid other than Arg. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RXVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:200), where X is any amino acid other than Val. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVXAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:201), where X is any amino acid other than Val. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAXEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:202), where X is any amino acid other than Gly. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGXGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:203), where X is any amino acid other than Glu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEXSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:204), where X is any amino acid other than Gly. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGXGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:205), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVXLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:206), where X is any amino acid other than Asp. In some cases, X is Ala;
  • GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:207), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLXPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:208), where X is any amino acid other than Pro. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPAXS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:209), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASX EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:210), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS XARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:211), where X is any amino acid other than Glu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EAXNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:212), where X is any amino acid other than Arg. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARXSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:213), where X is any amino acid other than Asn. In some cases, X is Ala; [00381] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNXAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:214), where X is any amino acid other than Ser. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAXGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:215), where X is any amino acid other than Phe. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGX RLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:216), where X is any amino acid other than Gin. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ XLGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:217), where X is any amino acid other than Arg. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RXGVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:218), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLXVHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:219), where X is any amino acid other than Gly. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGXHLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:220), where X is any amino acid other than Val. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVXLHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:221), where X is any amino acid other than His. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHXHTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:222), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLXTEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:223), where X is any amino acid other than His. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHXEA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:224), where X is any amino acid other than Thr. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTXA RARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:225), where X is any amino acid other than Glu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA XARHAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:226), where X is any amino acid other than Arg. In some cases, X is Ala;
  • GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:227), where X is any amino acid other than Arg. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARXAWQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:228), where X is any amino acid other than His. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAXQLTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:229), where X is any amino acid other than Trp. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQXTQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:230), where X is any amino acid other than Leu. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLXQ GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:231), where X is any amino acid other than Thr. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTX GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:232), where X is any amino acid other than Gin. In some cases, X is Ala;
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ XATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:233), where X is any amino acid other than Gly. In some cases, X is Ala; [00401] PAGLLDLRQG MFAQLVAQNV LLIGGPLSWY SDPGLAGVSL TGGLSYKEDT
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GAXVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:234), where X is any amino acid other than Thr. In some cases, X is Ala; and
  • KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATXLGLFRV TPEIPAGLPS PRSE (SEQ ID NO:235), where X is any amino acid other than Val. In some cases, X is Ala.
  • a variant immunomodulatory polypeptide present in a TMAPP of the present disclosure is a variant IL-2 polypeptide. Wild-type IL-2 binds to IL-2 receptor (IL-2R).
  • IL-2R IL-2 receptor
  • a wild-type IL-2 amino acid sequence can be as follows: APTSSSTKKT
  • Wild-type IL2 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-2Ra; also referred to as CD25), a beta chain (IL-2R ; also referred to as CD122: and a gamma chain (IL-2Ry; also referred to as CD 132).
  • IL-2Ra alpha chain
  • IL-2R also referred to as CD122
  • IL-2Ry also a gamma chain
  • Amino acid sequences of human IL-2Ra, IL2R , and IL-2Ry can be as follows.
  • Human IL-2Ra ELCDDDPPE IPHATFKAMA YKEGTMLNCE CKRGFRRIKS
  • Human IL-2R VNG TSQFTCFYNS RANISCVWSQ DGALQDTSCQ
  • Human IL-2Ry LNTTILTP NGNEDTTADF FLTTMPTDSL SVSTLPLPEV
  • TMAPP of the present disclosure comprises a variant IL-2
  • a "cognate co-immunomodulatory polypeptide” is an IL-2R comprising polypeptides comprising the amino acid sequences of SEQ ID NO: 16, 17, and 18.
  • a variant IL-2 polypeptide exhibits reduced binding affinity to IL-2R, compared to the binding affinity of a IL-2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15.
  • 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 set forth in SEQ ID NO: 15 for an IL-2R (e.g., an IL-2R comprising polypeptides comprising the amino acid sequence set forth in SEQ ID NO:
  • a variant IL-2 polypeptide has a binding affinity to IL-2R that is from 100 nM to 100 ⁇ .
  • a variant IL-2 polypeptide has a binding affinity for IL-2R (e.g., an IL-2R comprising polypeptides comprising the amino acid sequence set forth in SEQ ID NOs: 16-18) 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
  • a variant IL-2 polypeptide has a single amino acid substitution compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has from 2 to 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has 2 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has 3 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15.
  • a variant IL-2 polypeptide has 4 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has 5 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has 6 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has 7 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has 8 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15.
  • a variant IL-2 polypeptide has 9 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15. In some cases, a variant IL-2 polypeptide has 10 amino acid substitutions compared to the IL-2 amino acid sequence set forth in SEQ ID NO: 15.
  • Suitable IL-2 variants include a polypeptide that comprises an amino acid sequence having 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:
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 236), where X is any amino acid other than Phe. In some cases, X is Ala;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:237), where X is any amino acid other than Asp. In some cases, X is Ala;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:238), where X is any amino acid other than Glu. In some cases, X is Ala;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:239), where X is any amino acid other than His.
  • X is Ala.
  • X is Arg.
  • X is Asn.
  • X is Asp.
  • X is Cys.
  • X is Glu.
  • X is Gin.
  • X is Gly.
  • X is He. I n some cases, X is Lys.
  • X is Leu.
  • X is Met.
  • X is Phe. In some cases, X is Pro. In some cases, X is Ser. In some cases, X is Thr. In some cases, X is Tyr. In some cases, X is Trp. In some cases, X is Val;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 240), where X is any amino acid other than Tyr. In some cases, X is Ala;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCXSIIS TLT (SEQ ID NO:241), where X is any amino acid other than Gin. In some cases, X is Ala;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 242), where Xi is any amino acid other than His, and where X2 is any amino acid other than Phe.
  • Xi is Ala.
  • X2 is Ala.
  • Xi is Ala; and X2 is Ala;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:243), where Xi is any amino acid other than Asp; and where X2 is any amino acid other than Phe.
  • Xi is Ala.
  • X2 is Ala.
  • Xi is Ala; and X2 is Ala;
  • Xi is any amino acid other than Glu
  • X2 is any amino acid other than Asp
  • X3 is any amino acid other than Phe.
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • Xi is any amino acid other than His; where X 2 is any amino acid other than Asp; and where X 3 is any amino acid other than Phe.
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WI TFCX 3 S I IS TLT (SEQ ID NO:246), where Xi is any amino acid other than Asp; where X 2 is any amino acid other than Phe; and where X 3 is any amino acid other than Gin.
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • Xi is Ala; X 2 is Ala; and X 3 is Ala;
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO: 247), where Xi is any amino acid other than Asp; where X 2 is any amino acid other than Phe; and where X 3 is any amino acid other than Tyr.
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT (SEQ ID NO:248), where Xi is any amino acid other than His; where X 2 is any amino acid other than Asp; where X 3 is any amino acid other than Phe; and where X 4 is any amino acid other than Tyr.
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • X 4 is Ala.
  • Xi is Ala; X 2 is Ala; X 3 is Ala; and X 4 is Ala;
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • X 4 is Ala.
  • Xi is any amino acid other than His; where X 2 is any amino acid other than Asp; where X 3 is any amino acid other than Phe; where X 4 is any amino acid other than Tyr; and where X5 is any amino acid other than Gin.
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • X 4 is Ala.
  • X5 is Ala.
  • Xi is Ala.
  • X 2 is Ala.
  • X 3 is Ala.
  • Xi is Ala; X 2 is Ala; and X 3 is Ala.
  • an antigen-presenting polypeptide of the present invention is an antigen-presenting polypeptide of the present invention.
  • an antigen-presenting polypeptide of the present disclosure (including a TMAPP of the present disclosure) is a multimeric polypeptide comprising at least a first and a second polypeptide, in some cases, the first polypeptide comprises a first member of a dimerization pair, and the second polypeptide comprising a second member of the dimerization pair.
  • Dimerization peptides are known in the art; and any known dimerization peptide is suitable for use.
  • Dimerization peptides include polypeptides of the collectin family (e.g., ACRP30 or ACRP30-like proteins) which contain collagen domains consisting of collagen repeats Gly-Xaa-Xaa.
  • Other dimerization peptides include coiled-coil domains and leucine - zipper domains.
  • a collagen domain can comprise (Gly-Xaa-Xaa) n , where Xaa is any amino acid, and where n is an integer from 10 to 40.
  • a collagen domain comprises (Gly-Xaa- Pro) n , where Xaa is any amino acid and n is an integer from 10 to 40.
  • Dimerization peptides are well known in the art; see, e.g., U.S. Patent Publication No. 2003/0138440.
  • a dimerization pair includes two leucine zipper polypeptides that bind to one another.
  • leucine-zipper polypeptides include, e.g., a peptide of any one of the following amino acid sequences: RMKQIEDKIEEILSKIYHIENEIARIKKLIGER (SEQ ID NO:252); LSSIEKKQEEQTSWLIWISNELTLIRNELAQS (SEQ ID NO:253); LSSIEKKLEEITSQLIQISNELTLIRNELAQ (SEQ ID NO:254);
  • a leucine zipper polypeptide comprises the following amino acid
  • a collagen oligomerization peptide can comprise the following amino acid sequence:
  • SNP SEQ ID NO:260.
  • Coiled-coil dimerization peptides are known in the art.
  • a coiled-coil dimerization peptide is known in the art.
  • dimerization peptide can be a peptide of any one of the following amino acid sequences:
  • a dimerization peptide comprises at least one cysteine residue.
  • Examples include, e.g.: VDLEGSTSNGRQCAGIRL (SEQ ID NO:266);

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CA3071881A1 (en) 2019-03-14
EP3678677A1 (en) 2020-07-15
KR20200064083A (ko) 2020-06-05

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